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https://openalex.org/W2072082955	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0099003&type=printable	English	null	Human Retinal Transmitochondrial Cybrids with J or H mtDNA Haplogroups Respond Differently to Ultraviolet Radiation: Implications for Retinal Diseases	PloS one	2,014	cc-by	10,889	Human Retinal Transmitochondrial Cybrids with J or H mtDNA Haplogroups Respond Differently to Ultraviolet Radiation: Implications for Retinal Diseases Deepika Malik1, Tiffany Hsu1, Payam Falatoonzadeh1, Javier Ca´ceres-del-Carpio1, Mohamed Tarek1,2, Marilyn Chwa1, Shari R. Atilano1, Claudio Ramirez1, Anthony B. Nesburn1,3, David S. Boyer4, Baruch D. Kuppermann1, S. Michal Jazwinski5, Michael V. Miceli5, Douglas C. Wallace6, Nitin Udar1, M. Cristina Kenney1,7* 1 Gavin Herbert Eye Institute, University California Irvine, Irvine, California, United States of America, 2 Department of Ophthalmology, El-Minya University, El-Minya, Egypt, 3 Cedars-Sinai Medical Center, Los Angeles, California, United States of America, 4 Retina-Vitreous Associates Medical Group; Beverly Hills, California, United States of America, 5 Tulane Center for Aging, Tulane University, New Orleans, Louisiana, United States of America, 6 Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia and Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 7 Department of Pathology and Laboratory Medicine, University California Irvine, Irvine, California, United States of America Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Discovery Eye Foundation, Guenther Foundation, Beckman Initiative for Macular Research, Polly and Michael Smith Foundation, Max Factor Family Foundation, Skirball Foundation, Lincy Foundation, Iris and B. Gerald Cantor Foundation, and unrestricted grant from Research to Prevent Blindness. National Institute on Aging grant (AG006168) to SMJ and MVM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mkenney@uci.edu * E-mail: mkenney@uci.edu Nutrition Examination Survey (NHANES 2005–2008 report), the estimated prevalence of any AMD was 6.5% and of late AMD was 0.8% in the US population 40 years and older [2]. Aging and oxidative damage to retinal pigment epithelial (RPE) cells, a monolayer of cells located between the neural retina and Bruch’s membrane contributes to AMD [3]. Early AMD has sub-retinal drusen, and can progress to advanced AMD, which includes dry (non-exudative, atrophic) and wet (exudative, neovascular) forms Editor: Neena B. Haider, Schepens Eye Research Institute/Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, United States of America Abstract Background: It has been recognized that cells do not respond equally to ultraviolet (UV) radiation but it is not clear whether this is due to genetic, biochemical or structural differences of the cells. We have a novel cybrid (cytoplasmic hybrids) model that allows us to analyze the contribution of mitochondrial DNA (mtDNA) to cellular response after exposure to sub-lethal dose of UV. mtDNA can be classified into haplogroups as defined by accumulations of specific single nucleotide polymorphisms (SNPs). Recent studies have shown that J haplogroup is high risk for age-related macular degeneration while the H haplogroup is protective. This study investigates gene expression responses in J cybrids versus H cybrids after exposure to sub-lethal doses of UV-radiation. Methodology/Principal Findings: Cybrids were created by fusing platelets isolated from subjects with either H (n = 3) or J (n = 3) haplogroups with mitochondria-free (Rho0) ARPE-19 cells. The H and J cybrids were cultured for 24 hours, treated with 10 mJ of UV-radiation and cultured for an additional 120 hours. Untreated and treated cybrids were analyzed for growth rates and gene expression profiles. The UV-treated and untreated J cybrids had higher growth rates compared to H cybrids. Before treatment, J cybrids showed lower expression levels for CFH, CD55, IL-33, TGF-A, EFEMP-1, RARA, BCL2L13 and BBC3. At 120 hours after UV-treatment, the J cybrids had decreased CFH, RARA and BBC3 levels but increased CD55, IL- 33 and EFEMP-1 compared to UV-treated H cybrids. Conclusion/Significance: In cells with identical nuclei, the cellular response to sub-lethal UV-radiation is mediated in part by the mtDNA haplogroup. This supports the hypothesis that differences in growth rates and expression levels of complement, inflammation and apoptosis genes may result from population-specific, hereditary SNP variations in mtDNA. Therefore, when analyzing UV-induced damage in tissues, the mtDNA haplogroup background may be important to consider. Citation: Malik D, Hsu T, Falatoonzadeh P, Ca´ceres-del-Carpio J, Tarek M, et al. (2014) Human Retinal Transmitochondrial Cybrids with J or H mtDNA Haplogroups Respond Differently to Ultraviolet Radiation: Implications for Retinal Diseases. PLoS ONE 9(6): e99003. doi:10.1371/journal.pone.0099003 Editor: Neena B. Haider, Schepens Eye Research Institute/Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, United States of America Received December 17, 2013; Accepted May 8, 2014; Published June 11, 2014 Received December 17, 2013; Accepted May 8, 2014; Published June 11, 2014 lik et al. Cybrids Respond Differently to Ultraviolet Radiation [4]. The dry form of advanced AMD is characterized by geographic atrophy involving the macula and accounts for approximately 25% of cases with central vision loss [5]. The wet form accounts for 75% of the central loss of vision and is characterized by choroidal neovascularization and if untreated, eventually a disciform scar in the macular region [5]. [4]. The dry form of advanced AMD is characterized by geographic atrophy involving the macula and accounts for approximately 25% of cases with central vision loss [5]. The wet form accounts for 75% of the central loss of vision and is characterized by choroidal neovascularization and if untreated, eventually a disciform scar in the macular region [5]. migration to other continents, mtDNA SNP variations due to adaptations to different environmental conditions evolved. At this time, geographic origins of populations can be identified by their haplogroup patterns. In general, the mtDNA SNP variations in coding and non-coding regions are associated with different rates of replication, transcription and metabolic processes [16] and it has been proposed that haplogroup-related variants may lead to differential responses for cell functions. Solar radiation consists of variable wavelengths: UV-C (100– 280 nm), UV-B (280–315 nm), UV-A (315–400 nm) and visible spectrum (400–700 nm) [6]. Light with shorter wavelengths has higher energy and can produce intense tissue damage. The human cornea filters out most of the radiation below 295 nm [7]. The human lens in younger individuals can transmit UV-B radiation but by adulthood, the lens blocks almost all UV-A and UV-B and most of the blue light [8]. However, exposure to both UV-A and UV-B are known to produce cataract and UV-radiation imposes increased damage to both pediatric and adult retina. Damage induced by UV-radiation is cumulative and depends on the duration, wavelength and intensity of exposure. Both haplogroups H and J represent European-Caucasian populations. However, J haplogroup originated from northern Europe and the H haplogroup was from southern Europe. Recent studies have shown that individuals with the J haplogroup have higher susceptibility to developing AMD and that the H haplogroup is protective [17]. Our work, among others, has used the cybrid (cytoplasmic hybrid) model to demonstrate that the H and J haplogroups have differences in production of ATP, ROS and lactate levels, as well as cell growth rates and expressions of genes involved in the complement pathway [18–21]. Ethics Statement All research involving human participants was approved by the Institutional Review Board of University of California, Irvine (#2003-3131). Written informed consents were obtained and all clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Materials and Methods Aging is associated with increased DNA damage, including both mitochondrial (mtDNA) and nuclear DNA (nDNA), and de- creased efficacy of the DNA repair mechanisms [11]. The DNA repair process becomes less efficient in presence of risk factors such as UV exposure and environmental toxins [11]. The repair mechanisms for nDNA are more efficient than mtDNA and over time, the accumulation of DNA damage can be associated with age-related vascular dysfunctions [12], which may correlate to the wet form of AMD. Increased DNA damage can also trigger apoptosis and autophagy [13], which can contribute to degener- ative retinal diseases. The production of reactive oxygen/nitrogen species (ROS/RNS), such as superoxide radicals, hydrogen peroxide and peroxynitrites, are key factors as they lead to oxidative damage and apoptosis. Mitochondria are a major site of ROS production via the electron transport chain and because of this close proximity, the mtDNA is very susceptible to oxidative damage. Mitochondria are present in large numbers in retinal tissue since photoreceptors are one of the highest consumers of oxygen in the body. In 2006, Feher et al, reported significant decreases in numbers and size of mitochondria and an increase in peroxisomes in AMD retinas [14]. Harman theorized that mtDNA mutations and production of free radicals played a large role in the aging process [15]. Cybrids Respond Differently to Ultraviolet Radiation However, it is not known if the different mtDNA haplogroups might influence the responses of the cells to stress factors. After 3 years of age, the yellow pigment 3-hydroxykynurenine, a tryptophan metabolite, gradually develops in the human lens and absorbs UV-radiation, which provides protection to the retina. Lutein and zeaxanthin are macular pigments and along with glutathione protect the retina against inflammatory and photo- oxidative damage. Antioxidants, antioxidant enzymes, and mela- nin pigments within RPE cells and the choroid provide protection against light-induced ocular damage. However, with age, these pigments and antioxidants are gradually depleted, thereby making the retina more susceptible to free radical damage. Acute exposure to UV-radiation, as in sunlight reflected from snow or an eclipse, can produce inflammatory damage and interleukin responses [9,10]. Over a lifetime, chronic exposure induces accumulative photo-oxidative damage via singlet oxygen and free radical production that leads to damage of DNA, proteins and lipids. In the present study, we have used the human RPE cybrids that contain either the H or J haplogroups to investigate their responses to environmental stress using UV-radiation, a potential risk factor for AMD. After exposure to sub-lethal dose of UV-radiation, the H cybrids and J cybrids, which have identical nuclei but differ only in their mtDNA haplogroups, show different rates of cell growth and expression levels for genes involved in inflammation, cell proliferation and apoptosis. Our findings support the hypothesis that in responses UV radiation, the inherited mtDNA variants (haplogroups) influence cells to have differential responses in major molecular pathways, which are associated with many aging diseases. Transmitochondrial Cybrid Cultures The preparations of transmitochondrial cybrids were performed as described in our previous manuscripts [22–24]. Cybrids were grown in DMEM/Ham’s F12 1:1 (Invitrogen-Gibco, Grand Island, NY) containing 24 mM sodium bicarbonate, 10% dialyzed fetal bovine serum and 1.0 mM sodium pyruvate. Mitochondrial haplogroups of each cybrid were confirmed by using PCR, restriction enzyme digestion and sequencing of mtDNA. All experiments utilized the H and J cybrids, which were at passage 5. Cybrids that contained mitochondria with haplogroup H (n = 3 different individuals) or J (n = 3 different individuals) were studied in 2 groups, the Control (untreated) group and Study group (treated with UV-radiation). Assays to determine the growth curve and gene expression analyses were performed for the individual cybrids in the Control and Study groups. The human mtDNA is a circular double stranded DNA that encodes 37 genes including 13 protein subunits essential for oxidative phosphorylation, and a non-coding region mtDNA D- loop which is critical for replication and transcription. The mtDNA have been classified into various haplogroups based on variable combinations of single nucleotide polymorphisms (SNPs) that have accumulated over 150,000 years. The oldest hap- logroups have been identified from Africa and with eventual Introduction Age-related macular degeneration (AMD) is a multifactorial disease, wherein multiple genetic variants and environmental risk factors, including diet, smoking, hypertension, light exposure, ultraviolet (UV) radiation, blue light and ionizing radiation, contribute to the disease [1]. Despite new medical and surgical interventions, AMD is one of the important causes of decreased vision in the United States. According to the National Health And June 2014 \| Volume 9 \| Issue 6 \| e99003 1 PLOS ONE \| www.plosone.org Cybrids Respond Differently to Ultraviolet Radiation Quantitative-PCR (Q-PCR) Analyses Expression of Genes Normalized to 0-Hour H Cybrids Complement pathway inhibition genes. Initially we nor- malized the H-untreated cybrids at 0 hour value to 1 and then compared the H and J cybrid values of gene expression at 0, 72 and 120 hours (Figure 2). At 0 hours, we found that the H- untreated cybrids had significantly higher expression levels of CFH than J-untreated cybrids (1 vs. 0.6960.05 fold, p = 0.003). In the H-untreated cybrids, there was an increase in CFH gene expression at 72 hours (6.960.19 fold, p,0.0001) and 120 hours (5.8560.07 fold, p,0.0001) compared to the 0 hours H-untreated cybrids. The J-untreated cybrids started at a lower level of CFH expression (0.6960.05 fold, p,0.01) and increased to 4.460.1 fold, p,0.0001 and 5.260.03 fold, p,0.0001 at 72 and 120 hours, respectively. Compared to the 0-hour H cybrids, the H-treated cybrids cultured 72 hours and 120 hours had increased levels of CFH expression (4.860.15 fold, p,0.001 and 5.5260.12 fold, p,0.0001, respectively). At 72 hours and 120 hours, the J-treated cybrids also showed an increase expression of CFH compared to the 0-hour H-untreated cybrids (3.6360.2 fold, p,0.001 and 4.3160.13 fold, p,0.0001), respectively (Figure 2a). At 0-hours, the CD55 gene expression levels were significantly higher in the H-untreated cybrids versus J-untreated cybrids In our previous publication, we demonstrated that CFH, C3 and EFEMP-1 genes were differentially expressed in H versus J cybrids (at 0 hours) [18]. In the present study, Q-PCR was performed using primers (QuantiTect primer assay, Qiagen) for genes associated with alternate complement pathway- Comple- ment Factor-H (CFH), Decay Accelerating Factor for Comple- ment (CD55/DAF), Complement Regulatory Protein (CD59), genes related to inflammation and cellular proliferation- Interleu- kin-33 (IL-33), Transforming Growth Factor, alpha (TGF-A), EGF containing fibulin like extracellular matrix protein 1 (EFEMP-1) and pro-apoptotic genes- Retinoic acid receptor, alpha (RARA), BCL2 binding component 3 (BBC3), BCL2-like 13 (BCL2L13) (Table 1). The Q-PCR was performed on individual H cybrids (n = 3) and J cybrids (n = 3) using a QuantiFast SYBR Green PCR kit (Qiagen) on a Bio-Rad iQ5 iCycler detection system. Gene expression levels were standardized for all primers using Hypoxanthine-guanine phosphoribosyltransferase (HPRT1, NM_000194) as a reference gene. Each sample at individual time- points was assessed in triplicate. The selection of the genes analyzed in this study was based on our previous studies using the untreated H and J cybrids. Growth Curve Assay The growth curves of three different H cybrids and three different J cybrids in both Study and Control groups were analyzed over 6 days. For each individual cybrid, cells were plated and treated with UV-radiation as described above. Cells numbers were measured with the Cell Viability Analyzer (ViCell, Beckman Coulter, Miami, FL) using the trypan blue staining technique, which counts and averages cells over 50 images. The number of cells at the time of treatment (time-point 0 hours) was designated as 100% and percentage increase in growth of each cybrid were measured at 48, 96, 120 and 144 hours. The assays were run in triplicate and experiments were repeated twice. There were biological triplicates within each experiment. Statistical Analysis Data were subjected to statistical analysis by ANOVA using GraphPad Prism 5.0 version statistics program (GraphPad Software Inc., San Diego, CA). For the growth curve assays, the Control samples were normalized to 100% and then the cell count for the 48, 96, 120 and 144 hours samples were calculated accordingly. In the gene expression assays, the DCt was calculated as the difference between the Ct (threshold cycle) of primer and Ct of the housekeeper/reference gene. The DDCt was calculated as the mean difference of DCts of a specific primer at a single time point between H and J cybrids or DCts of the primer being compared at two different time points of either H or J cybrids. The fold values were calculated using the formula Fold = 22DDCt. Fold values were calculated relative to the readings for corresponding H cybrids at 0 hours. Data is presented as mean 6 standard error of mean (SEM). Experiments were performed in triplicate. P values ,0.05 (two-tail test) were considered statistically significant. Cybrids Respond Differently to Ultraviolet Radiation for cell viability 48 and 96 hours later to identify the sub-threshold dose. 5 mJ UV-radiation did not have any impact on growth rate in the study group when compared to the untreated cells (data not shown). When cybrid cultures were treated with 20 mJ of UV exposure, there was significant loss of cells in both the H and J cybrids (data not shown). However, ninety-six hours after a single pulse of 10 mJ UV-radiation, we found that the growth rates of cells were temporarily reduced but they increased by 120 hours indicating that the UV radiation had a sub-lethal effect on the cells. Therefore, we chose 10 mJ UV-radiation as the dosage to treat the cybrids. Individual H or J cybrids were plated at 300,000 cells per well onto six well plates with 2 ml of culture media. Twenty-four hours after plating, the culture media were removed and the cybrids were treated with a single 10 seconds pulse of 10 mJ of UV-radiation using Stratalinker 1800. For the Control group, no treatment was given but culture media were changed at that time. Thereafter, culture media were changed every 48 hours in both groups. cybrids were analyzed by the Affymetrix Human U133 Plus 2.0 Array, which is a comprehensive human genome expression array that allows for the analyses of over 40K transcripts. Then differences between the individual H and individual J cybrids were verified by Q-PCR analyses [22,24]. The CD59 were analyzed because it is a major inhibitor of the alternative pathway. Isolation of RNA and cDNA Synthesis RNA was isolated from both the Study and Control groups at 0 hours, 72 hours and 120 hours after UV exposure using the RNeasy Mini-Extraction kit (Qiagen, Inc.) as per the manufac- turer’s protocol. The RNA was quantified using NanoDrop 1000 (Thermoscientific, Inc.). RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen, Inc.). Growth Curve Assay The 0 hour cell counts were normalized to 100% in the both Control (untreated) and Study (UV-treated) groups (Figure 1). At each time point, J-untreated cybrids showed greater growth than H-untreated cybrids (48 hours- 141% vs. 122%, 96 hours- 183% vs. 157%, 120 hours- 254% vs. 192%, and 144 hours- 272% vs. 198%). In the UV-treated groups at time interval between 48 and 96 hours, there was initially a downslope followed by an upward trend in the growth patterns. The J-treated cybrids showed higher growth rates than H-treated cybrids (48 hours-116% vs. 98%, 96 hours- 113% vs. 72%, 120 hours- 188% vs. 156%, and 144 hours- 234% vs. 148%), but the growth rates were reduced in comparison to their corresponding untreated groups. UV-radiation Treatment A sub-lethal dose of UV-radiation was defined as the dose sufficient to decrease the growth rate of cells but not cause irreversible damage to cell viability. Initially the cybrids were treated with 5, 10, and 20 mJ of UV-radiation using the Stratalinker 1800 (Stratagene, Santa Clara, CA) and analyzed June 2014 \| Volume 9 \| Issue 6 \| e99003 PLOS ONE \| www.plosone.org 2 Cybrids Respond Differently to Ultraviolet Radiation Cybrids Respond Differently to Ultraviolet Radiation PLOS ONE \| www.plosone.org Quantitative-PCR (Q-PCR) Analyses With this category of genes, we again normalized the H cybrids at 0-hours to a value of 1 and then compared this against all other values (Figure 3). At 0-hours, Q-PCR analyses showed lower expression levels in J-untreated cybrids for IL-33 (0.3760.01 fold, p,0.0001) compared to the 0-hour H cybrids. At 120 hours, the J-untreated cybrids and the H-untreated cybrids showed significantly elevated levels of IL-33 compared to the 0- hour H cybrids (4.0960.09 fold, p,0.001 and 2.0960.09 fold, p,0.001, respectively, Figure 3a). At 120 hours after UV treatment, the IL-33 expression in the J-treated cybrids increased to 5.0160.1 fold (p,0.001) while the H-treated cybrids increased 2.9760.07 fold (p,0.001). Figure 1. Differential growth patterns were observed for H cybrids versus J cybrids in UV-treated and untreated groups. Cybrids containing J haplogroup mtDNA showed a steeper slope for the growth curve than H haplogroup mtDNA in both UV-treated and untreated groups. In untreated there were constant increases in growth patterns for both H and J cybrids. In the UV-treated samples, the growth rates slowed or declined until approximately 96 hours post treatment, and then increased again. Triplicate wells were run for each sample and the experiments were repeated twice. During the growth curve assay, each sample was run in triplicate. Grey solid line, untreated H cybrids; Grey broken line, UV-treated H cybrids; Black solid line, untreated J cybrids; Black broken line, UV-treated J cybrids. doi:10.1371/journal.pone.0099003.g001 A similar pattern was seen for the TGF-A expression levels, with the J-untreated cybrids having lower levels than the H-untreated cybrids at 0 hours (0.3860.02 vs. 1 fold, p,0.0001, Figure 3b). At 120 hours, the J-untreated (2.2660.08 fold, p,0.001) and H- untreated cybrids (1.9760.08 fold, p = 0.04) showed an elevated expression levels of TGF-A compared to the 0-hour H-untreated cybrids. After UV treatment, there was an increase for TGF-A expression in the J-treated cybrids (3.7160.14 fold, p,0.0001) and H cybrids, (2.760.05 fold, p,0.0001) compared to the 0-hour H cybrids. Figure 1. Differential growth patterns were observed for H cybrids versus J cybrids in UV-treated and untreated groups. Cybrids containing J haplogroup mtDNA showed a steeper slope for the growth curve than H haplogroup mtDNA in both UV-treated and untreated groups. In untreated there were constant increases in growth patterns for both H and J cybrids. In the UV-treated samples, the growth rates slowed or declined until approximately 96 hours post treatment, and then increased again. Quantitative-PCR (Q-PCR) Analyses Each of these genes were initially identified when cDNA from the H and J At 0-hours, the CD55 gene expression levels were significantly higher in the H-untreated cybrids versus J-untreated cybrids June 2014 \| Volume 9 \| Issue 6 \| e99003 3 3 PLOS ONE \| www.plosone.org Cybrids Respond Differently to Ultraviolet Radiation Table 1. Gene Functions and Accession Numbers. Gene Gene name Gene Bank Accession No. Function CFH Complement Factor H NM_000186. Essential in regulation of complement activation. CD55/DAF Decay accelerating factor for complement NM_000574, NM_001114543, NM_001114544, NM_00111475. Involved in regulation of complement cascade by accelerating decay of complement proteins and disrupting the cascade. CD59 CD59 Molecule, Complement Regulatory Protein NM_000611, NM_203329, NM_203331, NM_001127223, NM_001127225, NM_001127226, NM_001127227. Cell surface glycoprotein that regulates complement-mediated cell lysis, inhibits complement membrane attack complex and is involved in lymphocyte signal transduction. IL-33 Interleukin 33 NM_033439, NM_001127180, NM_001199640. Member of IL-1 family; Critical pro-inflammatory cytokine involved in production of T helper 2 associated cytokines. TGF-A Transforming Growth Factor, alpha NM_003236, NM_001099691. Ligand for EGFR that activates signaling pathway for cell proliferation, differentiation and activation. It is also upregulated in human cancers. EFEMP-1 EGF containing fibulin like extracellular matrix protein 1 NM_004105, NM_018894, NM_001039348, NM_001039349. High-risk gene associated with AMD, misfolded EFEMP-1 protein accumulates within RPE cells causing altered cellular function and inflammation. High EFEMP-1 levels are associated with tumor metastasis. RARA Retinoic acid receptor, alpha NM_000964, NM_001145301, NM_001033603. Nuclear retinoic acid receptor. Regulates transcription. Involved with apoptosis and differentiation. BBC3 BCL2 binding component 3 NM_014417, NM_00112741, NM_001127240. Member of BCL2 family, BH3 only pro-apoptotic subclass to induce mitochondrial outer membrane permeabilization, apoptosis, mitochondrial dysfunction and caspase activation. BLC2L13 BCL2-like 13 NM_015367 Mitochondria specific protein whose over-expression results in apoptosis. doi:10.1371/journal.pone.0099003.t001 (1.00 vs. 0.7760.05 fold, p = 0.002), however, both H and J cybrids did not increase greatly after UV treatment (Figure 2b). 0.8960.10 fold, p = 0.30) or after 120 hours of UV treatment (0.8660.08 fold, p = 0.14 vs 1.1260.04 fold, p = 0.06) (Figure 2c). (1.00 vs. 0.7760.05 fold, p = 0.002), however, both H and J cybrids did not increase greatly after UV treatment (Figure 2b). The CD59 expression levels at 0-hours were not significantly different between H-untreated and J-untreated cybrids (1 vs. Inflammation, cellular proliferation and angiogenesis genes. Cybrids Respond Differently to Ultraviolet Radiation Figure 2. Expression patterns of alternate complement inhib- itor genes after UV treatment. The 0-hour value for H-untreated cybrid was normalized to 1 and then compared to gene expression values at 0, 72 and 120 hours. Panel 2a: At 0-hour, the J-untreated cybrids had lower levels of CFH compared to H-untreated cybrids (p = 0.003). At 72 hours and 120 hours, the H-untreated cybrids (grey solid line) showed higher levels of CFH expression compared to the J- untreated cybrids (black solid line). After UV exposure, the H-treated cybrids (grey broken line) expressed increased CFH levels compared to the J-treated cybrids (black broken line) at both 72 and 120 hours. Panel 2b: At 0-hours the H-untreated cybrids showed higher expression levels for CH55 (grey solid line) compared to J-untreated cybrids (black solid line, p = 0.002). At 72 and 120 hours post-UV treatment, there were no significant differences in the CD55 expression levels. Panel 2c: The CD59 expression levels were similar for the untreated (grey solid and black solid lines) and UV-treated (grey broken and black broken lines) H and J cybrids. There were triplicate wells for each sample and the experiments were repeated twice. n = 3 different individuals for the H cybrids and n = 3 different individuals for the J cybrids. doi:10.1371/journal.pone.0099003.g002 values at 0, 72 and 120 hours. Panel 2a: At 0-hour, the J-untreated cybrids had lower levels of CFH compared to H-untreated cybrids (p = 0.003). At 72 hours and 120 hours, the H-untreated cybrids (grey solid line) showed higher levels of CFH expression compared to the J- untreated cybrids (black solid line). After UV exposure, the H-treated cybrids (grey broken line) expressed increased CFH levels compared to the J-treated cybrids (black broken line) at both 72 and 120 hours. Panel 2b: At 0-hours the H-untreated cybrids showed higher expression levels for CH55 (grey solid line) compared to J-untreated cybrids (black solid line, p = 0.002). At 72 and 120 hours post-UV treatment, there were no significant differences in the CD55 expression levels. Panel 2c: The CD59 expression levels were similar for the untreated (grey solid and black solid lines) and UV-treated (grey broken and black broken lines) H and J cybrids. There were triplicate wells for each sample and the experiments were repeated twice. n = 3 different individuals for the H cybrids and n = 3 different individuals for the J cybrids. Cybrids Respond Differently to Ultraviolet Radiation doi:10.1371/journal.pone.0099003.g002 showed significant increases after UV treatment (Figure 4). All samples were normalized to the H-untreated cybrid at time point 0-hours. The gene expression levels for the 0-hour J-untreated cybrids were significantly lower compared to the 0-hour H- untreated cybrids (RARA, 0.88+0.03 fold, p = 0.003, Figure 4a; BBC3, 0.7160.2 fold, p,0.01, Figure 4b; and BCL2L13; 0.8160.05 fold, p = 0.002, Figure 4c). At 120 hours after UV treatment, the H-treated and J-treated cybrids showed increased expression levels for RARA (1.9660.05 fold, p,0.001 and 1.6460.03 fold, p = 0.009, respectively); BBC3 (3.7860.15, p, 0.001 fold and 3.2160.09 fold, p,0.0001); and BCL2L13 (2.3960.05 fold, p,0.001 and 2.2060.15 fold, p,0.0001, re- spectively) compared to the 0-hour H-untreated cybrids. Quantitative-PCR (Q-PCR) Analyses Triplicate wells were run for each sample and the experiments were repeated twice. During the growth curve assay, each sample was run in triplicate. Grey solid line, untreated H cybrids; Grey broken line, UV-treated H cybrids; Black solid line, untreated J cybrids; Black broken line, UV-treated J cybrids. doi:10.1371/journal.pone.0099003.g001 At 0-hour, Q-PCR analyses showed lower expression levels for EFEMP-1 in J-untreated cybrids (0.2660.02 fold, p = 0.001) compared to the 0-hour H cybrids (Figure 3c). At 120 hours, compared to the 0-hours H cybrids, the EFEMP-1 gene expres- sion for both the J-untreated cybrids, (3.360.17 fold, p,0.0001) and J-treated cybrids (4.060.09 fold, p,0.0001) increased to a greater degree than H-untreated cybrids (1.5460.11 fold, p, 0.0001) and H-treated cybrids (2.3960.08 fold, p,0.0001). Expression levels for apoptosis genes. We analyzed 3 pro-apoptotic genes and found that only RARA and BBC3 June 2014 \| Volume 9 \| Issue 6 \| e99003 4 PLOS ONE \| www.plosone.org Cybrids Respond Differently to Ultraviolet Radiation Cybrids Respond Differently to Ultraviolet Radiation showed higher levels of IL-33 expression compared to the H-treated cybrids. Panel 3b: At 0-hours, the TGF-A expression levels were lower for the J-untreated cybrids (black solid line) compared to the H-untreated cybrids (grey solid line, p,0.0001). At 120 hours after UV-treatment, the TGF-A levels for both H-treated (grey broken line) and J-treated (black broken line) were higher than the untreated H (grey solid line) or J (black solid line) cybrids. Panel 3c: At 0-hours, the expression levels for EFEMP1 were lower in the J-untreated (black solid lines) compared to H- untreated cybrids (grey solid lines, p = 0.0001). By 120 hours, the J- treated cybrids (black broken line) and J-untreated cybrids (black solid line) showed greater fold increases of EFEMP1 expression compared to the H-treated (grey broken line) and H-untreated (grey solid line) cybrids. There were triplicate wells for each sample and the experiments were repeated twice. J cybrids, n = 3 different individuals; H cybrids, n = 3 different individuals. doi:10 1371/journal pone 0099003 g003 Figure 3. Expression patterns of inflammation, cellular pr eration and angiogenesis genes after UV treatment. The 0- values for H-untreated cybrids were normalized to 1 and compared to gene expression values at 0, 72 and 120 hours. P 3a: At 0-hour, the J-untreated cybrids (black solid line) had lo expression levels of IL-33 compared to H-untreated cybrids (grey line, p,0.0001). At 120 hours, the H-untreated cybrids (grey solid showed higher levels of IL-33 expression compared to the J-untre cybrids (black solid line). At both 72 and 120 hours, the J-treated cy doi:10.1371/journal.pone.0099003.g003 H-treated cybrids. However, the increases in TGF-A gene expression levels from their respective baseline values were quantitatively similar in both H-treated and J-treated cybrids. In Figure 5c, we analyzed the expression levels for the three pro- apoptosis genes comparing H-treated cybrids to J-treated cybrids at 120 hours. The J-treated cybrids consistently showed lower mean change in DDCt values compared to the H-treated cybrids for the RARA (DDCt 0.7160.07 vs. DDCt 1.0160.03, p = 0.02) and BBC3 genes (DDCt 1.6760.06 vs. DDCt 1.9460.04, p = 0.004) but not the BCL2L13 gene (DDCt 1.1260.07 vs. DDCt 1.2560.05, p = 0.28). This indicates that after UV radiation, the H-treated cybrids showed greater increases of expression levels of pro-apoptotic genes than J-treated cybrids. Cybrids Respond Differently to Ultraviolet Radiation In summary, our findings demonstrate that at different time periods after UV sub-lethal radiation, the J cybrids maintains higher rates of growth, higher expression levels for inflammation genes but lower expression levels for apoptosis-related genes (Figure 6). Comparison of Gene Expression Levels from 0 to 120 Hours The analyses performed above compared the gene expression values for all samples to the 0-hour H-untreated cybrids. However, in all cases the 0-hours J-untreated cybrids showed lower gene expression levels at baseline than the 0-hour H-untreated cybrids. Therefore, in order to measure the total expression increase of each of the genes, we needed to compare the values at the 120- hour period compared to the original 0-hour baseline for the H cybrids and J cybrids (represented as DDCt values). Figure 5 shows the DDCt values of the 120-hour UV-treated H cybrids compared to the 0-hour H-untreated cybrids and the UV-treated 120-hour J cybrids compared to the 0-hour J-untreated cybrids. Figure 2. Expression patterns of alternate complement inhib- itor genes after UV treatment. The 0-hour value for H-untreated cybrid was normalized to 1 and then compared to gene expression At 120 hours after UV treatment, there was a significant increase in CFH gene expression from their respective baseline levels in the H-treated cybrids compared to the J-treated cybrids (DDCt 2.4460.11 vs. DDCt 2.0960.07, p,0.02, Figure 5a). The DDCt values for CD55 expression were decreased in the H-treated cybrids compared to the J-treated cybrids, (DDCt 0.7860.09 vs. DDCt 1.1260.11, p,0.03, Figure 5a). There were no significant differences in DDCt values of CD59 expression for the H-treated cybrids compared to J-treated cybrids (DDCt 20.2360.2 vs. DDCt 0.1560.13, p = 0.064). The mean DDCt values in the H-treated cybrids were significantly lower for the IL-33 gene (1.5660.12, p,0.001 vs. 2.2960.1, p,0.0008) and the EFEMP-1 gene (DDCt 1.3460.004 vs. DDCt 1.9960.03, p,0.001) compared to the J- treated (Figure 5b). There were no statistically significant differences in DDCt values for the TGF-A expression (H cybrids value was DDCt 1.4360.05 vs. J cybrids value of DDCt 1.860.22, p = 0.14,). This demonstrates that when H and J cybrids were treated with the same amount of UV radiation under identical conditions, the J-treated cybrids showed significantly greater IL-33 and EFEMP1 expression levels from baseline, when compared to Figure 2. Expression patterns of alternate complement inhib- itor genes after UV treatment. The 0-hour value for H-untreated cybrid was normalized to 1 and then compared to gene expression June 2014 \| Volume 9 \| Issue 6 \| e99003 PLOS ONE \| www.plosone.org 5 Discussion The study investigates the response of cybrids, with identical nuclei but either H haplogroup mtDNA or J haplogroup mtDNA, to sub-lethal UV-radiation, which is an oxidative stressor and a risk factor for AMD [25]. Our previous study has shown that J cybrids have higher growth rates than H cybrids when cultured in similar environmental conditions [18]. In addition, we reported that although both H and J cybrids have similar copy numbers of mtDNA, H cybrids produce higher ATP levels and utilize oxidative phosphorylation (OXPHOS) while the J cybrids utilize predominantly glycolysis [18]. In our study, we observed that J haplogroup cybrids, with or without UV treatment, had more rapid growth rates even though the J cybrids are less energy efficient [18]. After 144 hours in vitro, the J-untreated cybrids had approximately 75% higher numbers of cells than the H-untreated cybrids. After UV treatment, the J- treated cybrids showed an approximately 85% higher cell count levels compared to the H-treated cybrids. The increased growth rates may be related in part to increased expression of IL-33 and EFEMP-1 genes, which play key roles in cell proliferation. The mechanisms by which the J haplogroup mtDNA affects rapid growth rates and glycolysis are not understood. One can speculate that the phenomenon might be similar to Warburg effect, described in 1956 by Dr. Otto Warburg [26], which suggested that tumor cells with damaged respiratory mechanisms have uncontrolled proliferation. These cancer cells had adapted to hypoxic conditions by shifting to glycolysis for ATP production, and even if returned to normoxic conditions, the cells continued with glycolysis as their main source of energy production. Figure 3. Expression patterns of inflammation, cellular prolif- eration and angiogenesis genes after UV treatment. The 0-hour values for H-untreated cybrids were normalized to 1 and then compared to gene expression values at 0, 72 and 120 hours. Panel 3a: At 0-hour, the J-untreated cybrids (black solid line) had lower expression levels of IL-33 compared to H-untreated cybrids (grey solid line, p,0.0001). At 120 hours, the H-untreated cybrids (grey solid line) showed higher levels of IL-33 expression compared to the J-untreated cybrids (black solid line). At both 72 and 120 hours, the J-treated cybrids June 2014 \| Volume 9 \| Issue 6 \| e99003 6 PLOS ONE \| www.plosone.org Cybrids Respond Differently to Ultraviolet Radiation hours. Similarly, the J haplogroups represent a subgroup of the Caucasian population which originated in Northern Europe, and over thousands of years, in response to extremely cold climatic conditions, may have developed adaptations within the mtDNA which preferentially uses glycolysis rather than OXPHOS. Others have shown that J haplogroups have accumulated higher levels of non-synonymous SNPs compared to other haplogroups, suggest- ing either a high mutation rate or a continuous selection process in the lineage [27]. We speculate these divergent energy efficiencies, as seen in the J versus H haplogroups, may influence the mitochondrial-nuclear interactions, thereby causing different expression levels of genes in major pathways. Further studies are underway to clarify the relationships between mtDNA variants and nuclear responses. Complement system activation and inflammation are major events contributing to AMD pathogenesis [28]. Studies have reported accumulation of immunoglobulin and complement components within drusen [29–31], associations between genetic variants of CFH, C2/CFB, C3, and CFI with increased risk for AMD [32,33], and elevated serum CRP levels in AMD patients [34]. Studies of European mtDNA variants have revealed that J, T and U haplogroups are associated with AMD [17,35–38] while the H haplogroup is protective [39]. Our previous paper shows that at resting state, the J cybrids express lower levels of CFH than H cybrids. After UV-treatment, J cybrids still showed lower CFH levels at 72 and 120 hours in both the untreated and UV-treated samples. Since CFH is a negative regulator of the alternative complement pathway, the lower levels in J haplogroup subjects would likely lead to increased activation of the complement pathway and higher levels of inflammation. Alternatively, the higher CFH levels in H cybrids, as seen at all time points, may be associated with the protective effects associated between H haplogroup and AMD. In cells from the outer retinal layers, photo-oxidative stress induces complement activation and de- creases the level of CFH, which results in increased inflammatory responses [40]. Interestingly, we found less than 2-fold increase of CD55 expression after UV treatment, suggesting that CFH was the major inhibitor mediated by mtDNA. This makes the CFH gene an attractive target for future therapies to treat AMD and other complement-related diseases. Figure 4. Discussion Panel 4a: At 0-hour, the J-untreated cybrids (black solid line) had lower expression levels of RARA compared to H-untreated cybrids (grey solid line, p = 0.0036). Panel 4b: At 0-hour, the J-untreated cybrids (black solid line) had lower expression levels of BBC3 compared to H- untreated cybrids (grey solid line, p,0.01). At 120 hours after UV treatment, the H-treated cybrids (grey broken line) and J-treated cybrids (black broken line) showed higher expression levels of BBC3 compared to H-untreated (grey solid line) and J-untreated (black solid line). Panel 4c: At 0-hours, the J-untreated cybrids (black solid line) showed lower expression levels of BCL2L13 compared to the H-untreated cybrids (grey solid line, p = 0.002). After 120 hour of UV treatment, the H- treated (grey broken line) and J-treated (black broken line) cybrids showed an increase in BCL2L13 expression levels. There were triplicate wells for each sample and the experiments were repeated twice. doi:10.1371/journal.pone.0099003.g004 Figure 4. Expression patterns of pro-apoptosis genes after UV treatment. The 0-hour values for H-untreated cybrids were normalized to 1 and then compared to gene expression values at 0, 72 and 120 The open bars represent the DDCt values of the J-untreated cybrids at 0 hours compared to J-treated cybrids at 120 hours. Panel 5a: At 120 hours after UV treatment, the H-treated cybrids have higher expression levels for CFH compared to the J-treated cybrids (p,0.02) but lower expression for the CD55 gene (p,0.03). There was no significant difference for the CD59 levels in the H-treated versus the J-treated cybrids. Panel 5b: At 120 hours after UV treatment, the J-treated cybrids have higher expression levels for IL-33 and EFEMP1 genes compared to the H-treated cybrids (p,0.02) but similar expression levels for the TGF-A gene. (p = 0.14). Panel 5c: At 120 hours after UV treatment, the H-treated cybrids have higher expression levels for RARA (p = 0.02) and BBC3 (p = 0.004) genes compared to the J- treated cybrids. There expression levels for the BCL2L13 gene were similar for both cybrids (p = 0.28). doi:10.1371/journal.pone.0099003.g005 noting that the TGF-A and IL-33 may interact at some level because their expression patterns closely followed each other in our experiments. inflammation, innate immunity and/or angiogenesis, which are pathologic processes of AMD. IL-33, which can be found in RPE cells, is a cytokine belonging to the IL-1 superfamily, and can regulate inflammatory cytokines including IL-6, IL-8, IL-1B and TNF-alpha. Alipoprotein E (ApoE), a high-risk gene associated with AMD, mediates the distribution of lipids and cholesterol. Abnormal functions of ApoE can lead to hypercholesteremia [46], and increased production of amyloid, a protein associated with both AMD (geographic atrophy) and Alzheimer’s disease. It has been reported that in RPE cells, beta-amyloid can stimulate IL-33 mediated inflammatory responses [47], which suggests that there may be a correlation between amyloid production, IL-33 and ApoE levels related to RPE cells responses to oxidative stressors. Increased expression of EFEMP-1 genes was also found in untreated and UV-treated J cybrids. EFEMP-1 is another important cancer-related gene that was differentially expressed in UV-treated J cybrids. EFEMP-1, a favorable prognostic marker for glioblastoma multiforme tumors [52], modulates the extracel- lular environment and influences important signaling pathways. Song and coworkers transfected EFEMP-1 into HeLa cells and observed increased VEGF expression, acceleration of angiogene- sis, increased growth and proliferation of cervical carcinoma in vivo [53]. It is reported that misfolded EFEMP-1 accumulates within endoplasmic reticulum and up-regulates VEGF expression in pancreatic adenocarcinoma [54]. Expression patterns of pro-apoptosis genes after UV treatment The 0 hour values for H untreated cybrids were normalized RPE cells, with their high oxygen demands and metabolic rates [41], are susceptible to UV radiation, which can cause loss of cell viability, mitochondrial dysfunction, DNA damage and apoptosis [42]. UV exposure can increase ROS production in RPE cells that can trigger inflammation [43–45]. In our study, we found similar expression patterns for IL-33 and EFEMP-1 genes, whereby at resting stages, the J-untreated cybrids had levels lower than H- untreated cybrids but after UV stress, the J-treated cybrids showed increased expression levels of each gene compared to H-treated cybrids. This is important because these genes play a role in Figure 4. Expression patterns of pro-apoptosis genes after UV treatment. The 0-hour values for H-untreated cybrids were normalized to 1 and then compared to gene expression values at 0, 72 and 120 June 2014 \| Volume 9 \| Issue 6 \| e99003 PLOS ONE \| www.plosone.org 7 Cybrids Respond Differently to Ultraviolet Radiation Figure 5. Comparison of gene expression levels from 0 to 120 hours after UV treatment. The solid black bars represent the DDCt values of H-untreated cybrids at 0 hour compared to H-treated cybrids at 120 hours. The open bars represent the DDCt values of the J-untreated cybrids at 0 hours compared to J-treated cybrids at 120 hours. Panel 5a: At 120 hours after UV treatment, the H-treated cybrids have higher expression levels for CFH compared to the J-treated cybrids (p,0.02) but lower expression for the CD55 gene (p,0.03). There was no significant difference for the CD59 levels in the H-treated versus the J-treated cybrids. Panel 5b: At 120 hours after UV treatment, the J-treated cybrids have higher expression levels for IL-33 and EFEMP1 genes compared to the H-treated cybrids (p,0.02) but similar expression levels for the TGF-A gene. (p = 0.14). Panel 5c: At 120 hours after UV treatment, the H-treated cybrids have higher expression levels for RARA (p = 0.02) and BBC3 (p = 0.004) genes compared to the J- treated cybrids. There expression levels for the BCL2L13 gene were similar for both cybrids (p = 0.28). doi:10.1371/journal.pone.0099003.g005 Figure 5. Comparison of gene expression levels from 0 to 120 hours after UV treatment. The solid black bars represent the DDCt values of H-untreated cybrids at 0 hour compared to H-treated cybrids at 120 hours. Cybrids Respond Differently to Ultraviolet Radiation After H2O2–induced oxidative stress, the SIRT3 gene was downregu- lated in osteosarcoma cybrids with the J haplogroup compared to H cybrids [66]. Lin and coworkers used cybrids created with Taiwanese haplogroups and showed that the B4b haplogroup had increased susceptibility to H2O2 compared to other ethnic Chinese backgrounds. Bellizzi and co-workers used the osteosarcoma cybrid model to demonstrate that at baseline the H cybrids had significantly lower levels of heat shock protein (HSP)60 and HSP75 but after oxidative stress, the H cybrids accumulated HSP60 within mitochondria at a much greater rate than other cybrids [67]. In another series of experiments, they showed that in basal and H2O2-stressed conditions, the H and J cybrids showed different levels of interleukin (IL)-6, IL-1beta and tumor necrosis factor receptor 2, which are stress–related cytokines [68]. Finally, an osteosarcoma cybrid model showed that the H cybrids differed significantly from Uk cybrids with respect to mitochondrial RNA levels, protein synthesis, membrane potential and growth capacity [69]. These findings, along with ours, strongly support our hypothesis that mtDNA haplogroups can mediate molecular pathways that influence the complement/inflammation responses of cells. y doi:10.1371/journal.pone.0099003.g006 With respect to AMD, EFEMP-1 expressed in RPE cells is a high-risk gene associated with AMD and Doyne honeycomb retinal dystrophy. Misfolded EFEMP-1 protein accumulates within RPE cells causing altered cellular function and inflamma- tion [55]. EFEMP-1 protein is present between RPE layer and drusen [56,57]. Roybal et al. studied the response of transfected EFEMP-1 wild type and mutant EFEMP-1 R345W on VEGF expression in ARPE-19 cell line. R345W mutation in EFEMP-1 is linked to two inherited early–onset macular degenerative retinal diseases Malattia Leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD). There was an abnormal accumulation and retention of EFEMP-1 R345W in the endoplasmic reticulum that induced the unfolded protein response (UPR) activation followed by increased VEGF expression. Activation of UPR and VEGF expression may be correlated with RPE dysfunction and choroidal neovascularization in AMD patients [58]. In our study, the UV- treated J cybrids showed a 4-fold increase in EFEMP-1 levels while the UV-treated H cybrids had a 2.56-fold increase. Even though the mechanisms by which EFEMP-1 might contribute to AMD is not known, based upon the cancer studies, it is reasonable to speculate that numerous downstream pathways are activated by increased EFEMP-1 levels. Cybrids Respond Differently to Ultraviolet Radiation The pro-apoptosis genes (RARA and BBC3) were expressed at a lower level in J-treated cybrids after UV treatment. doi:10.1371/journal.pone.0099003.g006 In our study, we investigated expression levels for three apoptosis-related genes and found significant changes in RARA and BBC3. In general, BBC3 is involved in BAX-dependent cell death related to developing retina and brain [59] but in the adult nervous system the cell death process is mediated by BIM. BBC3 expression can be altered in response to various insults and is involved in adult retinal cell degeneration [60–62]. In a mouse model, UV exposure can increase BIM expression, which in turn can increase BBC3 expression and caspase-dependent cell death in retinal photoreceptor cells [63]. Our study demonstrates a 3.78- fold increase expression for BBC3 in UV-treated H cybrids occurring in response to UV-radiation stress. We need to be cautious because although the BBC3 expression levels are elevated, this may or may not translate into higher apoptotic activities in the UV-treated H cybrids. A second apoptotic gene, RARA, was differentially expressed in the H versus J cybrids after UV treatment. RARA, which is present in the bipolar cells and retinal ganglion cells, is an important regulator for development [64]. In addition, RARA upregulates expression of glial cell-derived neurotrophic factors (GNDF), which inhibits vascular permeability factor-VEGF [65]. We observed increased expression of RARA in UV-treated H cybrids as compared to J cybrids. If RARA indirectly inhibits VEGF expression, then this may correlate with lower incidence of wet AMD in individuals having H haplogroup mtDNA. Figure 6. Schematic summary of the changes found in cybrids after UV treatment. In response to identical UV conditions, cybrids with identical nuclei but different mtDNA haplogroups (H vs. J) showed differential responses of cell growth and expression patterns genes related to inflammation and apoptosis. After UV treatment, the J- treated cybrids had increased cell growth rates but a gene expression pattern consistent with higher levels of inflammation (elevated IL-33 and EFEMP1 along with decreased expression of CFH, a complement pathway inhibitor) compared to H-treated cybrids. The pro-apoptosis genes (RARA and BBC3) were expressed at a lower level in J-treated cybrids after UV treatment. d i 10 1371/j l 0099003 006 g p g p The cybrid model using different Rho0 cells as the host cell line has provided similar findings to ours by showing that the mtDNA haplogroup profiles influence cellular/molecular responses. Cybrids Respond Differently to Ultraviolet Radiation Most importantly, it must be pointed out that (1) the J and H cybrids showed a differential response to the UV treatment, and (2) these cybrids differed only in their mtDNA but had identical nuclei and treatment conditions. This is important because it strongly suggests that within an individual, the mtDNA haplogroup background may provide a baseline for expression of major signaling pathways, which are then influenced to different degrees by identical stressors, depending upon which haplogroup the individual possesses. One can speculate that this may explain in part why some individuals are more susceptible to environmental stressors, such as smoking or UV light, while others are not. Therefore, the finding that EFEMP-1 expression is mediated by mtDNA haplogroups is exciting because it implies that the mtDNA can greatly influence this important pathway and that it is responsive to sub-lethal UV radiation. TGF-A plays a role in both angiogenesis and wound healing [48]. In addition, elevated levels of TGF-A have been consistently observed in gastrointestinal cancers, breast cancers and primary epithelial ovarian cancers [49–51]. While an association between TGF-A and AMD has not been reported, it is a relatively newly described gene and little is known about its functions. It is worth June 2014 \| Volume 9 \| Issue 6 \| e99003 PLOS ONE \| www.plosone.org 8 Cybrids Respond Differently to Ultraviolet Radiation With respect to AMD, EFEMP-1 expressed high-risk gene associated with AMD and D retinal dystrophy. Misfolded EFEMP-1 pro within RPE cells causing altered cellular functi tion [55]. EFEMP-1 protein is present betwee drusen [56,57]. Roybal et al. studied the respo EFEMP-1 wild type and mutant EFEMP-1 R expression in ARPE-19 cell line. R345W mutat linked to two inherited early–onset macular d diseases Malattia Leventinese (ML) and Doyne dystrophy (DHRD). There was an abnormal retention of EFEMP-1 R345W in the endoplas induced the unfolded protein response (UPR) a by increased VEGF expression. Activation of expression may be correlated with RPE dysfunc neovascularization in AMD patients [58]. In o treated J cybrids showed a 4-fold increase in EFE the UV-treated H cybrids had a 2.56-fold incr the mechanisms by which EFEMP-1 might con not known, based upon the cancer studies, i speculate that numerous downstream pathway increased EFEMP-1 levels. Most importantly, out that (1) the J and H cybrids showed a diffe the UV treatment, and (2) these cybrids diff mtDNA but had identical nuclei and treatment important because it strongly suggests that wit the mtDNA haplogroup background may prov expression of major signaling pathways, which a to different degrees by identical stressors, depe haplogroup the individual possesses. One can may explain in part why some individuals are m environmental stressors, such as smoking or UV Figure 6. Schematic summary of the changes after UV treatment. In response to identical UV with identical nuclei but different mtDNA haplogro differential responses of cell growth and express related to inflammation and apoptosis. After UV treated cybrids had increased cell growth rates bu pattern consistent with higher levels of inflamma and EFEMP1 along with decreased expression of pathway inhibitor) compared to H-treated cybrids genes (RARA and BBC3) were expressed at a low cybrids after UV treatment. doi:10.1371/journal.pone.0099003.g006 Figure 6. Schematic summary of the changes found in cybrids after UV treatment. In response to identical UV conditions, cybrids with identical nuclei but different mtDNA haplogroups (H vs. J) showed differential responses of cell growth and expression patterns genes related to inflammation and apoptosis. 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Increasing our understanding of nuclear-mitochondrial interactions, and their relationship to environmental stimuli, will lead to an entirely new era of disease management in which the mitochondria, with its unique DNA, will become a target for therapy. June 2014 \| Volume 9 \| Issue 6 \| e99003 PLOS ONE \| www.plosone.org 9 Cybrids Respond Differently to Ultraviolet Radiation 54. Seeliger H, Camaj P, Ischenko I, Kleespies A, De Toni EN, et al. (2009) EFEMP1 expression promotes in vivo tumor growth in human pancreatic adenocarcinoma. Mol Cancer Res 7: 189–198. 62. Doonan F, Donovan M, Gomez-Vicente V, Bouillet P, Cotter TG (2007) Bim expression indicates the pathway to retinal cell death in development and degeneration. J Neurosci 27: 10887–10894. 63. Gomez-Vicente V, Doonan F, Donovan M, Cotter TG (2006) Induction of BIM(EL) following growth factor withdrawal is a key event in caspase-dependent apoptosis of 661W photoreceptor cells. Eur J Neurosci 24: 981–990. 55. 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Ramkumar HL, Zhang J, Chan CC (2010) Retinal ultrastructure of murine models of dry age-related macular degeneration (AMD). Prog Retin Eye Res 29: 169–190. 66. D’Aquila P, Rose G, Panno ML, Passarino G, Bellizzi D (2012) SIRT3 gene expression: a link between inherited mitochondrial DNA variants and oxidative stress. Gene 497: 323–329. 58. Roybal CN, Marmorstein LY, Vander Jagt DL, Abcouwer SF (2005) Aberrant accumulation of fibulin-3 in the endoplasmic reticulum leads to activation of the unfolded protein response and VEGF expression. Invest Ophthalmol Vis Sci 46: 3973–3979. 67. Bellizzi D, Taverna D, D’Aquila P, De Blasi S, De Benedictis G (2009) Mitochondrial DNA variability modulates mRNA and intra-mitochondrial protein levels of HSP60 and HSP75: Experimental evidence from cybrid lines. Cell Stress Chaperones 14: 265–271. 59. Ren D, Tu HC, Kim H, Wang GX, Bean GR, et al. (2010) BID, BIM, and PUMA are essential for activation of the BAX- and BAK-dependent cell death program. Science 330: 1390–1393. 68. References Charbel Issa P, Chong NV, Scholl HP (2011) The significance of the complement system for the pathogenesis of age-related macular degeneration - June 2014 \| Volume 9 \| Issue 6 \| e99003 10 PLOS ONE \| www.plosone.org Cybrids Respond Differently to Ultraviolet Radiation Bellizzi D, Cavalcante P, Taverna D, Rose G, Passarino G, et al. (2006) Gene expression of cytokines and cytokine receptors is modulated by the common variability of the mitochondrial DNA in cybrid cell lines. Genes Cells 11: 883– 891. 60. Yu JS, Koujak S, Nagase S, Li CM, Su T, et al. (2008) PCDH8, the human homolog of PAPC, is a candidate tumor suppressor of breast cancer. Oncogene 27: 4657–4665. 69. Gomez-Duran A, Pacheu-Grau D, Lopez-Gallardo E, Diez-Sanchez C, Montoya J, et al. (2010) Unmasking the causes of multifactorial disorders: OXPHOS differences between mitochondrial haplogroups. Hum Mol Genet 19: 3343–3353. 61. Marti A, Hafezi F, Lansel N, Hegi ME, Wenzel A, et al. 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https://openalex.org/W2612882336	https://documentserver.uhasselt.be//bitstream/1942/24335/1/carroll%201.pdf	English	null	Extensions to Multivariate Space Time Mixture Modeling of Small Area Cancer Data	International journal of environmental research and public health/International journal of environmental research and public health	2,017	cc-by	8,900	Extensions to Multivariate Space Time Mixture Modeling of Small Area Cancer Data Rachel Carroll 1,, Andrew B. Lawson 1, Christel Faes 2, Russell S. Kirby 3, Mehreteab Aregay and Kevin Watjou 2 Rachel Carroll 1,, Andrew B. Lawson 1, Christel Faes 2, Russell S. Kirby 3, Mehreteab Aregay 1 and Kevin Watjou 2 Department of Public Health Sciences, Medical University of South Carolina, 135 Cannon St, Charleston, SC 29425, USA; lawsonab@musc.edu (A.B.L.); mhrete ab@yahoo.com (M.A.) p , y , , , SC 29425, USA; lawsonab@musc.edu (A.B.L.); mhrete_ab@yahoo.com (M.A.) 2 Interuniversity Institute for Statistics and Statistical Bioinformatics, Hasselt University, 3500 Hasselt, Belgium; christel.faes@uhasselt.be (C.F.); kevin.watjou@uhasselt.be (K.W.) 3 Department of Community and Family Health, University of South Florida, Tampa, FL 33620, USA; rkirby@health.usf.edu * C d h l ll@ ih , ; ( ); y ( ) 2 Interuniversity Institute for Statistics and Statistical Bioinformatics, Hasselt University, 3500 Hasselt, Belgium; christel.faes@uhasselt.be (C.F.); kevin.watjou@uhasselt.be (K.W.) 2 Interuniversity Institute for Statistics and Statistical Bioinformatics, Hasselt University, 3500 Hasselt, Belgium; christel.faes@uhasselt.be (C.F.); kevin.watjou@uhasselt.be (K.W.) 3 Department of Community and Family Health, University of South Florida, Tampa, FL 33620, USA; rkirby@health.usf.edu 3500 Hasselt, Belgium; christel.faes@uhasselt.be (C.F.); kevin.watjou@uhasselt.be (K.W.) 3 Department of Community and Family Health, University of South Florida, Tampa, FL 33620, USA rkirby@health.usf.edu , g ; ( ); j ( ) 3 Department of Community and Family Health, University of South Florida, Tampa, FL 33620, USA; rkirby@health.usf.edu * Correspondence: rachel.carroll@nih.gov Academic Editors: Louise Ryan and Craig Anderson Academic Editors: Louise Ryan and Craig Anderson y g Received: 6 February 2017; Accepted: 5 May 2017; Published: 9 May 2017 Abstract: Oral cavity and pharynx cancer, even when considered together, is a fairly rare disease. Implementation of multivariate modeling with lung and bronchus cancer, as well as melanoma cancer of the skin, could lead to better inference for oral cavity and pharynx cancer. The multivariate structure of these models is accomplished via the use of shared random effects, as well as other multivariate prior distributions. The results in this paper indicate that care should be taken when executing these types of models, and that multivariate mixture models may not always be the ideal option, depending on the data of interest. Keywords: lung and bronchus cancer; melanoma cancer of the skin; oral cavity and pharynx cancer; incidence; mixture model; spatio-temporal; disease mapping International Journal of Environmental Research and Public Health Int. J. Environ. Res. Public Health 2017, 14, 503; doi:10.3390/ijerph14050503 www.mdpi.com/journal/ijerph 1. Introduction Many cancers share risk factors but differ in incidence. Previous studies have demonstrated beneﬁts for rarer diseases via the implementation of multivariate modeling with a more prevalent disease that presents common risk factors [1,2]. Oral cavity and pharynx cancer (hereafter referred to as oral/pharynx) (ICD-9-CM codes: 145.9, 146.2, 146.3, 146.5–146.9, 147.0–147.3, 147.8, 147.9, 148.9, 149.0, 149.8, 149.9) and lung and bronchus cancer (henceforth referred to as lung) (ICD-9-CM codes: 162.2–162.5, 162.8, and 162.9) share tobacco smoke as an exposure, while lip cancer, which is included with oral/pharynx cancer, and melanoma cancer of the skin (referred to as melanoma from here) (ICD-9-CM codes: 172.2, 172.3, 172.5–172.7, and 172.9), have the common risk factor of ultraviolet light exposure [3–6]. Additionally, oral/pharynx is quite rare in comparison to lung and melanoma, which are among the most common types of cancer in the United States [7]. Thus, it is of interest to explore the beneﬁts of employing multivariate modeling for these relationships by including all three diseases in a single model. Oral/pharynx, melanoma, and lung all display spatio-temporal (ST) patterns in their incidences [8–12], and a Bayesian multivariate ST mixture model proposed by Lawson et al. [2] can offer an ideal, ﬂexible model for risk that offers improvements over a more standard multivariate model introduced by Knorr-Held [13–15]. The real data example in that paper also involved the three diseases of interest and illustrated that care should be taken in implementing types of models. In that example, there was evidence that modeling was improved for oral/pharynx, but only up to a certain point in time, year 10, in the multivariate setting. To deal with this, they simply restricted to the ﬁrst Int. J. Environ. Res. Public Health 2017, 14, 503; doi:10.3390/ijerph14050503 www.mdpi.com/journal/ijerph www.mdpi.com/journal/ijerph 2 of 13 Int. J. Environ. Res. Public Health 2017, 14, 503 10 years of the study time to create a working example. However, in this paper we wish to explore extensions to the model that could offer greater temporal ﬂexibility for situations of this nature so that all of the data can be utilized and no information is lost. These extensions will involve imposing more ﬂexibility in the mixture model, as well as bivariate ﬁts of only oral/pharynx with lung because that relationship is more grounded in the literature. Often, some important risk factors may be unavailable because they are difﬁcult to measure or obtain. 1. Introduction A beneﬁt of employing an ST model involves the models’ ability to account for such unmeasured exposures that may be common among the diseases considered. Examples of these exposures include: health service availability, environmental inﬂuences, or demographic factors. In this study, we do not have the ability to measure many of these exposures since data are aggregated at the county level. This paper is developed as follows: First, we describe the case study and the statistical methods. Following that, we present the results. Finally, we discuss the ﬁndings and draw conclusions. 2. Materials and Methods The outcome data of interest is in the form of the annual South Carolina county level incidences for oral/pharynx, lung, and melanoma over the years 1996–2009. A conditionally-independent Poisson distribution is a commonly-used model for small area counts in disease mapping [16–18], and is described as follows: yijk ∼Pois µijk yijk ∼Pois µijk µijk = eijkθijk µijk = eijkθijk with yijk, the incidence of cancer k in county i at time j, and µijk, the mean of the Poisson distribution deﬁned as the product of the known expected rate of disease, eijk, and the relative risk, θijk. In this framework, we model the logarithm of the relative risk; these modeling options are described in Section 3. with yijk, the incidence of cancer k in county i at time j, and µijk, the mean of the Poisson distribution deﬁned as the product of the known expected rate of disease, eijk, and the relative risk, θijk. In this framework, we model the logarithm of the relative risk; these modeling options are described in Section 3. 2.1. Case Study The outcome data for this analysis was gathered from the South Carolina Assessment Network [19]. In this data, the managers placed thresholds on small counts of disease such that an observed count between 1 and 4 for a particular county is given the value 5 and an observed count between 5 and 9 is given the value 10. To make the distribution of counts more appropriate, we imputed values based on the appropriate interval as indicated by the threshold value. To do this, we calculate an expected count of diseases for each county and year by multiplying the overall rate of disease (based on the threshold values) by the county population. Next, we sample from a Poisson distribution with the expected rate as the mean restricted such that the imputed value falls within the appropriate threshold. The code for accomplishing this is included in the supplemental ﬁles of Lawson et al. [2]. The numbers and percentages of zeros and imputed data for oral/pharynx, melanoma, and lung are displayed in Table 1. Table 1. Number and percent of zeros and imputed data per threshold. Disease Number (%) of Data in Each Threshold 0 1–4 5–10 Oral/pharynx 16 (2.5%) 200 (31.1%) 190 (29.5%) Melanoma 13 (2.0%) 128 (19.9%) 151 (23.4%) Lung 0 (0.0%) 4 (0.6%) 15 (2.3%) Table 1. Number and percent of zeros and imputed data per threshold. 3 of 13 Int. J. Environ. Res. Public Health 2017, 14, 503 Following the imputation, the expected rate of disease (eijk) is calculated. The subsequent statistics and ﬁgures are based on the imputed dataset. Figure 1 displays a histogram of incidences per study year segmented by disease. From this, it is easy to see that the incidences of melanoma and lung are increasing over time while oral/pharynx remains steady. This could be part of why the previous multivariate model did not perform well in the later study years. For the 7718 diagnosed cases of oral/pharynx in South Carolina for these study years, there are approximately 168 (range: 19–651) cases per county per year leading to a rate of disease equal to 0.0001. Similarly, there were 20,156 and 44,595 diagnosed cases of melanoma and lung with an average of 438 (range: 25–2726) and 969 (range: 115–3885) cases per county per year leading to rates of disease equal to 0.0003 and 0.0008, respectively. 2.1. Case Study These rates of disease are calculated as the total incidence over the entire study time divided by the total population at risk over the entire study time. Int. J. Environ. Res. Public Health 2017, 14, 503 3 of 13 cases of oral/pharynx in South Carolina for these study years, there are approximately 168 (range: 19–651) cases per county per year leading to a rate of disease equal to 0.0001. Similarly, there were 20,156 and 44,595 diagnosed cases of melanoma and lung with an average of 438 (range: 25–2726) and 969 (range: 115–3885) cases per county per year leading to rates of disease equal to 0.0003 and 0.0008, respectively. These rates of disease are calculated as the total incidence over the entire study time divided by the total population at risk over the entire study time. Figure 1. Bar plot of incidences of all cancers over time. Figure 1. Bar plot of incidences of all cancers over time. Figure 1. Bar plot of incidences of all cancers over time. Figure 1. Bar plot of incidences of all cancers over time. Standardized incidence rate (SIR) calculations are often a first step in data analysis. The SIRs per county over time for each of the diseases are displayed in Figures 2–4. The SIR was calculated as a ratio of the observed cancer incidences to the expected rates of disease per county [20]. For interpretation purposes, a SIR of one indicates that the observed incidence is equal to that of the expected count. The SIRs for oral/pharynx do present some spatial structure, but are somewhat scattered as well. melanoma has a clear presence in the eastern coastal counties and the rates appear to increase over time within the same spatial pattern. Like oral/pharynx, lung appears to have a spatial structure in its distribution, albeit weak, and, like melanoma, the rates appear to increase over time. Standardized incidence rate (SIR) calculations are often a ﬁrst step in data analysis. The SIRs per county over time for each of the diseases are displayed in Figures 2–4. The SIR was calculated as a ratio of the observed cancer incidences to the expected rates of disease per county [20]. For interpretation purposes, a SIR of one indicates that the observed incidence is equal to that of the expected count. The SIRs for oral/pharynx do present some spatial structure, but are somewhat scattered as well. 2.1. Case Study melanoma has a clear presence in the eastern coastal counties and the rates appear to increase over time within the same spatial pattern. Like oral/pharynx, lung appears to have a spatial structure in its distribution, albeit weak, and, like melanoma, the rates appear to increase over time. Int. J. Environ. Res. Public Health 2017, 14, 503 I t J E i R P bli H lth 2017 14 503 Int. J. Environ. Res. Public Health 2017, 14, 503 4 of 13 4 of 13 Figure 2. SIR of oral cavity and pharynx cancer. Figure 2. SIR of oral cavity and pharynx cancer. Figure 2. SIR of oral cavity and pharynx cancer. Figure 2. SIR of oral cavity and pharynx cancer. Figure 2. SIR of oral cavity and pharynx cancer. Figure 2. SIR of oral cavity and pharynx cancer. Figure 2. SIR of oral cavity and pharynx cancer. Figure 2. SIR of oral cavity and pharynx cancer. Figure 2. SIR of oral cavity and pharynx cancer. Figure 2. SIR of oral cavity and pharynx cancer. Figure 2. SIR of oral cavity and pharynx cancer. Figure 2. SIR of oral cavity and pharynx cancer. Figure 3 SIR of melanoma cancer of the skin Figure 3. SIR of melanoma cancer of the skin. Figure 3. SIR of melanoma cancer of the skin. Fi 3 SIR f l f th ki Figure 3. SIR of melanoma cancer of the skin. Figure 3. SIR of melanoma cancer of the skin. The predictors for this analysis are demographic and environmental measures obtained from a variety of sources with known oral/pharynx, melanoma, and lung, as well as general oncological associations [4–6,21–27]. The demographic predictors come from the Area Health Resources Files dataset [28] while the environmental predictors come from the National Oceanic and Atmospheric Administration [29], South Carolina Department of Health and Environmental Control (SCDHEC) [30], and the North America Land Data Assimilation System [31]. Int. J. Environ. Res. Public Health 2017, 14, 503 I E bl l h 5 of 13 Figure 4. SIR of lung and bronchus cancer. Figure 4. SIR of lung and bronchus cancer. Figure 4. SIR of lung and bronchus cancer. Figure 4. SIR of lung and bronchus cancer. The predictors for this analysis are demographic and environmental measures obtained from a variety of sources with known oral/pharynx, melanoma, and lung, as well as general oncological associations [4–6,21–27]. 2.1. Case Study The demographic predictors come from the Area Health Resources Files dataset [28] while the environmental predictors come from the National Oceanic and Atmospheric Administration [29], South Carolina Department of Health and Environmental Control (SCDHEC) [30], and the North America Land Data Assimilation System [31]. For these methods, it is important to note which of these variables are spatial, temporal, or, S.T.; and predictors were designated as such based on either their availability or apparent amount of spatial and/or temporal variation. The proportion of persons with health insurance (pHI), median household radon level (radon), and proportion of African American population (pAA) are spatial where the two socioeconomic covariates are census data from the year 2000 and the measure of radon is a county-level average of in-home test kit results analyzed by the SCDHEC laboratory. The single l di i id l i f ll Fi ll h ST di d il For these methods, it is important to note which of these variables are spatial, temporal, or, S.T.; and predictors were designated as such based on either their availability or apparent amount of spatial and/or temporal variation. The proportion of persons with health insurance (pHI), median household radon level (radon), and proportion of African American population (pAA) are spatial where the two socioeconomic covariates are census data from the year 2000 and the measure of radon is a county-level average of in-home test kit results analyzed by the SCDHEC laboratory. The single temporal predictor is statewide average annual rainfall. Finally, the ST predictors are average daily sunlight (sun), unemployment rate of those 16 years or older (UER), and the proportion of persons in poverty (pppov). Note that the predictors are standardized for model-ﬁtting purposes. Additionally, some of these variables are correlated, but each do still offer their own attributes for our risk model. For example, rainfall and statewide average sunlight are correlated, but rainfall could also offer information about humidity. p p sunlight (sun), unemp ( ) 2.2. Statistical Methods in poverty (pppov). Note that the predictors are standardized for model-fitting purposes. Additionally, some of these variables are correlated, but each do still offer their own attributes for our risk model. For example, rainfall and statewide average sunlight are correlated, but rainfall could also offer information about humidity. 2.2. Statistical Methods Here, we describe the methodology associated with the bivariate and multivariate mixture models utilized in this exploration. These methods are implemented via the R package R2WinBUGS which calls WinBUGS from R [32–35]. All models reached convergence with two chains for the Markov chain Monte Carlo sampler running for a total burn-in of 45,000 iterations and a sample of 5000. Trace plots of the deviance and some random effect standard deviations, as well as model BUGS code, are included in the supplemental materials. Here, we describe the methodology associated with the bivariate and multivariate mixture models utilized in this exploration. These methods are implemented via the R package R2WinBUGS which calls WinBUGS from R [32–35]. All models reached convergence with two chains for the Markov chain Monte Carlo sampler running for a total burn-in of 45,000 iterations and a sample of 5000 T a e lot of the de ia e a d o e a do effe t ta da d de iatio a ell a odel BUGS We are interested in exploring the relationships between the two or three cancers in this bivariate or multivariate setting via a mixture model that was ﬁrst introduced in Carroll et al. [36] as a form of model selection. These methods were then further improved and tested via a simulation study in Lawson et al. [2]. The general structure of this model for disease k in county i at time j is as follows: nd some random effect standard ental materials. the relationships between the t re model that was first introduc were then further improved an ture of this model for disease ݇ log θijk = α0k + ∑ h ph ikMh ijk ∑ h ph ik = 1; ph ik ∈[0, 1] model that was first introd e then further improved e of this model for disease ∑ h ph ik = 1; ph ik ∈[0, 1] Int. J. Environ. Res. p p sunlight (sun), unemp ( ) 2.2. Statistical Methods vi ∼N 1 ni ∑ i∼l vl, 1 niτv is a correlated spatial random effect following an intrinsic conditional autoregressive (CAR) model, where ni is the number of neighbors for county i, and i ∼l indicates that the two counties i and l are neighbors (i ̸= l) [37,38]. γj ∼N γj−1, τ−1 γ is the temporal random effect modeled by a temporal random walk prior, φijk ∼N 0, τ−1 φk is the uncorrelated ST interaction term, and all precisions are deﬁned such that τ−1/2 ∼Uni f (0, C). The random effects included here are typical of ST models such as the commonly applied Knorr-Held model [13,14]. Finally, the mixture parameter is made up of two parameters, zi and ai (univariate) or zik and aik (multivariate), that are correlated and uncorrelated in space, respectively. However, when three mixture components and parameters are needed, as in Alternative 2 (Alt2), constraints must be imposed such that the sum of the three mixture parameters is equaled to one. This is accomplished with the following structure: logit qh i = zh i + ah i and ph i = qh i / ∑ h qh i or h h h h h h uik ∼N 0, τ−1 uk is the uncorrelated spatial random effect. vi ∼N 1 ni ∑ i∼l vl, 1 niτv is a correlated uik ∼N 0, τ−1 uk is the uncorrelated spatial random effect. vi ∼N 1 ni ∑ i∼l vl, 1 niτv is a correlated spatial random effect following an intrinsic conditional autoregressive (CAR) model, where ni is the number of neighbors for county i, and i ∼l indicates that the two counties i and l are neighbors (i ̸= l) [37,38]. γj ∼N γj−1, τ−1 γ is the temporal random effect modeled by a temporal random walk prior, φijk ∼N 0, τ−1 φk is the uncorrelated ST interaction term, and all precisions are deﬁned such that τ−1/2 ∼Uni f (0, C). The random effects included here are typical of ST models such as the commonly applied Knorr-Held model [13,14]. Finally, the mixture parameter is made up of two parameters, zi and ai (univariate) or zik and aik (multivariate), that are correlated and uncorrelated in space, respectively. p p sunlight (sun), unemp ( ) 2.2. Statistical Methods Public Health 2017, 14, 503 6 of 13 where α0k ∼Norm 0, τ−1 a is an intercept, ph ik is a mixture parameter, and Mh ijk is a mixture component. In the previous study, the set of mixture parameters and components was given by h = {S, ST}, thus containing a spatial and spatio-temporal parts. However, the temporal effect appeared to be causing model ﬁt issues with oral/pharynx in the multivariate setting as this parameter was shared between diseases. Thus, in this paper we wish to explore the beneﬁt of either keeping the same mixture component structure and allowing the temporal effect to vary between diseases or introducing a third mixture component for temporal effects. Hence, the three alternative mixture deﬁnitions for both the univariate and bivariate/multivariate cases are displayed in Table 2. The parameters displayed are such that Xi, Xj, and Xij are the ith, jth, and ijth values of the spatial, temporal, and ST covariates, respectively, β ∼N 0, τ−1 β for the general case are the ﬁxed effect parameter estimates, where α0k ∼Norm 0, τ−1 a is an intercept, ph ik is a mixture parameter, and Mh ijk is a mixture component. In the previous study, the set of mixture parameters and components was given by h = {S, ST}, thus containing a spatial and spatio-temporal parts. However, the temporal effect appeared to be causing model ﬁt issues with oral/pharynx in the multivariate setting as this parameter was shared between diseases. Thus, in this paper we wish to explore the beneﬁt of either keeping the same mixture component structure and allowing the temporal effect to vary between diseases or introducing a third mixture component for temporal effects. Hence, the three alternative mixture deﬁnitions for both the univariate and bivariate/multivariate cases are displayed in Table 2. The parameters displayed are such that Xi, Xj, and Xij are the ith, jth, and ijth values of the spatial, temporal, and ST covariates, respectively, β ∼N 0, τ−1 β for the general case are the ﬁxed effect parameter estimates, uik ∼N 0, τ−1 uk is the uncorrelated spatial random effect. p p sunlight (sun), unemp ( ) 2.2. Statistical Methods Public Health 2017, 14, 503 7 of 13 Table 2. Alternative mixture term deﬁnitions. p p sunlight (sun), unemp ( ) 2.2. Statistical Methods Table 2. Alternative mixture term deﬁnitions. Bivariate/Multivariate 1 logit(pik) = zik + αik zik ∼CAR τ−1 zk αik ∼Norm 0, τ−1 αk logit qh ik = zh ik + ah ik zh ik ∼CAR τ−1 pk ah ik ∼N 0, τ−1 ah k ph ik = qh ik/ ∑ h qh ik h = {S, T, ST} 1 Note that bivariate and multivariate expressions can be grouped together because the only difference in their structure is that k = 1, 2 for the former and k = 1, 2, 3 for the latter. p p sunlight (sun), unemp ( ) 2.2. Statistical Methods However, when three mixture components and parameters are needed, as in Alternative 2 (Alt2), constraints must be imposed such that the sum of the three mixture parameters is equaled to one. This is accomplished with the following structure: logit qh i = zh i + ah i and ph i = qh i / ∑ h qh i or logit qh ik = zh ik + ah ik and ph ik = qh ik/ ∑ h qh ik, where h = {S, T, ST}. The two correlated random effects, vi and γj, are deﬁned such that they are common between diseases when there is no k subscript written. This is how information is shared between diseases of interest in these mixture models. Thus, Alternative 1 (Alt1) allows the temporally-correlated random effect to vary across diseases while the spatially-correlated random effect is shared amongst diseases. In the univariate setting, Alt1 is the same as F2PRED from the previous study [2], and the multivariate version of F2PRED offered the best ﬁt for oral/pharynx. On the other hand, Alt2 has the interpretation that the temporally correlated effect is common between diseases, but the relationship between disease and temporal effect is different depending on the cancer of interest through the addition of pT ik. Finally, Alternative 3 (Alt3) is like Alt1, but it allows for a shared temporal random effect that is scaled by a function which is related to the disease of interest. Alt3 was only applied in the bivariate and multivariate settings as an attempt to further improve model ﬁts by offering even more temporal ﬂexibility. The two functions, leading to Alt3a and Alt3b, are such that: Alt3a : fk γj, ρk = ρkγk, ρk ∼Norm 0, τρk Alt3b : fk γj, ρk = γj ρk, ρk ∼Gamma(2, 1) Thus, Alt3a offers a scaling coefﬁcient that differs per disease to relate to the temporal random effect while Alt3b offers a power term that differs per disease for relating to the temporal random effect. These alternatives offer different ways of incorporating more model ﬂexibility relating to the temporal random effect. For notation, a “U,” “B,” or “M” following Alt1, Alt2, Alt3a, or Alt3b indicates that these models are ﬁt in the univariate (each disease ﬁtted separately), bivariate (only considers oral/pharynx with lung), or multivariate setting. Int. J. Environ. Res. p p sunlight (sun), unemp ( ) 2.2. Statistical Methods Model Spatial Temporal Spatio-Temporal Mixture Parameter Univariate F2PRED piMS i MS i = X′ iβS + ui + vi - (1 −pi)MST ij MST ij = X′ ijβST j + X′ jβT+ γj + φij logit(pi) = zi + αi zi ∼CAR τ−1 z αi ∼Norm 0, τ−1 α Alt1 piMS i MS i = X′ iβS + ui + vi - (1 −pi)MST ij MST ij = X′ ijβST j + X′ jβT+ γj + φij logit(pi) = zi + αi zi ∼CAR τ−1 z αi ∼Norm 0, τ−1 α Alt2 pS i MS i MS i = X′ iβS + ui + vi pT i MT j MT j = X′ jβT + γj pST i MST ij MST ij = X′ ijβST j + φij logit qh i = zh i + ah i zh i ∼CAR τ−1 p ah i ∼N 0, τ−1 ah ph i = qh i / ∑ h qh i h = {S, T, ST} Bivariate/Multivariate 1 F2PRED pikMS ik MS ik = X′ iβS k + uik + vi - (1 −pik)MST ijk MST ijk = X′ ijβST jk + X′ jβT k + γj + φijk logit(pik) = zik + αik zik ∼CAR τ−1 zk αik ∼Norm 0, τ−1 αk Alt1 pikMS ik MS ik = X′ iβS k + uik + vi - (1 −pik)MST ijk MST ijk = X′ ijβST jk + X′ jβT k + γjk + φijk logit(pik) = zik + αik zik ∼CAR τ−1 zk αik ∼Norm 0, τ−1 αk Alt2 pS ikMS ik MS ik = X′ iβS k + uik + vi pT ikMT jk MT jk = X′ jβT k + γj pST ik MST ijk MST ijk = X′ ijβST jk + φijk logit qh ik = zh ik + ah ik zh ik ∼CAR τ−1 pk ah ik ∼N 0, τ−1 ah k ph ik = qh ik/ ∑ h qh ik h = {S, T, ST} Alt3a pikMS ik MS ik = X′ iβS k + uik + vi - (1 −pik)MST ijk MST ijk = X′ ijβST jk + X′ jβT k + ρkγρk + φijk logit(pik) = zik + αik zik ∼CAR τ−1 zk αik ∼Norm 0, τ−1 αk Alt3b pikMS ik MS ik = X′ iβS k + uik + vi - (1 −pik)MST ijk MST ijk = X′ ijβST jk + X′ jβT k + γj ρk + φijk logit(pik) = zik + αik zik ∼CAR τ−1 zk αik ∼Norm 0, τ−1 αk 1 Note that bivariate and multivariate expressions can be grouped together because the only difference in their structure is that k = 1, 2 for the former and k = 1, 2, 3 for the latter. 3. Results However, it is worth noting that the other diseases’ temporal random effect estimates are much larger in magnitude than that of oral/pharynx for Alt1M. Additionally, while this parameter is shared between all diseases in Alt2M and Alt3bM, as well as all of the bivariate ﬁts, the large jump does not appear for those situations. Supplemental Figures S2 and S3 display the uncorrelated and correlated heterogeneity terms that relate to oral cavity and pharynx cancer. Note here that the correlated random effect is shared between diseases for all bivariate and multivariate models. The pattern for the uncorrelated random effect appears to be similar across all model speciﬁcations, however, the variances associated with Alt2U and Alt3bM are much larger than for any of the other models. Alternatively, for Alt2U, the variance for the correlated random effect is smaller and appears to follow a different pattern compared to the other models while this is not the case for Alt3bM. In general, the variance of the spatially-correlated random effect is greater than that of the uncorrelated random effect; however, when only considering Alt2U, the ranges for ui and vi are nearly identical. Supplemental Figures S4 and S5 display the oral/pharynx related mixture parameter estimates for Alt1, Alt2, and Alt3 in the univariate, bivariate, and multivariate settings as labeled. The results here vary widely for all models. The univariate setting of Alt1 strongly selects the spatial mixture component with mixture parameter estimate very close to 1. This selection indicates that a better ﬁt may arise from only employing the spatial component of Alt1U, and this is the case as the WAIC estimate associated with this reﬁt is 3222.13 with a pD of 28.25. On the other hand, the MSPE is not improved by only using the spatial component. The bivariate model results are all alike and around the 0.5 range indicating that an equal mixture between the spatial and ST components is best. The estimates associated with Alt1M, Alt3aM, and Alt3bM agree and show much more variation, indicating that the central part of the state is slightly better explained by the spatial mixture component while the counties along western and eastern borders are better explained by the ST mixture component. For the Alt2 parameterization, the univariate and bivariate setting appears to prefer an equal mixture of the different components as a large majority of the counties are in the range of 0.3 to 0.4. 3. Results Table 3 presents the goodness of ﬁt results for oral/pharynx with the models described in Section 2. Supplemental Tables S1–S3 present results for melanoma and lung as well as the overall model ﬁt. The measurements here include Watanabe-Akaike information criterion (WAIC) and mean squared predictive error (MSPE). Both of these estimates indicate a superior model with a lower value. WAIC was proposed by Watanabe et al. as an alternative to the deviance information criterion and proves to be a better option of model assessment for mixture models [39,40]. These results indicate that Alt2B offers the best ﬁt for oral/pharynx in terms of WAIC for all year classiﬁcations as well as MSPE for most of the year classiﬁcations. When examining WAIC for the multivariate models, there is evidence of improvement over the univariate models when comparing the results associated with Alt2. Further, the MSPE values are very large for the multivariate setting while the WAIC estimates remain fairly stable. This is largely driven by oral/pharynx indicating that prediction is poor, particularly for 8 of 13 Int. J. Environ. Res. Public Health 2017, 14, 503 the ﬁnal four years of the study period under these modeling scenarios. The MSPE estimates that only consider the ﬁrst 10 years show that the multivariate models do perform best up to that point. Finally, these estimates demonstrate that Alt1, Alt2, and Alt3 show improvements over F2PRED for oral/pharynx with respect to multivariate model ﬁt and model prediction, respectively. Supplemental Figure S1 displays the temporal random effect (γj or γjk) estimates from univariate and multivariate ﬁts of Alt1 and Alt2 and the f γj, ρk estimates for Alt3aM and Alt3bM. Note that this parameter is shared between all diseases in bivariate and multivariate versions of Alt2, Alt3a, and Alt3b and the ﬁgure only displays the estimate associated with oral/pharynx for Alt1B and Alt1M. The estimates from Alt2U, Alt1B, Alt3bB, and Alt3bM appear to be essentially zero for the entire study period. All of the multivariate models except Alt3bM show the estimates increasing over time with a dramatic increase at year 10 (2005) for Alt1M and Alt3aM. This is much alike the jumps that were present in the example from Lawson et al., thus allowing the temporal random effect to be disease speciﬁc does not appear to ﬁx that issue. 3. Results The multivariate setting of Alt2 appears to indicate some selection relating to each of the mixture components for different counties. Both multivariate settings appear to agree somewhat in that central counties indicate better explanation from the spatial component while the counties on the eastern and western borders indicate a better explanation from the ST component. It could be the case that this relationship is an inﬂuence from the more common melanoma and lung that is not necessarily true for oral/pharynx. 9 of 13 Int. J. Environ. Res. Public Health 2017, 14, 503 Table 3. Goodness of ﬁt results for oral cavity and pharynx cancer.1 Measure Years Univariate Bivariate 2 Multivariate 2 F2PRED Alt1 Alt2 Alt1 Alt2 Alt3a Alt3b F2PRED Alt1 Alt2 Alt3a Alt3b WAIC ’96–‘09 3241.74 3239.09 4448.71 3196.02 3192.77 3194.97 3192.18 3431.50 3426.94 3520.64 3470.28 4184.67 ’96–‘05 2298.34 2296.40 3130.11 2265.29 2265.94 2264.57 2262.46 2285.45 2479.40 2322.88 2473.00 2954.48 ’06–‘09 943.40 942.69 1318.60 930.73 926.83 930.40 929.72 1146.05 1000.37 1197.76 997.28 1230.20 pD ’96–‘09 105.89 105.29 157.31 94.31 96.66 93.44 93.76 131.08 131.07 141.69 149.43 501.02 ’96–‘05 75.39 74.35 96.14 65.53 68.85 66.34 65.84 74.29 103.97 73.92 106.24 344.19 ’06–‘09 30.50 30.94 61.17 28.79 27.80 27.09 27.92 56.78 42.40 67.78 43.18 156.83 MSPE ’96–‘09 153.44 154.03 117.00 117.93 111.64 116.81 115.23 14,050.59 14,037.08 13,995.61 13,953.98 13,678.78 ’96–‘05 103.93 103.75 84.43 84.53 80.58 82.13 82.00 73.51 71.79 79.94 74.44 73.05 ’06–‘09 49.51 50.28 32.57 33.40 31.06 34.68 33.22 13,977.08 13,965.29 13,915.67 13,879.53 13,605.73 1 Values in bold indicate the model with superior ﬁt for that measure and time period. If multiple measures are indicated as superior for the same measure and time period combination, they are statistically equivalent. 2 The bivariate and multivariate results are the goodness of ﬁt estimates related speciﬁcally to oral/pharynx. Even though multiple diseases were ﬁt, these measures can be calculated in a way such that they only pertain to the ﬁt of the individual diseases. Table 3. Goodness of ﬁt results for oral cavity and pharynx cancer.1 Table 3. Goodness of ﬁt results for oral cavity and pharynx cancer.1 1 Values in bold indicate the model with superior ﬁt for that measure and time period. If multiple measures are indicated as superior for the same measure and time period combination, they are statistically equivalent. 2 The bivariate and multivariate results are the goodness of ﬁt estimates related speciﬁcally to oral/pharynx. 3. Results Even though multiple diseases were ﬁt, these measures can be calculated in a way such that they only pertain to the ﬁt of the individual diseases. 10 of 13 10 of 10 of 13 10 of Int. J. Environ. Res. Public Health 2017, 14, 503 Int. J. Environ. Res. Public Health 2017, 14, x Since our goal is, ultimately, to improve the modeling of risk related to oral/pharynx cancer, Figure 5 displays the overall risk (θij) of lung and oral/pharynx associated with the best model, Alt2B. The ﬁxed effect parameter estimates used for this calculation are included in Supplemental Table S4. Only 1996, 2002, and 2009 are included for brevity. From this ﬁgure, it can be deduced that lung has high risk in the northern portion of the state, as well as a lone county of elevated risk in the southern tip. For oral/pharynx cancer, the overall risk is higher in the eastern counties, particularly for some along the borders. This could be some edge effect which occurs in models of this nature. Temporally, the risk for both diseases appears to be increasing slightly over the years represented. In general, the trend in risk appears to be consistent across both space and time. Since our goal is, ultimately, to improve the modeling of risk related to oral/pharynx cance Figure 5 displays the overall risk (ࣂ࢏࢐) of lung and oral/pharynx associated with the best model, Alt2 The fixed effect parameter estimates used for this calculation are included in Supplemental Table S Only 1996, 2002, and 2009 are included for brevity. From this figure, it can be deduced that lung ha high risk in the northern portion of the state, as well as a lone county of elevated risk in the souther tip. For oral/pharynx cancer, the overall risk is higher in the eastern counties, particularly for som along the borders. This could be some edge effect which occurs in models of this nature. Temporall the risk for both diseases appears to be increasing slightly over the years represented. In general, th trend in risk appears to be consistent across both space and time. Figure 5. Overall posterior mean risk estimate (ࣂ࢏࢐) for lung and bronchus cancers, as well as oral cavity and pharynx cancer from model Alt2B for a selection of years. Figure 5. 4. Discussio 4. Discussion The multivariate modeling of diseases offers many benefits; however, issues can arise wh models fail to offer the appropriate amount of flexibility or inappropriate diseases are combined. T results above illustrate the importance of choosing the best model, as well as the best set of disease to incorporate to gain the desired improvement in fit for the less common disease. In this case, t bivariate modeling combining oral/pharynx with lung furnishes the best fit. The multivariate modeling of diseases offers many beneﬁts; however, issues can arise when models fail to offer the appropriate amount of ﬂexibility or inappropriate diseases are combined. The results above illustrate the importance of choosing the best model, as well as the best set of diseases, to incorporate to gain the desired improvement in ﬁt for the less common disease. In this case, the bivariate modeling combining oral/pharynx with lung furnishes the best ﬁt. g g p y g The example from the paper in which these methods are introduced simply removes the da for the years where the multivariate model does not perform well with respect to oral/pharynx. this exploration, we have successfully produced a slight variation on their methods that allow th entire data to be used. This is important because removing data results in a loss of information, an that is not an ideal solution. Ultimately, the results indicate that bivariate rather than multivaria methods are ideal for producing the best fit associated with oral/pharynx across the entire study tim The example from the paper in which these methods are introduced simply removes the data for the years where the multivariate model does not perform well with respect to oral/pharynx. In this exploration, we have successfully produced a slight variation on their methods that allow the entire data to be used. This is important because removing data results in a loss of information, and that is not an ideal solution. Ultimately, the results indicate that bivariate rather than multivariate methods are ideal for producing the best ﬁt associated with oral/pharynx across the entire study time. As far as interpretation is concerned, these models do offer some difficulties. For the best mod Alt2B, the ideal interpretation of the random component comes from the information provided in th term: ݌௜௞ ௌ(ݑ௜௞+ ݒ௜) + ݌௜௞ ்ߛ௝+ ݌௜௞ ௌ்߶௜௝௞. 3. Results Overall posterior mean risk estimate (θij) for lung and bronchus cancers, as well as oral cavity and pharynx cancer from model Alt2B for a selection of years. Figure 5. Overall posterior mean risk estimate (ࣂ࢏࢐) for lung and bronchus cancers, as well as or cavity and pharynx cancer from model Alt2B for a selection of years. Figure 5. Overall posterior mean risk estimate (θij) for lung and bronchus cancers, as well as oral cavity and pharynx cancer from model Alt2B for a selection of years. 4. Discussio 4. Discussion This estimate represents the total of the confounding adjuste for in the fitted model and is displayed in Supplemental Figure 4. From this estimate, it is clear th the overall risk unexplained by the fixed effects becomes more variable over the study perio Increased risk appears in the northwestern and northeastern corners of the state; these areas are the mountains and along the coast respectively. Interpretation of the fixed effects is similar in that th measure of interest would be ݌௜௞ ்ߚ௞ ் for the particular county and disease of interest. Beyond that, a interpretation of the mixture parameter can be ascertained wherein, for example, if a particul As far as interpretation is concerned, these models do offer some difﬁculties. For the best model, Alt2B, the ideal interpretation of the random component comes from the information provided in the term: pS ik(uik + vi) + pT ikγj + pST ik φijk. This estimate represents the total of the confounding adjusted for in the ﬁtted model and is displayed in . From this estimate, it is clear that the overall risk unexplained by the ﬁxed effects becomes more variable over the study period. Increased risk appears in the northwestern and northeastern corners of the state; these areas are in the mountains and along the coast respectively. Interpretation of the ﬁxed effects is similar in that the measure of interest would be pT ikβT k for the particular county and disease of interest. Beyond that, an interpretation of the mixture parameter can be ascertained wherein, for example, if a particular county in Alt2B has Int. J. Environ. Res. Public Health 2017, 14, 503 11 of 13 pS ik = 0.1, pT ik = 0.5, and pST ik = 0.4, then 10% of the overall variability in the risk is explained by spatial effects, 50% is explained by temporal effects, and 40% is explained by ST effects. If the entire state and all diseases have a fairly low estimate for one of the mixture parameters, as with Alt1U for oral/pharynx, it might be of interest to ﬁt the model without that component to determine if it is necessary for explaining the overall variability in the risk of disease. There are several explanations regarding the poor ﬁt of the multivariate model. 4. Discussio 4. Discussion The ﬁrst is that, while melanoma is also a more prevalent disease than oral/pharynx with common risk factors, they may be too different as far as spatial and temporal patterns are concerned to aid in the modeling of oral/pharynx in these data. Additionally, the multivariate models add even more random effects of the same type, and this could be leading to identiﬁcation issues and, thus, poor ﬁtting models. This is an issue common to all mixture model structures; however, the hierarchical structure and prior dependence, even when uninformative choices are made, that is characteristic of these Bayesian methods minimizes this issue as much as possible. We have assumed non-informative priors in this example, but even stronger prior assumptions could continue to minimize the issue of identiﬁability. Finally, there could be collinearity between the ﬁxed and random effects. This issue could also lead to poorly-ﬁtting models, as well as inaccurate estimates of the effects. Some, or all, of these explanations could impact the individual estimates presented in this paper, but when considering the overall risk of disease, which is our main goal, the random effects take on the role of representing the confounding variables to potentially produce the optimal ﬁt. 5. Conclusions The best model for explaining the overall risk of oral/pharynx is Alt2B. This model includes separate spatial, temporal, and ST mixture components, meaning that it is important to separate the temporal and ST ﬁxed and random effects. Further, this model only combines oral/pharynx with lung, and that suggests that melanoma is not the best disease choice for modeling with oral/pharynx. Alternatively, multivariate models Alt3b and Alt3a offer improved ﬁts for lung and melanoma, respectively. Supplementary Materials: The following are available online at www.mdpi.com/1660-4601/14/5/503/s1, Figure S1: γjk, γj, and f γj, ρk posterior mean estimates for univariate and multivariate ﬁts of Alt1, Alt2, and Alt3 for oral cavity and pharynx cancer, Figure S2: Posterior mean ui estimates for Alt1 and Alt2 in the univariate and multivariate framework, Figure S3: Posterior mean vi estimates for Alt1 and Alt2 as well as univariate and multivariate, Figure S4: Alt1 oral cavity and pharynx cancer posterior mean mixture parameter estimates in the univariate, bivariate, and multivariate settings, Figure S5: Alt2 oral/pharynx posterior mean mixture parameter estimates (pS i , pT i , pST i , pS ik, pT ik, and pST ik ) in the univariate, bivariate, and multivariate settings, Figure S6: Sum of the posterior mean random effects times posterior mean mixture parameters (pS ik(uik + vi) + pT ikγj + pST ik φijk) associated with oral cavity and pharynx cancer for Alt2B, Table S1: Overall goodness of ﬁt results for all cancers, Table S2: Goodness of ﬁt results for melanoma cancer of the skin, Table S3: Goodness of ﬁt results for lung and bronchus cancer, Table S4: Posterior mean ﬁxed effect coefﬁcient estimates for the best ﬁtting model, Alt2B. 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https://openalex.org/W2563488180	https://flore.unifi.it/bitstream/2158/1079784/1/20000-40403-2-PB%20%281%29.pdf	Italian	null	Il lavoro atipico e la dimensione soggettiva: il ruolo della formazione permanente	DOAJ (DOAJ: Directory of Open Access Journals)	2,016	cc-by	8,051	Il lavoro atipico e la dimensione soggettiva: il ruolo della formazione permanente1 Maria Rita Mancaniello Abstract. The widespread presence of atypical forms of work in our society is not only due to a change of the possible types of work, but is also emptying the same work of its social function. There are more at risk psychological variables. The insta- bility of the work connected to a personal perception of losing their jobs; the prob- lems that arise on the construction of professional identity of persons; on the interac- tions between the subject and its organization of work and the satisfaction and quality of life. Especially the private life. In this essay I will put my attention on the meaning that this working model involves a psychological level. The goal is to promote a learn- ing process which can give the subject of resilience potential and creative resources to respond to the suffering that these types of work involved. For the education and training that attention is becoming increasingly urgent. Until now the goal was per- haps more to build knowledge and basic skills and specific, without taking into seri- ous consideration the specific aspect of identity of the worker and his personal stabil- ity.Today it is necessary to pay attention to all the human sciences - and pedagogy before any other - on the problems that the atypical work and job instability fee for the subject and for all the people who share his sphere of life. Keywords. Atypical jobs, lifelong learning, job stress, work psychology, pedagogy of work Abstract. La presenza diffusa di forme atipiche di lavoro nella nostra società non solo è causa di un cambiamento delle possibili tipi di lavoro, ma sta anche svuotando lo stesso lavoro della sua funzione sociale. Ci sono più variabili psicologiche a rischio dal momento che, se stiamo parlando di instabilità del lavoro collegato ad una percezione personale di perdere il posto di lavoro, o ad una condizione contrattuale precaria vera e propria, le tipologie di problemi che sorgono si ripercuotono sulla costruzione dell’i- dentità professionale delle persone, sulle interazioni tra il soggetto e la sua organiz- zazione del lavoro e sulla soddisfazione e la qualità della vita, in particolare della vita privata. DOI: 10.13128/ccselap-20000 \| ISSN 2531-9884 (online) Comparative Cultural Studies: European and Latin America Perspectives 2: 89-102, 2016 DOI: 10.13128/ccselap-20000 \| ISSN 2531-9884 (online) Comparative Cultural Studies: European and Latin America Perspectives 2: 89-102, 2016 1 Alcuni dei temi qui trattati sono stati discussi in precedenza in Mancaniello (2012a; 2012). Per la stesura di questo articolo sono state rivisti e aggiornati. 22 Uno studio di László e collaboratori ha indagato, con un metodo auto-valutativo, quali sono gli effetti del lavo- ro temporaneo sulle condizioni di salute di lavoratori di età compresa fra i 45 e i 70 anni di 16 paesi europei: Repubblica Ceca, Danimarca, Germania, Grecia, Ungheria, Israele, Paesi Bassi, Polonia, Russia, Austria, Francia, Italia, Spagna, Svizzera, Belgio e Svezia. Gli intervistati sono tutti soggetti con impieghi precari e dai dati emersi, risulta che siano soggetti con discreto rischio di malattia nella maggior parte dei paesi inclusi nell’analisi. I paesi si differenziano per la portata delle norme sul mercato del lavoro, l’assistenza sanitaria e il grado di sindacalizza- zione. Questi fattori possono potenzialmente fungere da buffer o aumentare ulteriormente il rischio di malattia a causa della precarietà del lavoro. La correlazione significativa tra precarietà del lavoro e condizioni negative di salute è risultata presente in diversi paesi, compresi quelli con uno Stato sociale ben sviluppato, fattore che sem- bra indicare che la precarietà del lavoro ha conseguenze negative sul soggetto non solo per l’aspetto economico, ma relativamente alla percezione di sé. Una sintesi del lavoro di ricerca è consultabile nel sito: <http://www.ncbi. nlm.nih.gov/pmc/articles/PMC2845821/> (10/2016). Il lavoro atipico e la dimensione soggettiva: il ruolo della formazione permanente1 In questo saggio porrò la mia attenzione sul significato che questo modello di lavoro comporta, dal punto di vista psicologico, per tentare di promuovere un proces- so formativo che possa offrire al soggetto potenzialità di resilienza e risorse creative per reagire alla sofferenza che questi tipi di lavoro comportano. Per il mondo dell’i- struzione e della formazione questa attenzione sta diventando sempre più urgente. Fino ad oggi l’obiettivo è stato forse più quello di costruire conoscenze e competenze di base e specifiche, senza prendere in seria considerazione lo specifico aspetto dell’i- 1 Alcuni dei temi qui trattati sono stati discussi in precedenza in Mancaniello (2012a; 2012). Per la stesura di questo articolo sono state rivisti e aggiornati. Copyright © 2016 The Author(s). Open Access. This in an open access article published by Firenze University Press (www.fupress.com/bae) and distributed under the terms of the Creative Commons Attribution 4.0 Inter- national License. The Creative Commons Public Domain Dedication waiver applies to the data made available in this article, unless otherwise stated. 90 Maria Rita Mancaniello dentità del lavoratore e la sua stabilità personale. Oggi è necessario porre l’attenzione di tutte le scienze umane - e della pedagogia prima di ogni altra - sui problemi che il lavoro atipico e l’instabilità lavorativa comportano per il soggetto e per tutte le perso- ne che condividono la sua sfera di vita. Parole chiave. Formazione permanente, lavoro atipico, pedagogia del lavoro, psicolo- gia del lavoro, stress lavorativo. Comparative Cultural Studies: European and Latin America Perspectives 2: 89-102, 2016 2. Uno sguardo alla recente riforma del lavoro Prima di entrare più nel merito della questione soggettiva, credo importante fare un breve accenno alla recente riforma sul lavoro, meglio conosciuta come Jobs act, che ha cambiato in modo sensibile le forme di assunzione e la tipologia dei contratti di lavoro, riportando al centro il contratto a tempo indeterminato, scelta che è stata pensata come funzionale alla riduzione delle forme di instabilità, seppur permangano incertezze nell’oc- cupazione, come mostrano i dati sulle assunzioni, in aumento in un primo periodo e poi ristabilizzate su dati preoccupanti di disoccupazione. Tale decreto di riforma ha introdotto anche la variabile di “flessibilità” delle mansioni, con un potenziale di cambiamento nei diversi ruoli lavorativi, in caso di crisi aziendale o di necessità di riorganizzare l’impresa. In tal senso, il nuovo incarico all’interno della stessa azienda/area di lavoro, potrà essere anche un “de-mansionamento” di un livello contrattuale, ma sempre nella stessa categoria di inquadramento (operaio, impiegato, quadro), seppur debba rimanere invariato lo sti- pendio della precedente qualifica. l i Relativamente al focus della nostra riflessione, altra variabile introdotta nella normati- va che sembra ricadere maggiormente sul mondo del lavoro femminile è l’incremento dei periodi in cui è possibile richiedere i congedi parentali, con l’obiettivo di conciliare il lavo- ro con le esigenze familiari, ma sicuramente, la parte più innovativa e interessante e dove un nuovo modello di formazione potrebbe divenire un reale e significativo supporto, è l’introduzione del “contratto di ricollocamento”, che riguarda il lavoratore che perde il pro- prio lavoro e nella sua nuova veste di disoccupato, deve essere preso in carico da un uffi- cio del lavoro che ne traccia il “profilo di occupabilità”. Tale percorso prevede che, dopo la firma di un patto di attivazione, al quale è condizionata la corresponsione dei sussidi di disoccupazione, al lavoratore venga assegnato un voucher che gli permetta di usufruire di servizi di formazione e ricollocazione in un altro posto di lavoro e la scelta se avvalersi di servizi per l’impiego pubblici o delle agenzie per il lavoro private accreditate, ai quali verrà corrisposto il compenso relativo solo a risultato ottenuto, rimane del lavoratore. Diversi studi di tipo multidisciplinare stanno mettendo in rilievo risultati contrastanti sull’efficacia di questa legge, tanto che, mentre i dati istituzionali sottolineano con forza la diminuzione del tasso di disoccupazione, altri autori, con la stessa forza, ne evidenziano invece un aumento. ra personalità del soggetto con tutte le sue peculiari caratteristiche. Come sottolinea Emilio Reyneri (2005, p. 127): ra personalità del soggetto con tutte le sue peculiari caratteristiche. Come sottolinea Emilio Reyneri (2005, p. 127): finora, almeno nel contesto italiano, accadeva che il lavoratore poteva avere un periodo, quello gio- vanile, ove la precarietà di diritto e/o di fatto era alta. Poi però, ad una certa età, raggiungeva una stabilità o di fatto o di diritto. Ma in futuro cosa accadrà? Quello che dovrebbe preoccuparci non è tanto la precarietà del ventenne, che ha ancora una vita personale e familiare davanti a sé e può farvi fronte bene anche sul piano emotivo. La grande questione è cosa accadrà successivamente nel suo corso di vita, quando si avvierà lungo i suoi trent’anni e avrà sempre più bisogno di sicurezze, sul piano sia economico che emotivo.3 finora, almeno nel contesto italiano, accadeva che il lavoratore poteva avere un periodo, quello gio- vanile, ove la precarietà di diritto e/o di fatto era alta. Poi però, ad una certa età, raggiungeva una stabilità o di fatto o di diritto. Ma in futuro cosa accadrà? Quello che dovrebbe preoccuparci non è tanto la precarietà del ventenne, che ha ancora una vita personale e familiare davanti a sé e può farvi fronte bene anche sul piano emotivo. La grande questione è cosa accadrà successivamente nel suo corso di vita, quando si avvierà lungo i suoi trent’anni e avrà sempre più bisogno di sicurezze, sul piano sia economico che emotivo.3 3 Per il dibattito in corso si veda anche Bauman (2002); Boeri, Garibaldi (2008). 1. Il lavoro atipico e le sue caratteristiche Nel presente contributo l’analisi si focalizza sulle forme del lavoro atipico e sulle que- stioni che si pongono quando tale tipologia di lavoro non è determinato da una libera scelta del soggetto, ma obbligato dall’offerta del mercato, con una specifica attenzione ai bisogni di formazione che questo status comporta e richiede (Mancaniello, 2012).fi La possibilità di inserirsi in un mondo del lavoro precario attiva una serie di difficoltà su diversi piani dell’esistenza, ma sembra che sia in particolare la dimensione psicologica ad essere maggiormente in sofferenza, a causa della frammentazione delle azioni da svol- gere, dal doversi continuamente adattare a nuovi contesti organizzativi e relazionali e dalla frustrazione che provoca la necessità di dover orientare le proprie aspettative alle possibili- tà offerte, senza poter scegliere sulla base dei propri desideri e delle proprie aspirazioni, e, spesso, anche della formazione compiuta.(László, Pikhart, Kopp, Bobak, Pajak, Malyutina, Salavecz, Marmot, 2010, pp. 867-874)2. Le tipologie di lavoro a cui stiamo facendo riferimento sono quelle del lavoro part- time, apprendistato, lavoro temporaneo, formazione lavoro, lavoro a progetto, collabora- zione occasionale, collaborazione coordinata e continuativa, forme di lavoro che hanno introdotto una trasformazione radicale nel mercato del lavoro, ma che sono diventate fon- te di preoccupazione per giovani e meno giovani. Forme “atipiche” in quanto non abituali, diverse dai tradizionali contratti di lavoro dipendente a tempo indeterminato e dalle for- me di lavoro autonomo, ma che hanno portato ad una rilevante trasformazione nel tessuto sociale e personale del soggetto di oggi (Andreoni, 2005). L’attenzione che vorremmo porre in questa analisi è legata al fatto che viene coinvol- ta nel processo lavorativo la personalità dei lavoratori e delle lavoratrici, tanto che oggi, quando ci si riferisce al “mercato del lavoro”, si parla di un vero e proprio mercato della vita, ovvero si guarda a quella dimensione lavorativa in cui si scambia non solo una qual- siasi capacità lavorativa, qualificata da capacità acquisite di ordine professionale, ma l’inte- Comparative Cultural Studies: European and Latin America Perspectives 2: 89-102, 2016 Il lavoro atipico e la dimensione soggettiva 91 3. Precarietà e stabilità a confronto Riportando l’attenzione sulle forme di lavoro con contratti atipici, si nota immedia- tamente come spesso tali contratti si caratterizzino per salari bassi e discontinui, con la conseguente insicurezza economica ed un alto rischio di essere più facilmente in balia del dominio del committente, ma questo aspetto è legato a una situazione che richiede una maggiore attenzione da parte di chi deve garantire i diritti del lavoratore e diventa difficile da gestire in ambito formativo. Un ulteriore problema che non va sottovalutato è sicuramente l’avere un reddito instabile, presunto ma non prevedibile, che è la caratteristica che accomuna tutti coloro che hanno contratti a tempo determinato nelle diverse forme previste dal lavoro atipico. Questo piano ricade in modo immediato sulla gestione del tempo, poiché l’incertezza del reddito comporta l’incapacità di rinunciare a qualsiasi opportunità e l’ansia dei possibili periodi di inattività porta alla sovrapposizione di incarichi, con la necessità di dover lavo- rare molte ore al giorno, con conseguenti ricadute su tutti gli altri aspetti della propria vita. Se da una parte, la possibilità di un alto grado di autonomia organizzativa ha i carat- teri della libertà, dall’altra può portare ad una ricerca smodata di lavori da fare per tutelar- si da eventuali periodi in cui le opportunità di lavoro potrebbero venir meno. Una sorta di insicurezza che può portare a fagocitare ogni opportunità, senza più valutare quantità, qualità e possibilità reali di farcela senza rimanere soffocati rispetto a tutti gli altri bisogni quotidiani (Bologna, Banfi, 2011). il i Assume, però, un valore importante ai fini della riflessione sulle conseguenze psi- cologiche del lavoro atipico e sulle possibili risposte formative, perché pone al centro le necessità del soggetto, alle quali può essere data una risposta più adeguata e più signifi- cativa nella strutturazione dei percorsi formativi, nelle diverse dimensioni in cui questi si sviluppano. Affidabilità, elasticità, disponibilità, costanza, capacità di saper gestire il rischio, concretezza e capacità di relazionarsi con gli altri, sono tutti aspetti a partire dai quali deve essere progettato il processo formativo e devono essere sviluppate le metodo- logie di lavoro. 4 Solo da qualche anno il lavoro atipico e divenuto interesse di ricerca anche in ambito psicologico. In partico- lare, nel saggio di Ferrari e Veglio, si ritrovano molte delle variabili correlate al lavoro atipico quali: la formazi- one, le differenze di genere, l’identità lavorativa, la soddisfazione lavorativa, l’orientamento professionale. 2. Uno sguardo alla recente riforma del lavoro Come sostiene già Gallino da diversi anni, l’unico dato certo è che il rapporto tra flessibilità e occupabilità non è certo (Gallino, 2001; 2007). 3 Per il dibattito in corso si veda anche Bauman (2002); Boeri, Garibaldi (2008). DOI: 10.13128/ccselap-20000 \| ISSN 2531-9884 (online) 92 Maria Rita Mancaniello Maria Rita Mancaniello Non è il contesto adatto per entrare in merito alle questioni normative, che ci por- terebbero lontani e su piani di discussione attualmente molto ideologizzati o, comunque, che richiedono una attenzione e una analisi puntuali per essere compresi fino in fondo nella loro poliedrica complessità. Qui intanto ci poniamo nell’ottica che il lavoro è al cen- tro delle questioni sociali e che i sistemi di formazione devono rivedere molti piani della propria offerta e progettazione per poter essere incisivi e significativi nella realtà del sog- getto, sia in chiave psicologica che sociale (Ferrari, Veglio, 2006)4. Comparative Cultural Studies: European and Latin America Perspectives 2: 89-102, 2016 4. Una specifica fatica del lavoro precario: essere donne immigrate in Italia Se il problema della precarietà fa parte del nostro tempo e di una gran parte di lavo- ratori senza distinzione di genere, età ed origini, uno sguardo sulla situazione della realtà femminile e specificatamente delle donne immigrate, credo ci aiuti a comprendere meglio il tempo che stiamo attraversando dal punto di vista del lavoro.i Parlare di femminile nell’immigrazione in Italia significa riferirsi a più della metà della popolazione straniera con regolare permesso di soggiorno presente nel nostro pae- se. I dati più recenti forniti dall’ISTAT, pubblicati a fine 2015 e relativi alle analisi effet- tuate nell’anno 2013-2014 mostrano che la componente femminile straniera è del 52,7% sul totale della popolazione. Una donna che si pone come il collante tra famiglia e realtà sociale, sia nelle relazioni con la scuola che con i pubblici servizi o nelle rappresentanze collettive, con un importante ruolo nei processi di integrazione della famiglia e dei grup- pi originari nel tessuto sociale. Come ben sappiamo e ormai ricorrentemente sottolineato da studi e ricerche, così come media e organi di informazione, non sono mai state attua- te in Italia politiche specifiche sull’immigrazione e, forse, ancor meno sull’immigrazione femminile come fenomeno strutturale e significativo. Tale disattenzione ad un processo migratorio inarrestabile e irreversibile, in un periodo di crisi nazionale e internazionale, ricade in modo estremamente significativo sulle identità straniere a più livelli, sicuramen- te in modo evidente sul riconoscimento delle professionalità femminili straniere, per le quali gli ambiti di occupazione sono precipuamente legate ai servizi alle famiglie. Secondo i dati del Dossier Statistico Immigrazione 2014, il trend di occupazione delle donne stra- niere segue in modo quasi identico quello delle donne italiane, con il 44,8% delle lavora- trici madri, rispetto alle donne senza figli che raggiungono il 69,2% e una donna su tre lavora nel settore dei servizi di assistenza alla persona. Non facili occupazioni, per le quali si sentono spesso discriminate e con una qualità della vita che risente in modo profondo di ritmi di lavoro pressanti, faticosi per le relazioni di cura con anziani non autosufficien- ti o con deficit che richiedono assistenza continua, senza la possibilità di una vita socia- le e relazionale adeguata. 3. Precarietà e stabilità a confronto La situazione che ormai si è creata tra la richiesta del mercato del lavoro, il sistema di sostegno sociale e le strutture familiari mostra il processo di indebolimento che è in atto nel legame sociale, ma anche come la reazione dei singoli sia quella di attuare strate- gie in grado di contrastare la perdita di sicurezza lavorativa, attivando proprie consistenti Il lavoro atipico e la dimensione soggettiva 93 risorse e molteplici energie per vivere comunque bene il proprio tempo di vita e la pro- pria identità professionale. 4. Una specifica fatica del lavoro precario: essere donne immigrate in Italia Se poi si aggiunge, per molte donne, la distanza dalle proprie famiglie e dai figli, per la maggior parte rimasti nei territori di provenienza, emerge che anche i processi di inclusione sono quasi annullati, con conseguenti forme di solitudine o di rapporti solo con i propri connazionali, senza un reale sviluppo del proprio vissuto sociale. Nei diversi studi si evidenzia proprio questa mancanza come una delle più sentite criticità del proprio progetto migratorio, incastrato tra il difficile procedimento per l’otte- nimento del permesso di soggiorno e il riconoscimento dei titoli di studio conseguiti nei paesi di origine e una quotidianità che non riesce a dare spazio alle diverse esigenze del soggetto. Anche i servizi a disposizione delle persone immigrate sono molto limitati e, a parte alcune realtà territoriali maggiormente organizzate che mostrano livelli di valore in tal senso, nella maggior parte delle realtà cittadine e di paese, mancano sistemi in grado di orientare, sostenere e dare risposte adeguate alla maggior parte dei bisogni legati alla propria identità di lavoratrici, così come a quella di donne e di madri e anche della salute, senza tralasciare il problema di supporti psicologici per tutto il cambiamento che com- DOI: 10.13128/ccselap-20000 \| ISSN 2531-9884 (online) 94 Maria Rita Mancaniello porta, in molte situazioni, lo sradicamento culturale. Sembra quasi semplicistico ribadire come le donne immigrate avrebbero bisogno di un maggiore riconoscimento sia in termi- ni salariali che di qualificazione dell’occupazione, almeno con pari diritti degli autoctoni, così come una maggiore differenziazione di ruoli nei diversi settori lavorativi. Non si deve tralasciare che la realtà delle donne immigrate che sono con uomini svolgono quasi sem- pre principalmente il ruolo di mogli e di madri, soprattutto quando provengono da paesi dove ancora è forte la centralità del “pater familias” o che provengono da matrici culturali e religiose per le quali la condizione femminile è assoggettata a quella maschile in ogni espressione. In tali situazioni tutto il processo di integrazione diventa una difficoltà, a par- tire dal contatto con le istituzioni e le pubbliche amministrazioni, così come i contesti sco- lastici e educativi territoriali che richiedono forme di partecipazione e di condivisione che impattano con tradizioni e modelli culturali profondamente diversi. Situazioni che richie- derebbero una grande formazione alle relazioni da parte di chi accoglie e di rilettura dei propri approcci culturali in chiave almeno dialogica. 4. Una specifica fatica del lavoro precario: essere donne immigrate in Italia p p pp g Un ulteriore aspetto da rilevare come potenzialmente tutto da sviluppare si evidenzia quello dell’imprenditorialità femminile, settore lavorativo che rimane largamente maschile L’imprenditorialità femminile straniera è sicuramente uno dei settori produttivi da valo- rizzare nei prossimi anni, poiché attualmente è caratterizzata da un 75,5% (pari a 53.700 imprenditori) di uomini stranieri, con una percentuale di imprenditrici femminili che si assesta al 24,5% (17.400 donne). Come si può ben comprendere, per potenziare lo svilup- po in tale direzione è necessario che si abbiano politiche e interventi istituzionali precisi e mirati, così come una chiara informazione su quelle che possono essere le opportunità e i servizi offerti dal settore bancario, l’accesso agli strumenti di credito, forme di garanzia per l’utilizzo corretto e efficace degli strumenti di tutela legale propri del nostro ordina- mento nazionale. 5 Per un approfondimento su questo tema si veda Farina (1995, pp. 117-133); Rossi (1997, pp. 45-68); Buzzi, Cav- alli, De Lillo (2007). Comparative Cultural Studies: European and Latin America Perspectives 2: 89-102, 2016 5. La fatica di vivere nell’instabilità L’aumento dei soggetti che vivono una condizione di instabilità e di condizioni di vita precarie si colloca in un periodo storico in cui aumenta l’incertezza a tutti i livelli, in cui ogni sistema è in piena trasformazione e ridefinizione. Se è vero che a questo processo si associa un rischio molto alto di difficoltà esistenziale del soggetto, è anche vero che questo comporta lo sviluppo di alti gradi di libertà. L’instabilità lavorativa ha i complessi connotati della preoccupazione per ciò che non è sicuro, ma anche il piacere del non essere dentro il vincolo. Quel rischio insito nella visio- ne di Ulrich Beck, per il quale l’incertezza porta l’uomo a focalizzare maggiormente l’at- tenzione su se stessi, con un progressivo aumento dell’individuazione e una conseguente tensione all’azione (Beck, 2000)5. Partendo dalla convinzione che l’identità del soggetto trova costrutto anche nella scel- ta lavorativa, è indubbio che il processo di individualizzazione e le nuove forme di struttu- razione dei percorsi di vita sono inscindibilmente connessi con le trasformazioni che sono avvenute nel mercato del lavoro. Al contrario di ciò che si verificava nella società fordista, Il lavoro atipico e la dimensione soggettiva 95 dove il lavoro era di per sé sufficiente a sviluppare una identità stabile e ad assicurare l’in- tegrazione sociale, nella nostra società, caratterizzata da lavori precari e transitori, il senso della propria identità e la percezione di stabilità sono sempre in divenire, così come la definizione di un determinato status sociale. Questo si verifica in un contesto storico dove l’incertezza e la trasformazione sono ricorrenti in tutti i sistemi di riferimento del sog- getto, motivo che comporta che nessuno sembra più essere in grado di fare programmi a lunga scadenza e tutti tendono a mantenere sempre aperte possibili alternative, guardando continuamente a nuovi scenari verso cui indirizzare al propria vita e permanendo in una condizione di centralità del presente (Laffi, 2000).l fi Anche Richard Sennet sottolinea come la vita del lavoratore flessibile sia un puzzle di frammenti al quale il soggetto non sempre riesce ad attribuire un senso progettuale (Sen- net, 1999). Nel contesto caratterizzato dal lavoro instabile, il soggetto ha davanti a sé una realtà sempre in divenire, che gli lascia ampi spazi di libertà, ma che può comportare un inde- bolimento della capacità di elaborare strategie e di determinare processi intenzionali di sviluppo di sé. 66 Nella teoria della complessità di Morin il soggetto umano è indissolubilmente legato all’ambiente in cui è inseri- to, reintegrato in senso naturale e biologico nel mondo fisico, oltre che in quello sociale e culturale. La conoscenza è un processo che si attiva a partire dal soggetto vivente e la conoscenza dell’umano, che afferisce al culturale, è tale se viene intesa come auto-conoscenza, divenendo lo stesso soggetto responsabile del suo stesso agire. 5. La fatica di vivere nell’instabilità Se alcuni riescono ad usare l’instabilità come una risorsa per definire auto- nomamente il proprio percorso professionale, sapendo gestire il rischio che deriva dall’in- certezza, per altri – per i quali è mancata una formazione di specifiche capacità – rimane molto difficile riuscire a combinare le risorse a disposizione per raggiungere una posizione nel mercato del lavoro, sicura e appagante. Questo comporta che nel soggetto odierno ci sia un potenziale di adattamento che, se supportato anche da un processo formativo ben strutturato e organizzato per accogliere e contenere l’incertezza e la precarietà di vita, pos- sano fargli ottenere – nonostante tutto –, gradi di benessere e di soddisfazione adeguati alle richieste del nostro tempo e della nostra realtà (Manghi, 2009)6. 6. Uno sguardo al possibile ruolo della formazione Per cercare di contenere questo senso di rottura, una risposta è già stata quella di strutturare un sistema di offerta in grado di essere uno strumento a sostegno dell’appren- dimento continuo. Un’altra è stata quella di sostenere la motivazione all’apprendimento autonomo, attraverso il quale la persona può riportare nell’alveo dei dispositivi formali tutto il proprio sapere acquisito informalmente nel tempo che, altrimenti, rischia di essere estromesso da percorsi estremamente centrati solo sull’acquisizione di competenze (Man- caniello, 2012). Più di recente, la teorizzazione nata dagli approcci post-moderno e critico ha posto l’attenzione soprattutto ai processi di “costruzione sociale della conoscenza” la quale assu- me una determinata rappresentazione per il soggetto in base a come la interiorizza, piut- tosto che essere una acquisizione di un processo che viene definito dall’esterno, da una realtà che sta fuori. Da questa ottica l’enfasi viene posta sulla dimensione del potere e dell’acquisizione di potere, uno dei processi chiave dell’autoformazione e dell’auto-orien- DOI: 10.13128/ccselap-20000 \| ISSN 2531-9884 (online) 96 Maria Rita Mancaniello tamento. Per meglio comprendere questo approccio post-moderno e critico all’apprendi- mento dobbiamo leggere il processo di costruzione della conoscenza e di se stessi a partire dalla ricezione e dalla creazione dei messaggi o di “punti di vista” sul mondo concrea- ti all’interno di contesti comunicativi. Secondo l’approccio critico, si apprende attraverso la riflessione critica e mediante lo sviluppo della propria coscienza rispetto ai processi di costruzione del sapere a livello sociale, mentre, secondo l’approccio post-moderno, l’ap- prendimento è il risultato del processo di decostruzione e di eclettismo. L’educazione degli adulti, l’apprendimento auto-diretto, l’apprendimento trasformativo, la prospettiva post- moderna e il paradigma critico, l’attenzione all’uso dei metodi narrativi e alla creazione di percorsi destrutturati, il ritorno al soggetto, l’enfasi posta sull’esperienza individuale e alla dimensione sociale, rappresentano i riferimenti fondamentali le interazioni e le dinamiche esistenti tra contesti di formazione, autoformazione, orientamento e auto-orientamento (Grimaldi, Quaglino, 2004, p. 23). La necessità di preparare ad apprendere, di valorizzare le esperienze individuali, così come il bisogno di stimolare capacità di ragionamento e di sviluppare autonome capaci- tà nella soluzione di problemi e nella ricerca di informazioni, impongono al mondo della formazione di dover destrutturare progressivamente quelle che sono state le soluzioni pre- cedentemente adottate, a favore di una crescente contestualizzazione, sostenuta da approc- ci di formazione flessibili, ricchi di capacità operative e con una forte caratterizzazione euristica. Comparative Cultural Studies: European and Latin America Perspectives 2: 89-102, 2016 Il lavoro atipico e la dimensione soggettiva 97 - a livello di analisi dei bisogni, poiché, con l’affermarsi della nuova logica, dove l’at- tenzione della formazione si sposta sui processi di costruzione di significato e di inter- pretazione del mondo, oltre che sul ruolo e la valenza delle emozioni nella conoscenza, l’analisi dei bisogni si focalizza sui contenuti informali e le azioni spontanee, di modo che la sperimentazione del singolo diviene ricchezza e patrimonio di riflessione collettiva e di soluzione di problemi per molti (Argyris, Schön, 1996; Schön, 1994). Questo richiede che l’analisi dei bisogni non sia separata e svincolata dal processo di formazione, ma, anzi, che sia centrale come parte anche di percorsi di analisi organizzati- va o di ricerca-azione, in tutte le diverse forme con cui questa di può declinare, tenendo fermo il principio che è necessaria una conoscenza del contesto che si costruisce attraver- so l’azione su problemi concreti che lo caratterizzano; - a livello di progettazione, in quanto le riflessioni sulla difficoltà di previsione data dal ritmo delle trasformazioni e dall’emergere della consapevolezza che i soggetti sono più dominati dai livelli emozionali che quelli razionali, porta a mettere al centro non tanto fasi e obiettivi, quanto sviluppo per ipotesi di lavoro. Il tempo è oggi una variabile complessiz- zata che ha assunto una concezione dinamica che tende sempre più a seguire le articola- zioni del processo riflessivo e di quello narrativo, del tutto privi della linearità seguita dal modello razionale. Il progetto formativo assume, quindi, una sequenza aperta di ipotesi, di idee e concetti che fanno da “briccole”7 (Trisciuzzi, 1995). - a livello di progettazione, in quanto le riflessioni sulla difficoltà di previsione data dal ritmo delle trasformazioni e dall’emergere della consapevolezza che i soggetti sono più dominati dai livelli emozionali che quelli razionali, porta a mettere al centro non tanto fasi e obiettivi, quanto sviluppo per ipotesi di lavoro. Il tempo è oggi una variabile complessiz- zata che ha assunto una concezione dinamica che tende sempre più a seguire le articola- zioni del processo riflessivo e di quello narrativo, del tutto privi della linearità seguita dal modello razionale. Il progetto formativo assume, quindi, una sequenza aperta di ipotesi, di idee e concetti che fanno da “briccole”7 (Trisciuzzi, 1995). 77 “Le briccole sono dei grossi pali che vengono piantati nelle lagune e servono per segnalare la navigazione sicu- ra ai naviganti. Chi esce dalla via indicata dalle briccole finisce sicuramente per incagliarsi in qualche basso fon- dale. E nella vita di ciascuno di noi esistono anche le briccole dell’educazione, che ci offrono sicurezza in quanto punti di riferimento per ogni decisione da prendere”. 6. Uno sguardo al possibile ruolo della formazione Fondamentale diviene la capacità di saper riconoscere e capire i contesti in cui ci si muove in una prospettiva che recupera come cardine e guida la logica della ricerca- azione (Lipari, 2002).i Pensare in una logica di ricerca-azione – ancor più in una logica ancor più specifica di ricerca azione partecipativa – è garanzia di una effettiva contestualizzazione dell’inter- vento formativo (Orefice, 2006). i Un criterio dell’agire che trasforma profondamente le dimensioni interne del processo formativo, un processo che diviene sempre più aperto, partecipato e adattabile, basato sul fluire di eventi il cui senso e la cui coerenza sono ricostruibili solo dopo la fine dell’espe- rienza. Un cambiamento che modifica la visione del processo formativo sotto tutti i punti chiave del percorso: - a livello di logica spostandosi da quella unilineare, sequenziale, razionale e degli obiettivi predefiniti, verso la legge della formazione come “apertura”, come fluire di even- ti. Una prospettiva basata proprio su un processo costruito sulla dinamica relazionale e sulla dimensione soggettiva, nella quale la titolarità del processo di formazione è equa- mente condiviso tra chi gestisce la formazione e chi ne fruisce, in una circolarità che men- tre si sviluppa il percorso si alimenta dell’esperienza lavorativa vissuta per rielaborarla e riportarla nell’alveo formativo. Ciò comporta una costruzione condivisa, un punto di vista sistemico, una razionalità sinergica con le emozioni vissute e le sensazioni provate, una visione fatta di idee intuitive attraverso le quali gettare il proprio sguardo su un futuro dall’orizzonte ampio e composto da molteplici sfumature. Tale logica trova delle imme- diate ricadute sul metodo, poiché legittima l’utilizzo di tecniche e strumenti finalizzati al recupero delle esperienze e alla loro interpretazione, sia nei risvolti soggettivi che in quelli interpersonali; Comparative Cultural Studies: European and Latin America Perspectives 2: 89-102, 2016 Il lavoro atipico e la dimensione soggettiva - al percorso di formazione, che orientano e segnano la navigazione apprenditiva, ma che assumono una forma definita solo attraverso i processi di elaborazione attivati durante il procedere del lavoro formativo; - a livello di valutazione, poiché rompendo la logica della linearità e della predomi- nanza degli obiettivi si deve passare da una concezione ingegneristica della valutazione ad una che si focalizzi sul percorso e sul processo di apprendimento, basandosi sulla rac- colta dei dati, più qualitativi che quantitativi e considerando centrale la dinamica che si crea tra le componenti presenti nell’azione, tra le relazioni di contesto e gli intrecci che in questo si creano. Nel momento in cui i bisogni individuali divengono la base da cui partire in quanto riconosciuti come fondamentali per accompagnare un soggetto a vivere, gestire e padro- neggiare la flessibilità e l’incertezza dettata dalla attuale società, e il contesto assume il ruolo di variabile centrale, la formazione non può che attrezzarsi pensando a processi di azione molto destrutturati, a ipotesi più che progetti, a metodologie, metodi e strumenti in grado di cambiare al mutare della configurazione della relazione tra i soggetti protago- nisti del processo di formazione, dando una forte centralità all’autoformazione (Manca- niello, 2012a). 77 “Le briccole sono dei grossi pali che vengono piantati nelle lagune e servono per segnalare la navigazione sicu- ra ai naviganti. Chi esce dalla via indicata dalle briccole finisce sicuramente per incagliarsi in qualche basso fon- dale. E nella vita di ciascuno di noi esistono anche le briccole dell’educazione, che ci offrono sicurezza in quanto punti di riferimento per ogni decisione da prendere”. DOI: 10.13128/ccselap-20000 \| ISSN 2531-9884 (online) Maria Rita Mancaniello 98 7. Per concludere Quello a cui oggi la formazione deve guardare e dare risposte in grado di produr- re benessere e sicurezza, è un soggetto che ha subito una perdita di senso, disorientato rispetto alle categorie del tempo e dello spazio (senza confini e senza limiti, se non quello certo della morte, ma che spesso è nascosta perfino a se stessi), frammentato, ma anche inquieto e ansioso di conoscere quali sono gli aspetti, le dinamiche che si creano nella propria storia formativa e desideroso di ritrovare le “coordinate” di orizzonte e orienta- re il cammino alla ricerca del proprio Senso. Un approccio pedagogico e formativo che richiede al soggetto un costante intreccio tra essere formato e essere formatore di se stes- so, nel quale, attraverso la relazione con il contesto e mediante la rappresentazione della propria esperienza esistenziale, il soggetto si de-costruisce e si ri-compone, dando vita ad una dimensione di crescita collettiva, in cui cogliere anche il senso universale del proprio operare e del proprio essere. Un processo formativo del quale il soggetto è protagonista anche nello “svelare” e nel chiarire il suo divenire, imparando a ri-organizzare e ri-defi- nire la propria esistenza sulla base di una conoscenza profonda di sé e della realtà che lo circonda. Comparative Cultural Studies: European and Latin America Perspectives 2: 89-102, 2016 8. Bibliografia AA. VV. (2010), Vita, identità, genere in equilibrio precario: Ricerche psicologiche sul merca- to del lavoro in Italia, Unicopli, Milano. AA.VV. 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https://openalex.org/W2321864780	https://academicjournals.org/journal/JPHE/article-full-text-pdf/EB34F0445458.pdf	English	null	Prevalence, associated risk factors and consequences of premarital sex among female students in Aletawondo High School, Sidama Zone, Ethiopia	Journal of Public Health and Epidemiology	2,014	cc-by	3,933	Vol. 6(7), pp. 216-222, July 2014 DOI: 10.5897/JPHE2014.0640 ISSN 2006-9723 Article Number: EB34F0445458 Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/JPHE Journal of Public Health and Epidemiology Vol. 6(7), pp. 216-222, July 2014 DOI: 10.5897/JPHE2014.0640 ISSN 2006-9723 Article Number: EB34F0445458 Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/JPHE Journal of Public Health and Epidemiology Vol. 6(7), pp. 216-222, July 2014 DOI: 10.5897/JPHE2014.0640 ISSN 2006-9723 Article Number: EB34F0445458 Copyright © 2014 Author(s) retain the copyright of this article http://www.academicjournals.org/JPHE Journal of Public Health and Epidemiology Journal of Public Health and Epidemiology Received 2 April, 2014; Accepted 9 May, 2014 Received 2 April, 2014; Accepted 9 May, 2014 Youth age is the period when majority of youths become sexually active. However, majority of the youths lack sufficient knowledge about (HIV/AIDS) and other reproductive health issues. As a result, youths have multitude of reproductive problems, including HIV, other sexually transmitted infections (STIs), unwanted pregnancy, induced abortion and dropping out of school due to unwanted pregnancy. The objective of this study was to assess the prevalence, associated risk factors and consequences of premarital sex among high school female students in Aletawondo town, Southern Ethiopia 2013. A cross-sectional school-based study was conducted using a facilitator guided self administered questionnaire. Study participants were female students attending regular school classes in high schools. The percentage of youths involved in pre-marital sex and those affected by its consequence were computed. Factors associated with pre-marital sex were assessed using bivariate and multivariable logistic regression. Among the total participants (n = 413), 394 were never married. Of the 394 never married study participants, 72 (18.3%) have in premarital sex. Of these, 11 (15.3%) had unwanted pregnancy and 82% had terminated their pregnancy (induced abortion). Predictors of premarital sex were: age [AOR (95% CI) = 1.43 (1.15, 1.77)], rural family residence [AOR (95% CI) = 2.12 (1.09, 4.12)], having a boy friend [AOR (95% CI) = 4.15 (2.34, 7.36)], going to night club [AOR (95% CI) = 2.92 (1.12, 7.65)]. Significant number of girls started having premarital sex due to various factors at different levels. Therefore, comprehensive sexual education at their earlier age could help reduce the consequences of pre-marital sexual intercourse. Key words: Premarital sex, consequences, high school, female students. Key words: Premarital sex, consequences, high school, female students. Full Length Research Paper Prevalence, associated risk factors and consequences of premarital sex among female students in Aletawondo High School, Sidama Zone, Ethiopia Emebet Tekletsadik1, Debebe Shaweno1 and Deresse Daka2* 1School of Public and Environmental Health, Hawassa University, Ethiopia 2Department of Medical Laboratory Sciences, Hawassa University, Ethiopia Full Length Research Paper 1School of Public and Environmental Health, Hawassa University, Ethiopia 2Department of Medical Laboratory Sciences, Hawassa University, Ethiopia METHODOLOGY A cross-sectional school based study was conducted to determine prevalence, associated risk factors and consequences of premarital sex among female students in Aletawondo High School in 2012. Aletawondo town is located in Southern Ethiopia, at a distance of 337 km South of Addis Ababa and 64 km South of Hawassa (the capital of the region). The town had only one high school. As a result, students, whose parents reside in rural areas, live away from their parents in rented houses alone or with other peers in the town. In 2013, Aletawondo High School had 2004 female students in grades ranging from 9-12. Youths tend to be sexually active and have more sexual partners, thus putting them at greater risk of having HIV infection and unwanted pregnancy. This in turn is associated with increased maternal morbidity and mortality due to attempts for induced abortion. Moreover, due to the fact that majority of higher education students and high school students are economically insecure, they are susceptible to economic incentives that can lead to higher risk sexual practices (Lamesgin, 2013). g g g A sample size of 420 was estimated after fixing proportion of premarital sexual practice to be 50%, 95% CI; margin of error, 5% and none response rate, 10%. A sampling frame which contained the lists of all 2004 female students from grades 9 to 12 was developed based on the lists obtained from students’ record office. Eventually, 420 students were selected using a computer gene- rated random numbers. Data were collected using structured self- administered questionnaires prepared in the official language (Amharic). Suitability of the questionnaires was checked by con- ducting pretest on 5% of the study participants. Students selected randomly from the list were asked to fill the questionnaires in separate rooms during break period. Questionnaires were handed out to the students who consented to participate in the study. The study information sheet was attached with the questionnaires. Data collection was facilitated by trained supervisors. Data were collected after securing informed consent. Previous studies in Ethiopia among students indicated that from 14 to 23.4% of female adolescents had premarital sexual intercourse (Oljira et al., 2012; Ahmed et al., 2012; Seme and Wirtu, 2008). The first sexual practices in Ethiopia are often unplanned (Mazengia and Worku 2009), increasing young peoples' risk of having HIV and other STIs. METHODOLOGY Prevalence of HIV among female youths ranges from 0.2 to 9% in the country by region (Centeral Statistical Agency, 2012). g The collected data were entered, cleaned and analyzed using Statistical Package for Social Sciences (SPSS) version 20. Socio- economic characteristics of the study participants, magnitude and consequences of premarital sex were analyzed using frequency distribution. Predictors of premarital sex were analyzed using logistic regression model after checking major assumptions for goodness of fit test (Hosmer and Lemeshow test), outliers and interaction between independent variables. All variables which were significant at a p-value of 0.3 in the binary logistic regression model (crude) were fitted into the final multiple logistic regression model. Predictors significant at a p-value of 0.05 in the multiple logistic regression models were retained as independent predictors of premarital sex among female students. Pre-marital sexual intercourse is more common among the following group of adolescents: those whose parents live in urban areas, those who receive higher pocket mo- ney per month, those who perceive low self-educational rank and those who live in rented houses (Centeral Statistical Agency, 2012; Oljira et al., 2012). Early sexual intercourse is also associated with chat chewing, wa- tching pornographic movies below 18 years, living away from parents and having boyfriends (Seme and Wirtu, 2008; Mazengia and Worku, 2009). It was also reported that in Ethiopia risk of women having HIV infection is particularly exacerbated by early sexual debut, lack of comprehensive HIV/AIDS knowledge and lack of access to health care services (Lamesgin, 2013). The young age of students, long periods of physical stay in the educa- tional environment and emotional contacts within school communities are reported to increase the collective vulnerability of these communities to HIV/AIDS (Federal ministry of Health, 2004). INTRODUCTION majority of them lack sufficient knowledge about human immunodeficiency virus (HIV) transmission (Population Adolescence and youth (ages 15 to 24) is the time when majority of youths become sexually active; however, Corresponding author. E-mail: drsdk200@gmail.com. Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 217 Tekletsadik et al. students in Aletawondo town, Southern Ethiopia. Reference Bureau, 2013). As a result, youths have multitude of reproductive problems, including HIV, other STIs, unwanted pregnancy, induced abortion and dropping out of school (Tamire and Enqueselassie, 2007; WHO, 2012a). This unwanted early pregnancy is asso- ciated with increased maternal and neonatal morbidity and mortality (WHO, 2012b). Lack of access to sexual education and contraceptives is reported to be the key issue contributing to the reproductive health problems faced by youths (WHO, 2012a). About 5% of never married women in Ethiopia have had premarital sexual intercourse and 5% have had sexual intercourse within the prior one year (Centeral Statistical Agency, 2012). Prevalence and consequence of premarital sexual intercourse The proportion of pre-marital sex among female high school students is consistent with similar studies conducted among high school or university students in Nekemite and elsewhere in Ethiopia (Oljira et al., 2012; Seme and Wirtu, 2008; Molla et al., 2008). However, the notion of comparability between the Nekemte study (the zonal capital) where the proportion of female students with pre-marital sex was 21.1% and the magnitude in the current study (18.3%) should be cautiously interpreted as it appears that the latter study is more of rural in nature (Seme and Wirtu, 2008). As such, this study may corro- borate with a previous study conducted in Ethiopia where there was no difference in the rate of having sex between urban and rural (Mazengia and Worku 2009). However, the pre-marital sex magnitude observed in our study is relatively much higher than the finding from a Malasian study, conducted in 2006 (Lee et al. 2006). Among 394 female students who were never married, 72 (18.3%) had premarital sexual intercourse. And 51.4% of the students who had premarital sexual intercourse did so without condom. The prevalence of premarital sexual intercourse was higher among grade 9 students, with magnitude of 83 (32.4%). When the relationship of the study population to their sexual practices was examined, about 37 (51.4%) of the study participants who had premarital sex had no boyfriends. The main reasons for having sexual intercourse were lack of confidence to refuse 26 (6.6%), peer-pressure 14 (3.6%), desire to practice sexual intercourse and falling in love 10 (2.5%), money 7 (1.8%) and rape 4 (1%). Out of those who had premarital sexual intercourse, 15.3% of the women faced unwanted pregnancy and those with unwanted pregnancy, 82% had an induced abortion. Similarly, 19 (20.9%) reported symptoms of sexually transmitted disease such as vaginal discharge 13 (14.3%), genital ulcer 5 (5.5%) and inguinal swelling 1 (1.1%). Of 19 students with symptoms of sexually trans- mitted disease, 68% had vaginal discharge (Figure 1). y Unprotected sexual intercourse was reported by more than 50% of female participants in this study. As a result, the magnitude of the negative consequences of pre- marital sex is much higher in this study. WHO attributes the high rate of pregnancy and its complications to lack of effective sexuality education (WHO 2012b). Prevalence and consequence of premarital sexual intercourse According to WHO, 24% of young women aged 15 to 24 years in developing regions have comprehensive and correct knowledge of HIV/AIDS (WHO 2012b). Other similar investigations have strengthened the link between repro- ductive health knowledge and premarital sex (Wong, 2012; Thin et al., 2013). As a result, dissemination of reliable SRH information among youths is emphasized by different authors. g g g Magnitude of premarital sex was higher among students whose residence is in rural areas compared to their counterparts from urban areas (Figure 2). J. Public Health Epidemiol. Residence of parents was associated with premarital sexual intercourse, with lower odds of premarital sexual intercourse among students whose parents are in the urban area where the school is found [AOR (95% CI) = 0.41 (0.22, 0.79)]. Similarly, the odds of premarital sex was statistically significantly lower by 78% among female students who did visit night clubs [AOR (95% CI) = 0.22 (0.10, 0.45)] (Table 2). primary school and above were about 68 and 55.7%, respectively. Majority of the study participants’ fathers were farmers (44%) and mothers were house wives (60%). More than two-thirds of the study participants do not get pocket money. Regarding living arrangement of the study participants, 59, 10 and 5% live with both parents friends (peers) and alone, respectively. Regar- ding having a boy friend, 68.8% of unmarried female students had no boy friend at the time of this survey (Table 1). Characteristics of study respondents A total of 413 female students were included in the study with a response rate of 98.3%. The mean age of the respondents was 16.4 ± 1.38 years. A large majority of study participants (95%) were never married. Regarding residence, higher proportion of the study participants (81%) were from urban areas. In Ethiopia, most HIV related interventions targeting the general public do not respond to the needs of students, because they are considered to have adequate knowledge about HIV/AIDS and other reproductive health issues (Lamesgin, 2013). So the aim of this study is to assess the prevalence, associated risk factors and con- sequences of premarital sex among high school female By religious affiliation, majority of the respondents (66%) were protestants, and about one fifth (21%) were orthodox. Muslims accounted for 3% of study participants. By ethnic composition, about three-fourth of respondents (79%) belong to the Sidama ethnic group, followed by Amhara (9%). Respondents’ fathers and mothers who completed 218 J. Public Health Epidemiol. Factors associated with premarital sex Four predictors namely age of the respondents, having a boy friend, going to night clubs and family residence emerged as independent predictors of premarital sexual intercourse from the logistic regression analysis. Logistic regression analysis showed that the odds of premarital sex increased with an increase in age of the study participants. For every year, increase in the age of the study participants, the odds of premarital sexual inter- course increase by 33%; [AOR (95% CI) = 1.33 (1.05, 1.7)]. The odds of having a boyfriend was more than four times higher among unmarried female students who had premarital sexual intercourse than unmarried female students without premarital sexual intercourse experience [AOR (95% CI) = 4.15 (2.34, 7.36)]. The finding that significant proportion of study parti- cipants had unwanted pregnancy is supported by a study conducted among undergraduate students of Addis Ababa University, Ethiopia, where 73.5% had unwanted pregnancy and almost all were terminated through induced abortion (Tamire and Enqueselassie 2007). The higher prevalence rate of induced abortion reported in the Addis Ababa study compared to the current study could be related to differences in the study areas, where the former is mostly urban. However, the reason why proportion of unwanted pregnancy remains higher among undergraduate university students in the Addis Ababa where contraceptive methods are widely available compared to the current study conducted in a sub-urban 219 Tekletsadik et al. Table 1. Socio-demographic characteristics of the study participants. Factors associated with premarital sex Variable Category Premarital sex COR 95% CI AOR 95% CI P-value Yes No Students’ residence Urban 54 267 0.62 (0.34, 1.13) 0.78 (0.37, 1.65) 0.52 Rural 18 55 1 1 Pocket money Yes 29 100 1.50 (0.88, 2.54) 1.43 (0.80, 2.6) 0.23 No 43 222 1 1 Having a boy friend Yes 54 88 3.9 (2.39, 6.30) 4.15 (2.34, 7.36) 0.00 No 37 234 1 1 Go to night club Yes 53 296 1 1 No 19 26 0.25 (0.13, 0.47) 0.22 (0.10, 0.45)* 0.00 Live with parents Yes 68 304 1.01 (0.33, 3.07) 1.43 (0.43, 4.74) 0.52 No 4 18 1 1 Age 15-25 years 72 322 1.2 (0.96, 1.43) 1.33 (1.05,1.7)* 0.00 Family residence Urban 30 191 0.49 (0.29, 0.82) 0.41 (0.22, 0.79)* 0.01 Rural 42 131 1 1 Mothers’ education Illiterate 36 138 0.75 (0.45, 1.25) 0.96 (0.49, 1.85) 0.90 Primary/above 36 184 1 1 Fathers’ education Illiterate 31 192 1.89 (1.12, 3.20) 1.71 (0.89, 3.30) 0.11 Primary/Above 41 230 1 1 Statistically significant. Figure 1. Symptoms of sexually transmitted infection among study participants (Aletawondo, 2013). Table 2. Predictors of premarital sexual intercourse in Aleta Wondo Town. Variable Category Premarital sex COR 95% CI AOR 95% CI P-value Yes No Students’ residence Urban 54 267 0.62 (0.34, 1.13) 0.78 (0.37, 1.65) 0.52 Rural 18 55 1 1 Pocket money Yes 29 100 1.50 (0.88, 2.54) 1.43 (0.80, 2.6) 0.23 No 43 222 1 1 Having a boy friend Yes 54 88 3.9 (2.39, 6.30) 4.15 (2.34, 7.36) 0.00 No 37 234 1 1 Go to night club Yes 53 296 1 1 No 19 26 0.25 (0.13, 0.47) 0.22 (0.10, 0.45)* 0.00 Live with parents Yes 68 304 1.01 (0.33, 3.07) 1.43 (0.43, 4.74) 0.52 No 4 18 1 1 Age 15-25 years 72 322 1.2 (0.96, 1.43) 1.33 (1.05,1.7)* 0.00 Family residence Urban 30 191 0.49 (0.29, 0.82) 0.41 (0.22, 0.79)* 0.01 Rural 42 131 1 1 Mothers’ education Illiterate 36 138 0.75 (0.45, 1.25) 0.96 (0.49, 1.85) 0.90 Primary/above 36 184 1 1 Fathers’ education Illiterate 31 192 1.89 (1.12, 3.20) 1.71 (0.89, 3.30) 0.11 Primary/Above 41 230 1 1 *Statistically significant. Figure 1. Symptoms of sexually transmitted infection among study participants (Aletawondo, 2013). Table 2. Predictors of premarital sexual intercourse in Aleta Wondo Town. Figure 1. Symptoms of sexually transmitted infection among study participants (Aletawondo, 2013). Figure 1. Factors associated with premarital sex g p y p p Variable Category Freq Age 15-19 3 20-24 Grade Level 9th 2 10th 1 Preparatory 3 Marital status Single 3 Married 1 Ethnicity Sidama 3 Amhara 3 Oromo 2 Guragae 1 Others 1 Religion Orthodox 8 Protestant 2 Muslim 1 Others 4 Education level of Fathers No formal education 5 Read or/and write only 7 Primary 9 Secondary 9 Tertiary 9 Maternal Education level No formal education 6 Read or/and write only 1 Primary 9 Secondary 7 Tertiary 6 Students’ residence Urban 3 Rural 7 Live with Father 1 Both 2 Relative 4 Friends 4 Alone 2 Other 1 Pocket money Yes 1 No 2 Fathers’ occupation Government employ 1 Farmer 1 Merchant 6 Others 3 Variable Category Frequency Percent Age 15-19 391 99.2 20-24 3 0.8 Grade Level 9th 250 60.5 10th 128 31 Preparatory 35 8.5 Marital status Single 394 95.4 Married 19 4.6 Ethnicity Sidama 325 78.7 Amhara 39 9.4 Oromo 24 5.8 Guragae 11 2.7 Others 14 3.4 Religion Orthodox 86 20.8 Protestant 273 66.1 Muslim 13 3.1 Others 41 9.9 Education level of Fathers No formal education 58 14 Read or/and write only 74 17.9 Primary 96 23.2 Secondary 93 22.5 Tertiary 92 22.3 Maternal Education level No formal education 69 16.7 Read or/and write only 114 27.6 Primary 91 22 Secondary 73 17.7 Tertiary 66 16 Students’ residence Urban 334 80.9 Rural 79 19.1 Live with Father 15 3.6 Both 245 59.3 Relative 40 9.7 Friends 43 10.4 Alone 22 5.3 Other 10 2.4 Pocket money Yes 138 33.4 No 275 66.6 Fathers’ occupation Government employ 135 32.7 Farmer 180 43.6 Merchant 68 16.5 Others 30 7.3 J. Public Health Epidemiol. J. Public Health Epidemiol. 220 Table 2. Predictors of premarital sexual intercourse in Aleta Wondo Town. Factors associated with premarital sex Symptoms of sexually transmitted infection among study participants (Aletawondo, 2013). 221 Tekletsadik et al. Figure 2. Magnitude of premarital sex by place of residence of study participants. Source: Aletawondo (2013). Figure 2. Magnitude of premarital sex by place of residence of study participants. Source: Aletawondo (2013). from parents. As observed in similar studies conducted in Nekemte town, East of Wollega and Tehran (Seme and Wirtu 2008; Khalaj et al. 2011), having a boy friend is positively associated with premarital sex in this study. The likely reason is that as persons of opposite sex stay together during the time of fire age they will be sexually attracted and have sexual intercourse. area among high school students with lower educational level is still a paradox. On the contrary, the influence of night clubs and peer pressure on pre-marital sexual encounter could explain the increased magnitude among the urban, like that observed in Addis Ababa. The protective association between urban parental residence (where the school is found) and pre-marital sexual intercourse among female students could be due to the opportunity for parental monitoring and guidance. Those students whose parents live in rural areas often attend their education in rented houses in urban areas. This living arrangement provides the opportunity of being free from parental supervision so that the students will have freedom of exercising sexual issues. However, this finding is in contrast with the findings of other studies in Ethiopia (Oljira et al 2012). Consistent with many studies (Mazengia and Worku 2009; Gyan and Basel 2013), parental education (both mother and father) showed no statistically significant association with premarital sex in the current study. Based on the potential effect of the validity of the study and its objective, data were collected using self- administered questionnaire. This attempt, however, might not totally avoid social desirability bias. The scope of our study (limited to school and female students) limits its generalizability to out-school youths and male youths. Consistent with a study conducted elsewhere, the odds of pre-marital sex increased with increasing age (Khalaj et al. 2011; Gyan and Basel 2013). This finding is supported by the explanation that as age increases the exposure duration for predictors will increase. J. Public Health Epidemiol. 222 Lee LK, Chen PC, Lee KK, Kaur J (2006). Premarital sexual intercourse among adolescents in Malaysia: a cross-sectional Malaysian school survey. Singapore Med. J. 47(6):476-81. clubs were independent predictors of premarital sex. Thus, effective sexuality education that considers the identified predictors could help alleviate the problem. There is also a need to conduct more rigorous study that addresses out-school youths as well. Mazengia F, Worku A (2009). Age at sexual initiation and factors associated with it among youths in North East Ethiopia. Ethiop J. Health Dev. 23(2):154-62. ( ) Molla M, Berhane Y, Lindtjørn B (2008). Traditional values of virginity and sexual behavior in rural Ethiopian youth: results from a cross- sectional study. BMC Public Health 8(9). The author(s) have not declared any conflict of interests. Population Reference Bureau (2013). The World's Youth: Data Sheet. Seme A, Wirtu D (2008). Premarital Sexual Practice among School Adolescents in Nekemte Town, East Wollega. Ethiop J. Health Dev. 22(2). Conflict of Interests Oljira L, Berhane Y, Worku A (2012). Pre-marital sexual debut and its associated factors among in-school adolescents in eastern Ethiopia. BMC Public Health 12:375. CONCLUSION AND RECOMMENDATION The study demonstrated that a significant number of female students are engaged in premarital sexual intercourse and suffer from its complications (unwanted pregnancy, abortion and STIs. Age of the student, having a boy friend, family rural residence and exposure to night In contrast with similar studies (Lee et al., 2006; Khalaj et al. 2011; Gyan and Basel 2013), the association between living with or away from parents and premarital sex was not statistically significant. This could be due to lower sample size for study participants who live away REFERENCES ( ) Tamire W, Enqueselassie F (2007). Knowledge, attitude, and practice on emergency contraceptives among female university students in Addis Ababa, Ethiopia. Ethiop. J. Health Dev. 21(2):111-6. Ahmed FA, Moussa KM, Petterson KO, Asamoah BO (2012). Assessing knowledge, attitude, and practice of emergency contraception: a cross- sectional study among Ethiopian undergraduate female students. BMC Public Health 12(110) Thin Zaw P, Liabsuetrakul T, McNeil E, Htay T (2013). Gender differences in exposure to SRH information and risky sexual debut among poor Myanmar youths. BMC Public Health 13(1122):http://www.biomedcentral.com/1471-2458/13/1122. Centeral Statistical Agency (2012). Ethiopia Demographic and Health Survey 2011. ICF International Calverton, Maryland, USA. Federal Ministry of Health (2004). Ethiopian strategic plan for intensifying Multi-Sectoral HIV/AIDS Response (2004 - 2008). FMOH, Addis Ababa, Ethiopia. WHO (2012a). From Evidence to Policy: Expanding access to Contraceptive services for adolescents. World Health Organization, Geneva. Gyan Bahadur BC, Basel PL (2013). Premarital Sex Behaviors among College Youths of Kathmandu, Nepal. Kathmandu Univ. Med. J. 41(1):27-31. WHO (2012b). Early marriages, adolescent and young pregnancies. World Health Organization, Geneva. Wong LP (2012). Qualitative Inquiry into Premarital Sexual Behaviours and Contraceptive Use among Multiethnic Young Women: Implications for Education and Future Research. PLoS ONE 7(12):e51745. doi:10.1371/journal.pone.0051745. Khalaj F, Farahani A, Cleland J, Hooshang A, Mehryar (2011). Associations between family factors and Premarital heterosexual relationships among female College Students in Tehran. Int. Perspect. Sexual Reprod. Health 37(1):30–9. Lamesgin A (2013). HIV/AIDS and Sexual reproductive Health among university students in Ethiopia: A policy intervention framework. Health Policy Project, Washington DC.
https://openalex.org/W2969448433	http://eprints.sunway.edu.my/1132/1/Tiekink%20Crystal%20structure%20of%20the%20co%20crystal.pdf	English	null	Crystal structure of the co-crystal 4-[(4-carboxyphenyl)disulfanyl]benzoic acid–(1E,4E)-1-N,4-N-bis(pyridin-4-ylmethylidene)cyclohexane-1,4-diamine (1/1), C14H10O4S2⋅C18H20N4	Zeitschrift für Kristallographie. New crystal structures/Zeitschrift für Kristallographie. New crystal structures	2,019	cc-by	2,434	Experimental details The C-bound H atoms were geometrically placed (C—H = 0.95–0.99 Å) and refined as riding with Uiso(H) = 1.2Ueq(C). The O-bound H atoms were located in difference Fourier maps but were refined with a distance restraint of O— H = 0.84 ± 0.01 Å, and with Uiso(H) set to 1.5Ueq(O). CCDC no.: 1945351 The molecular structures are shown in the figure. Table 1 contains crystallographic data and Table 2 contains the list of the atoms including atomic coordinates and displacement parameters. Comment The isolation and crystallographic characterization of the title co-crystal is a rare example formed by one of the isomeric Schiff bases, N,N′-bis((pyridine- n-yl)methylene)cyclohexane-1,4-diamine, for n = 2, 3 and 4 [5]. The co-crystal co-former is disulfide 4-[(4- carboxyphenyl)disulfanyl]benzoic acid (4-DTBA), whose crystal structure has been reported in the literature [6]. The observation of n-DTBA, or its derived di-anion, arises from the oxidation of the n-mercaptobenzene, for n = 2, 3 and 4, starting material, as has been commented upon previ- ously [7–9]; the species with a single sulfur bridge has also been found [10]. Up to the present time, a characteristic of the aforementioned Schiff bases is their breakdown dur- ing co-crystallization [11–14]. Often, this results in isolation Abstract C32H30N4O4S2, monoclinic, I2/a (no. 15), a = 21.2034(7) Å, b = 5.0614(2) Å, c = 27.5987(12) Å, β = 105.991(4)°, V = 2847.3(2) Å3, Z = 4, Rgt(F) = 0.0431, wRref(F2) = 0.1246, T = 100(2) K. C32H30N4O4S2, monoclinic, I2/a (no. 15), a = 21.2034(7) Å, b = 5.0614(2) Å, c = 27.5987(12) Å, β = 105.991(4)°, V = 2847.3(2) Å3, Z = 4, Rgt(F) = 0.0431, wRref(F2) = 0.1246, T = 100(2) K. C32H30N4O4S2, monoclinic, I2/a (no. 15), a = 21.2034(7) Å, Wavelength: ratio. Colourless crystals were obtained three days after crys- tallization by the layering of benzene (2 mL) onto an equiva- lent volume of DMSO containing the dissolved mixture. M.pt: 438–439 K. IR (ATR, cm−1): 3060(w) ν(N—H), 2940–2826(m) ν(C—H), 1675(m) ν(C=O), 1556(m) ν(C=N), 1321(m) ν(C—N), 815(s) δ(C=C). ratio. Colourless crystals were obtained three days after crys- tallization by the layering of benzene (2 mL) onto an equiva- lent volume of DMSO containing the dissolved mixture. M.pt: 438–439 K. IR (ATR, cm−1): 3060(w) ν(N—H), 2940–2826(m) ν(C—H), 1675(m) ν(C=O), 1556(m) ν(C=N), 1321(m) ν(C—N), 815(s) δ(C=C). https://doi.org/10.1515/ncrs-2019-0488 Received July 11, 2019; accepted August 5, 2019; available online August 17, 2019 Received July 11, 2019; accepted August 5, 2019; available online August 17, 2019 Open Access. © 2019 Sang Loon Tan et al., published by De Gruyter. License. Unauthenticated Download Date \| 10/10/19 2:17 AM Z. Kristallogr. NCS 2019; 234(5): 1121–1123 Sang Loon Tan and Edward R.T. Tiekink* Crystal structure of the co-crystal 4-[(4-carboxyphenyl)disulfanyl]benzoic acid–(1E,4E)-1-N,4-N-bis(pyridin-4-ylmethylidene)cyclohexane-1,4- diamine (1/1), C14H10O4S2·C18H20N4 Table 1: Data collection and handling. Crystal: Colourless prism Size: 0.16 × 0.10 × 0.06 mm Wavelength: Cu Kα radiation (1.54184 Å) µ: 2.07 mm−1 Diffractometer, scan mode: XtaLAB Synergy, ω θmax, completeness: 76.9°, >99% N(hkl)measured, N(hkl)unique, Rint: 34319, 2996, 0.063 Criterion for Iobs, N(hkl)gt: Iobs > 2 σ(Iobs), 2795 N(param)refined: 193 Programs: CrysAlisPRO [1], SHELX [2, 3], WinGX/ORTEP [4] Table 1: Data collection and handling. Table 1: Data collection and handling. Crystal: Colourless prism Size: 0.16 × 0.10 × 0.06 mm Wavelength: Cu Kα radiation (1.54184 Å) µ: 2.07 mm−1 Diffractometer, scan mode: XtaLAB Synergy, ω θmax, completeness: 76.9°, >99% N(hkl)measured, N(hkl)unique, Rint: 34319, 2996, 0.063 Criterion for Iobs, N(hkl)gt: Iobs > 2 σ(Iobs), 2795 N(param)refined: 193 Programs: CrysAlisPRO [1], SHELX [2, 3], WinGX/ORTEP [4] Table 1: Data collection and handling. Crystal: Colourless prism Size: 0.16 × 0.10 × 0.06 mm Wavelength: Cu Kα radiation (1.54184 Å) µ: 2.07 mm−1 Diffractometer, scan mode: XtaLAB Synergy, ω θmax, completeness: 76.9°, >99% N(hkl)measured, N(hkl)unique, Rint: 34319, 2996, 0.063 Criterion for Iobs, N(hkl)gt: Iobs > 2 σ(Iobs), 2795 N(param)refined: 193 Programs: CrysAlisPRO [1], SHELX [2, 3], WinGX/ORTEP [4] This work is licensed under the Creative Commons Attribution 4.0 Public 1122 Table 2: Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2). the presence of the acid. The carboxyl group is co-planar with the phenyl ring to which it is attached, as seen in the dihedral angle between the two planes of 1.38(8)°. The char- acteristic orthogonal relationship between the phenyl rings is evidenced by the dihedral angle of 88.41(41)° between them, giving rise to a twisted, U-shaped molecule. The Schiff base is disposed about a center of inversion, as found in the free Schiff base [5]. However, a difference between the molecules is apparent in that in the free form [5], the C=C(H)C5H4N residue is almost orthogonal to the best plane through the cyclohexyl ring (which adopts a chair conformation in both structures) as seen in the dihedral angle of 86.01(5)°. This compares with the comparable angle in the title co-crystal of 38.93(3)° which arises from a twist about the C10—C13 bond, with the C9—C10—C13—N2 torsion angle being −24.0(3)°. isotropic displacement parameters (Å ). Atom x y z Uiso/Ueq S1 0.70397(2) 1.25411(8) 0.47699(2) 0.02786(16) O1 0.61712(6) 0.3255(3) 0.30000(5) 0.0291(3) H1O 0.6164(12) 0.211(4) 0.2777(7) 0.044 O2 0.72569(6) 0.2874(3) 0.31164(5) 0.0329(3) N1 0.60383(7) −0.4412(3) 0.73461(5) 0.0272(3) N2 0.52646(7) 0.1957(3) 0.59005(6) 0.0306(3) C1 0.70173(8) 0.9950(3) 0.43315(6) 0.0242(3) C2 0.63918(8) 0.9125(3) 0.40682(6) 0.0259(3) H2 0.601792 0.989443 0.413862 0.031* C3 0.63136(8) 0.7185(3) 0.37037(7) 0.0263(4) H3 0.588525 0.663798 0.352179 0.032* C4 0.68600(8) 0.6026(3) 0.36015(6) 0.0237(3) C5 0.74839(8) 0.6844(4) 0.38727(7) 0.0284(4) H5 0.785844 0.604956 0.380784 0.034* C6 0.75666(8) 0.8798(4) 0.42354(7) 0.0291(4) H6 0.799465 0.934929 0.441734 0.035* C7 0.67887(8) 0.3894(3) 0.32177(6) 0.0250(3) C8 0.54347(8) −0.3588(4) 0.70955(7) 0.0278(4) H8 0.506677 −0.441202 0.716538 0.033* C9 0.53257(8) −0.1595(4) 0.67407(7) 0.0277(4) H9 0.489132 −0.109413 0.656416 0.033* C10 0.58639(8) −0.0326(3) 0.66453(6) 0.0263(4) C11 0.64912(8) −0.1116(4) 0.69167(7) 0.0296(4) H11 0.686848 −0.024814 0.687188 0.035* C12 0.65532(8) −0.3200(4) 0.72544(7) 0.0301(4) H12 0.698118 −0.379035 0.742789 0.036* C13 0.57796(9) 0.1793(4) 0.62631(7) 0.0297(4) H13 0.611930 0.305537 0.629081 0.036* C14 0.52312(9) 0.4082(4) 0.55304(7) 0.0319(4) H14 0.546158 0.568047 0.570713 0.038* C15 0.55623(10) 0.3195(4) 0.51338(8) 0.0380(5) H15A 0.536724 0.150136 0.498537 0.046* H15B 0.603466 0.289115 0.529606 0.046* C16 0.54863(9) 0.5243(4) 0.47160(8) 0.0370(4) H16A 0.568568 0.455127 0.445665 0.044* H16B 0.572529 0.687005 0.485848 0.044* The key hydrogen bonding interactions in the crystal are of the type hydroxy-O—H· · · N(pyridyl) [O1—H1o· · · N1i: H1o· · · N1i = 1.78(2) Å, O1· · · N1i = 2.615(2) Å with angle at H1o = 170(2)° for symmetry operation (i) x, −1/2 −y, −1/2 + z]. The hydrogen bonding interactions lead to the formation of a supramolecular chain along the c axis direction, having a zigzag topology, being propagated by glide symmetry. The repeat distance of 35.1 Å is rather long as, owing to the kink in the 4-DTBA molecule, the edges are defined by a full Schiff base molecule and two half 4-DTBA molecules; the pitch is 53.4 Å. The 4- DTBA molecules stack along the b axis and are connected into a column by side-on C=O· · · π(phenyl) interactions [C7—O2· · · Cg(C1—C6)ii: O2· · · Cg(C1—C6)ii = 3.6026(16) Å, C7· · · Cg(C1—C6)ii = 3.6015(17) Å with angle at O2 = 80.22(10)° for (ii) x, 1 + y, z]. Source of material The title co-crystal was prepared through physical mixing of 4-mercaptobenzoic acid (Acros, Geel, Antwerp, Belgium) (0.154 g, 1 mmol) and N,N′-bis((pyridine-4-yl)methylene)- cyclohexane-1,4- diamine [5] (0.292 g, 1 mmol) in a 1:1 molar *Corresponding author: Edward R.T. Tiekink, Research Centre for Crystalline Materials, School of Science and Technology, Sunway University, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia, e-mail: edwardt@sunway.edu.my. https://orcid.org/0000-0003- 1401-1520 Sang Loon Tan: Research Centre for Crystalline Materials, School of Science and Technology, Sunway University, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia Unauthenticated Download Date \| 10/10/19 2:17 AM 1122 \| Tan and Tiekink: C14H10O4S2·C18H20N4 Reflecting the change in chemical composition, contributions from H· · · H [52.0%], C· · · H/H· · · C [17.4%] and N· · · H/H· · · N [12.3%] contacts have increased significantly. Complementing this observation are the decreases in the contributions from O· · · H/H· · · O [9.6%] S· · · H/H· · · S [3.1%], O· · · C/C· · · O [1.0%] and C· · · C [2.5%]. Of the minor surface contacts, contributions from O· · · N/N· · · O [0.6%] contacts remain the same, those from N· · · C/C· · · N [1.6%] increase while there are no O· · · O contacts for the Schiff base molecule. O· · · N/N· · · O [0.6%], N· · · C/C· · · N [0.4%] and O· · · O [0.1%]. Some significant differences are noted in the contributors to the surface of the Schiff base. Reflecting the change in chemical composition, contributions from H· · · H [52.0%], C· · · H/H· · · C [17.4%] and N· · · H/H· · · N [12.3%] contacts have increased significantly. Complementing this observation are the decreases in the contributions from O· · · H/H· · · O [9.6%] S· · · H/H· · · S [3.1%], O· · · C/C· · · O [1.0%] and C· · · C [2.5%]. Of the minor surface contacts, contributions from O· · · N/N· · · O [0.6%] contacts remain the same, those from N· · · C/C· · · N [1.6%] increase while there are no O· · · O contacts for the Schiff base molecule. trans-1,2-bis(4-pyridyl)ethene and 1,2-bis(4-pyridyl)ethane. CrystEngComm 9 (2007) 1096–1109. 8. Broker, G. A.; Bettens, R. P. A.; Tiekink, E. R. T.: Co- crystallisation of 2,2′-dithiodibenzoic acid with the isomeric n-pyridinealdazines, n = 2, 3 and 4: supramolecular polymers and the influence of steric factors upon aggregation patterns. CrystEngComm 10 (2008) 879–887. 9. Rowland, C. E.; Cantos, P. M.; Toby, B. H.; Frisch, M.; Deschamps, J. R.; Cahill, C. L.: Controlling disulfide bond formation and crystal growth from 2-mercaptobenzoic acid. Cryst. Growth Des. 11 (2011) 1370–1374. 10. Tan, S. L.; Tiekink, E. R. T.: A 1:2 co-crystal of 2,2′-thiodibenzoic acid and triphenylphosphane oxide: crystal structure, Hirshfeld surface analysis and computational study. Acta Crystallogr. E74 (2018) 1764–1771. 11. Tan, S. L.; Tiekink, E. R. The layers thus formed are connected laterally to form a three-dimensional architecture by pyridyl-C—H· · · O(carbonyl) interactions [C11—H11· · · O2iii: H11· · · O2iii = 2.43 Å, C11· · · O2iii = 3.357(2) Å with angle at H11 = 165° for (iii) 3/2 −x, y, 1 −z]. of cyclohexane-1,4-diammonium di-cation [11–13]. Intrigu- ingly, the original 2-pyridyl isomer of the Schiff base has also been converted to a 2-(4-ammoniocyclohexyl)-3-(pyridin-2- yl)imidazo[1,5-a]pyridin-2-ylium di-cation [14]. Herein, as a continuation of studies in this area and employing the same general procedure as previously, perhaps unexpectedly, the anticipated 1:1 co-crystal was formed from 4-mercaptobenzoic acid and the n = 4 isomer of the Schiff base. Given the nature of weak interactions sustaining the supramolecular chains into a three-dimensional architecture, the Hirshfeld surfaces as well as the full and delineated two- dimensional fingerprint plots were calculated for the individ- ual (symmetry expanded) co-crystal co-formers, using liter- ature procedures [15] and Crystal Explorer 17 [16], in order to determine the most important contacts in the molecular packing. The molecular structures of the two components of the title crystal are shown in the figure (70% probability displacement ellipsoids with unlabelled atoms for the 4- DTBA molecule being related by 3/2 −x, y, 1 −z and for the Schiff base, by 1 −x, 1 −y, 1 −z). The 4-DTBA molecule exhibits crystallographic 2-fold asymmetry. The disparity in the C7—O1, O2 bond lengths [1.321(2) and 1.218(2) Å] confirm Eleven different contacts were revealed in the analy- sis of the 4-DTBA molecule with the prominent contacts being H· · · H [39.4%] followed by O· · · H/H· · · O [14.4%], S· · · C/C· · · S [10.0%] and C· · · H/H· · · C [9.2%]. Signifi- cant contributions are also made by N· · · H/H· · · N [6.7%], S· · · H/H· · · S [6.6%], O· · · C/C· · · O [6.5%] and C· · · C [6.0%] contacts. Fractions of a percentage contributions are made by Unauthenticated Download Date \| 10/10/19 2:17 AM 1123 Tan and Tiekink: C14H10O4S2·C18H20N4 O· · · N/N· · · O [0.6%], N· · · C/C· · · N [0.4%] and O· · · O [0.1%]. Some significant differences are noted in the contributors to the surface of the Schiff base. T.: Crystal structure of hemikis (cyclohexane-1,4-diammonium) (pyridine-2-carboxylate), [C6H16N2]0.5[C6H4NO2]. Z. Kristallogr. NCS 234 (2019) 749–751. Acknowledgements: Sunway University Sdn Bhd is thanked for financial support of this work through Grant No. STR- RCTR-RCCM-001–2019. 12. Tan, S. L.; Tiekink, E. R. T.: Crystal structure of cyclohexane- 1,4-diammonium 2-[(2-carboxylatophenyl)disulfanyl] benzoate — dimethylformamide — monohydrate (1/1/1), [C6H16N2][C14H8O4S2] · C3H7NO·H2O. Z. Kristallogr. NCS 234 (2019) 903–905. Unauthenticated Download Date \| 10/10/19 2:17 AM References 1. Rigaku Oxford Diffraction. CrysAlisPRO. Rigaku Corporation, Oxford, UK (2018). 13. Tan, S. L.; Tiekink, E. R. T.: Crystal structure of bis(cyclohexane- 1,4-diammonium) 4-[(4-carboxylatophenyl)disulfanyl] benzoate dimethylsulphoxide hydrate (1/1/1/1), [C6H16N2]2+[C14H8O4S2]−2·C2H6OS·H2O. Z. Kristallogr. NCS 234 (2019) doi: 10.1515/ncrs-2019-0487. 2. Sheldrick, G. M.: A short history of SHELX. Acta Crystallogr. A64 (2008) 112–122.i 3. Sheldrick, G. M.: Crystal structure refinement with SHELXL. Acta Crystallogr. C71 (2015) 3–8. 14. Tan, S. L.; Tiekink, E. R. T.: Crystal structure of 2-(4- ammoniocyclohexyl)-3-(pyridin-2-yl)imidazo[1,5-a]pyridin-2- ium 2-[(2-carboxylatophenyl)disulfanyl]benzoate dihydrate, [C18H22N4][C14H8O4S2]·2H2O. Z. Kristallogr. NCS 234 (2019) 797–799. 4. Farrugia, L. J.: WinGX and ORTEP for Windows: an update. J. Appl. Crystallogr. 45 (2012) 849–854. 5. Lai, C. S.; Mohr, F.; Tiekink, E. R. T.: The importance of C— H· · · N, C-H· · · π and π· · · π interactions in the crystal pack- ing of the isomeric N1,N4-bis((pyridine-n-yl)methylene) cyclohexane-1,4-diamines, n = 2, 3 and 4. CrystEngComm 8 (2006) 909–915. 15. Tan, S. L.; Jotani, M. M.; Tiekink, E. R. T.: Utilizing Hirshfeld surface calculations, non-covalent interaction (NCI) plots and the calculation of interaction energies in the analysis of molecular packing. Acta Crystallogr. E75 (2019) 308–318.f 6. Rowland, C. E.; Belai, N.; Knope, K. E.; Cahill, C. L.: Hydrother- mal synthesis of disulfide-containing uranyl compounds: in situ ligand synthesis versus direct assembly. Cryst. Growth Des. 10 (2010) 1390–1398. 6. Rowland, C. E.; Belai, N.; Knope, K. E.; Cahill, C. L.: Hydrother- mal synthesis of disulfide-containing uranyl compounds: in situ ligand synthesis versus direct assembly. Cryst. Growth Des. 10 (2010) 1390–1398. 16. Turner, M. J.; Mckinnon, J. J.; Wolff, S. K.; Grimwood, D. J.; Spackman, P. R.; Jayatilaka, D.; Spackman, M. A.: Crystal Explorer v17. The University of Western Australia, Australia (2017). 7. Broker, G. A.; Tiekink, E. R. T.: Co-crystal formation between 2,2′-dithiodibenzoic acid and each of 4,4′-bipyridine, 7. Broker, G. A.; Tiekink, E. R. T.: Co-crystal formation between 2,2′-dithiodibenzoic acid and each of 4,4′-bipyridine, Unauthenticated Download Date \| 10/10/19 2:17 AM
https://openalex.org/W2805624612	https://iris.unibas.it/bitstream/11563/134256/2/IEEE%20Access.pdf	English	null	MultiDIC: An Open-Source Toolbox for Multi-View 3D Digital Image Correlation	IEEE access	2,018	cc-by	11,793	Received April 27, 2018, accepted May 22, 2018, date of publication June 4, 2018, date of current version June 26, 2018. Digital Object Identifier 10.1109/ACCESS.2018.2843725 DANA SOLAV 1, (Member, IEEE), KEVIN M. MOERMAN 1, (Member, IEEE), AARON M. JAEGER1, KATIA GENOVESE2, AND HUGH M. HERR 1, (Member, IEEE) 1MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 2School of Engineering, University of Basilicata, 85100 Potenza, Italy Corresponding author: Dana Solav (danask@mit.edu) This work was supported in part by NIH under Grant 1R01EB024531-01. The work of D. Solav was supported by the MIT-Technion Post-Doctoral Fellowship. ABSTRACT Three-dimensional Digital Image Correlation (3D-DIC) is a non-contact optical-numerical technique for evaluating the dynamic mechanical behavior at the surface of structures and materials, including biological tissues. 3D-DIC can be used to extract shape and full-ﬁeld displacements and strains with high resolution, at various length scales. While various commercial and academic 3D-DIC software exist, the ﬁeld lacks 3D-DIC packages which offer straightforward calibration and data-merging solutions for multi-view analysis, which is particularly desirable in biomedical applications. To address these limitations, we present MultiDIC, an open-source MATLAB toolbox, featuring the ﬁrst 3D-DIC software speciﬁcally dedicated to multi-view setups. MultiDIC integrates robust two-dimensional subset-based DIC software with specially tailored calibration procedures, to reconstruct the dynamic behavior of surfaces from multiple stereo-pairs. MultiDIC contains novel algorithms to automatically merge meshes from multiple stereo- pairs, and to compute and visualize 3D shape and full-ﬁeld motion, deformation, and strain. User interfaces provide capabilities to perform 3D-DIC analyses without interacting with MATLAB syntax, while stand- alone functions also allow proﬁcient MATLAB users to write custom scripts for speciﬁc experimental requirements. This paper discusses the challenges underlying multi-view 3D-DIC, details the proposed solutions, and describes the algorithms implemented in MultiDIC. The performance of MultiDIC is tested using a low-cost experimental system featuring a 360◦12-camera setup. The software and system are evaluated using measurement of a cylindrical object with known geometry subjected to rigid body motion and measurement of the lower limb of a human subject. The ﬁndings conﬁrm that shape, motion, and full- ﬁeld deformations and strains can be accurately measured, and demonstrate the feasibility of MultiDIC in multi-view in-vivo biomedical applications. INDEX TERMS Biomedical image analysis, full-ﬁeld 3-D deformation, material mechanical properties, open source software, skin shape and strain, soft tissue biomechanics, stereo-DIC, strain map. INDEX TERMS Biomedical image analysis, full-ﬁeld 3-D deformation, material mechanical properties, open source software, skin shape and strain, soft tissue biomechanics, stereo-DIC, strain map. This work is licensed under a Creative Commons Attribution 3.0 License. For more information, see http://creativecommons.org/licenses/by/3.0 I. INTRODUCTION ing its motion and deformation in a given target image. In three-dimensional DIC (3D-DIC), two cameras view an ROI from different angles (i.e. a stereo-camera pair), and capture a sequence of images representing a reference conﬁgura- tion (typically an undeformed conﬁguration) and a required number of current conﬁgurations (typically deformed con- ﬁgurations). Next, 2D-DIC is used to correlate a given set of points in the two stereo-views of the reference conﬁgu- ration (spatial correlation) and track these points through- out the sequence of stereo-images representing the current conﬁgurations (temporal correlation). The correlated sets of image points are then used to reconstruct and track the Digital Image Correlation (DIC) is a full-ﬁeld non-contact optical-numerical technique to measure shape, motion, and deformation, on almost any kind of material, even in extreme experimental settings, as long as the region of interest (ROI) on the sample’s surface is provided with a natural or syn- thetic speckle pattern [1], [2]. This allows the DIC algorithm to match dense sets of corresponding points in differ- ent images of the ROI, based on the local pixel inten- sity distribution. Speciﬁcally, local two-dimensional DIC (2D-DIC) [2] deﬁnes a pixel subset around a point of interest in a reference image and evaluates the parameters describ- 30520 VOLUME 6, 2018 VOLUME 6, 2018 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC FIGURE 1. Multi-view stereo system schematic. A circular array of cameras (in transparent color) surrounding the object, with highlighted two contiguous stereo-pairs (in solid color), sharing a common field of view on the object’s surface. The ROI for each pair is depicted on the object’s surfaces with dots, and the overlapping ROI for the two pairs is depicted with solid color. 3D position of the material points of the ROI over time, via stereo-triangulation [1], [3]. DIC was originally developed for experimental mechanics, where it has become a standard technique for characterizing material properties and the response of structures to loads [4]. The non-contact, full-ﬁeld, and large deformation capa- bilities of DIC make it particularly suited for shape and deformation measurements for a wide range of biomedical applications [5]–[11]. DIC methods have been used in com- bination with advanced inverse characterization methods to identify the mechanical properties of natural and synthetic biological materials and body parts [5]–[8]. I. INTRODUCTION Substantial work has been done in recent decades to improve the performance of computational algori- thms [12]–[17], to deﬁne good practices for performing experiments and calibration [18], to assess the measure- ment errors [19], [20], and to extend the capabilities of 3D-DIC [15], [21]. Furthermore, various commercial and academic software packages now exist. However, there is currently a lack of available 3D-DIC software speciﬁcally tai- lored for multi-view analysis, a feature which is highly desir- able for biomedical applications. It is challenging to study the shape, motion, and deformation of tissues and organs in-vivo, due to the often complex geometries, the large deformations that can occur, and the risk of undesired motions. To over- come these challenges, simultaneous multi-view imaging is often utilized [11], [22]. Moreover, in a clinical environment, a fast and ﬂexible image acquisition and calibration process is highly desirable [23]. FIGURE 1. Multi-view stereo system schematic. A circular array of cameras (in transparent color) surrounding the object, with highlighted two contiguous stereo-pairs (in solid color), sharing a common field of view on the object’s surface. The ROI for each pair is depicted on the object’s surfaces with dots, and the overlapping ROI for the two pairs is depicted with solid color. stereo-pair, calibration aims to ﬁnd the intrinsic parameters (deﬁning the geometric and optical characteristics of the cam- era) and the extrinsic parameters (deﬁning the position and orientation of the camera with respect to a reference coordi- nate system). Together, the intrinsic and extrinsic parameters serve to describe the transformation that maps each 3D mate- rial point P (X, Y, Z) in the global coordinate system into its image point on the camera sensor I (xP, yP), according to the pinhole optical model [3], [31]. Speciﬁcally, the coordinates (X, Y, Z) are ﬁrst rigidly transformed into the coordinates (XC, YC, ZC) in the camera coordinate system by Several multi-view DIC systems have been proposed in the literature (e.g. [22], [24]–[29]), where multiple cameras with partially overlapping ﬁelds of view (FOVs) allow for the imaging of large portions of an object surface. The multitude of cameras can be arranged in a 2D array when large quasi- planar objects need to be measured. Alternatively, for non- planar objects, the set of cameras are placed around the object, e.g. in a surrounding fashion, to obtain the 3D shape of the entire object (as illustrated in Fig. 1). I. INTRODUCTION In this work, we refer to the latter conﬁguration, which is suitable for many biomedical applications.   XC YC ZC  =   R11 R12 R13 R21 R22 R23 R31 R32 R33     X Y Z  +   TX TY TZ  , (1) where Rij (i, j = 1, 2, 3) and Tx, Ty, Tz are the components of the rotation matrix and the translation vector, respectively. Next, the (ideal) normalized image point coordinates are calculated as Furthermore, in scientiﬁc research reproducibility, veriﬁability, and validation are key [30]. Only open-source software tools offer full insight into the computational frame- work these aspects demand. Moreover, open-source software enable customizability, which is important since each sci- entiﬁc application offers its own unique computational and experimental challenges. There is hence a pressing need for open-source software implementing algorithms speciﬁcally tailored for multi-view 3D-DIC, which allow for customiza- tion and easy adaptation to different experimental settings. x = XC ZC; y = YC ZC. (2) (2) Then, the intrinsic camera parameters, i.e. the focal lengths in pixels fx, fy , the principal point (optical center) coordi- nates Cx, Cy , and the skew coefﬁcient s = fytanα, where α is the angle between the horizontal and vertical axes of the sensor (α = s = 0 if the axes are perpendicular), are used to calculate the image point coordinates I (xP, yP), in the sensor coordinate system, by B. OVERVIEW OF THE CURRENT WORK A speciﬁc aim of this work was to overcome the above- mentioned limitations of existing multi-view methods. To this end, we present MultiDIC, a free open-source software library for analyzing multi-view stereo images and obtaining 3D shapes and full-ﬁeld displacements, deformations, and strains. Speciﬁc consideration is given to develop a procedure for multi-view stereo calibration and data merging, which enables camera poses to be changed as needed, without requiring a long re-calibration process. The most common stereo calibration technique used in 3D- DIC methods is the Bundle Adjustment (BA) method [1]. BA is an explicit calibration method that allows for the estimation of both the intrinsic and extrinsic camera parameters by using repetitive observations of sparse scene points in different viewing directions [32]. BA allows for lens distortion cor- rection based on a non-linear distortion model. Speciﬁcally, the idealized image point coordinates (x, y) in Eq. (3), are replaced by the distorted normalized coordinates (xd, yd) deﬁned by MultiDIC utilizes the high-quality open-source 2D-DIC software Ncorr [38], which was previously veriﬁed and vali- dated by processing standard sets of synthetic and experimen- tal images [18]. In the present study, a combined approach for calibration was developed, whereby both BA and DLT are used in different steps, to harness the advantages of each method. Particularly, BA is used for obtaining the distortion and skew parameters, which can be used to correct the effect of distortion on the image points extracted from both the stereo calibration images and the speckled object images. BA is only required once per camera, as long as the cam- era’s internal settings remain unaltered. DLT is then used on the undistorted image points for retrieving the internal (distortion-free) and external (camera pose) parameters of the cameras, combined into the DLT parameters. The DLT parameters refer to a common global coordinate system asso- ciated with the 3D calibration object, which is seen simul- taneously from all the cameras. Since only a single image per camera is required, DLT can also be used to check the stability of the system whenever experiments are prolonged in time, and to quickly re-calibrate the system in case cam- eras are intentionally or accidentally moved. A. THEORETICAL BACKGROUND Two key issues entailed in the implementation of multi- view setups are system calibration (required for stereo trian- gulation) and data merging (required for combining results from multiple stereo-pairs). For each camera of a typical   xp yp 1  =   fx s Cx 0 fy Cy 0 0 1     x y 1  . (3)   xp yp 1  =   fx s Cx 0 fy Cy 0 0 1     x y 1  . (3) 30521 (3) VOLUME 6, 2018 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC Next, combining Eq. (1)-(3) and further assuming that s = 0 yields to approach is very time-consuming, especially in case a large number of cameras is required or when the camera poses change during a data collection session, potentially causing error accumulation. Furthermore, this method does not enable combining data from camera-pairs for which the FOV do not overlap. xp = fx R11X + R12Y + R13Z + Tx R31X + R32Y + R33Z + Tz + Cx yp = fy R21X + R22Y + R23Z + Ty R31X + R32Y + R33Z + Tz + Cy, (4) (4) In contrast, the DLT method can use a single image acqui- sition of a 3D (non-planar) calibration target placed in the scene, captured by all the cameras of the multi-view sys- tem. This arrangement allows one to calibrate each camera with respect to a common reference system associated to the 3D target, thus enabling an automatic data merging, even in cases where the FOV of different camera-pairs do not overlap. The main limitation of this approach is that the calibration target usually covers only a portion of the work- ing volume and this may affect the accuracy of the calibra- tion results, even when distortion correction procedures are adopted [35]–[37]. which can be rearranged into the form which can be rearranged into the form xp = L1X + L2Y + L3Z + L4 L9X + L10Y + L11Z + 1 yp = L5X + L6Y + L7Z + L8 L9X + L10Y + L11Z + 1. (5) (5) Eq. (5) represents the basis of the Direct Linear Transfor- mation (DLT) method [31] which allows for implicit camera calibration. The DLT method derives the (unphysical) param- eter set Lj (j = 1, 2, . . A. THEORETICAL BACKGROUND . , 11) by solving a linear system of equations, based on a single image containing a non-planar set of calibration points whose position s (X, Y, Z) are known with high accuracy. DLT, however, relies on an idealized distortion-free camera model which might yield inaccurate results, especially when high-distortion or low-quality lenses are used. B. OVERVIEW OF THE CURRENT WORK Moreover, this rationale allows one to eventually keep the original images and skip the distortion correction step in cases where images are obtained with high quality optics and sensors [25], or if the distortions are known to be negligible with respect to the required precision for the speciﬁc application. xd yd = 1 + k1r2 + k2r4 + k3r6 x y + 2p1xy + p2 r2 + 2x2 p1 r2 + 2y2 + 2p2xy , (6) (6) where r2 = x2 + y2, [k1, k2, k3] are the radial distortion parameters, [p1, p2] are the tangential distortion parameters, and the skew parameter in (3) can be non-zero. Successful BA typically requires a large number of images (approximately 50 [33]) of a planar calibration target acquired in different orientations and positions in the ﬁeld of view (FOV) of both cameras. Although BA is highly accurate, and is typically the ﬁrst choice for calibrating a two-camera system, the associated practice of calibrating one camera with respect to a given ‘master’ camera can limit its applicability to multi-view stereo systems. To calibrate a large number of camera-pairs, each camera-pair has to be calibrated separately. Then, the images of a given set of mark- ers [27], or of a speckled surface [34], which can be viewed by each contiguous pair of cameras, are used to: 1) ﬁnd the geometric transform between the pairs, and 2) combine the data into a mutual coordinate system [34]. This process must be repeated for each additional camera-pair [34]. Clearly, this 30522 VOLUME 6, 2018 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC FIGURE 2. MultiDIC algorithm workflow. Step 0 is optional, and is only required if distortion correction is necessary. Steps 1–3 comprise the core components of the 3D-DIC process. Moreover, step 3.2 is optional, and is only required if merging of surfaces into one continuous mesh is desired. FIGURE 2. MultiDIC algorithm workflow. Step 0 is optional, and is only required if distortion correction is necessary. Steps 1–3 comprise the core components of the 3D-DIC process. Moreover, step 3.2 is optional, and is only required if merging of surfaces into one continuous mesh is desired. non-code experts. For customization, proﬁcient MATLAB users can also use the stand-alone functions and data- structures to write custom scripts and analyze large data- sets semi-automatically. B. OVERVIEW OF THE CURRENT WORK Moreover, users are encouraged to extend the toolbox’s functionality by implementing addi- tional algorithms. When a large number of views is required, as is often the case in biomedical applications, imaging systems can become prohibitively expensive. In this study, we present an inexpensive system using low-cost camera modules, with which we test the capabilities of the toolbox. This paper describes the workﬂow of MultiDIC, the algorithms imple- mented in it, the experimental setup, and the data analysis procedures. Then, results of several tests that both validate it and showcase its capabilities are reported and discussed. Finally, measurement on the lower limb of a human subject demonstrates the typical shape and deformation information obtainable for in-vivo studies. MultiDIC integrates the 2D-DIC open-source soft- ware Ncorr [38] with the BA algorithm included in the MATLAB computer vision toolbox [39], and the DLT algo- rithm implemented in MATLAB by the authors, to perform the reconstruction of 3D surfaces from multiple stereo-image pairs. Moreover, it contains algorithms for merging surfaces, and for computing full-ﬁeld 3D displacements, deformations, strains, and rigid-body motion (RBM). In addition, extensive plotting and visualization functions are offered, which inte- grate tools from the open-source GIBBON toolbox [40]. A. OVERVIEW OF SOFTWARE ARCHITECTURE AND ALGORITHMS g p Fig. 2 outlines the workﬂow of the entire 3D-DIC proce- dure. In the optional step 0, the distortion and skew param- eters are calculated using BA on multiple images of a ﬂat checkerboard pattern. These parameters can be used to cor- rect for the effect of distortion on the points detected on both the calibration object images and the speckled object images. Step 1 comprises the calculation of the DLT cali- bration parameters of each camera, using images of a 3D calibration object seen simultaneously from all the views. Step 2 involves matching corresponding points on sets of stereo images of the speckled object using 2D-DIC. In step 3, the calibration parameters from step 1 are used to perform a stereo-triangulation which transforms the 2D points matched in step 2 for each stereo-pair into 3D triangular meshes. Furthermore, this step may optionally involve the merging together of multiple obtained surfaces. Lastly, step 4 includes MultiDIC is an open-source (Apache 2.0 licensed) library written in MATLAB (R2017b, The Mathworks Inc., USA), a programming language widely used among engineers and scientists. Users may visit the GitHub repository (http://www.github.com/MultiDIC) to obtain the most recent version (this paper refers to release version v1.0, which is also archived at http://doi.org/10.5281/zenodo.1256041), to join development, and to post issues. The toolbox is accompanied by a comprehensive instruction manual and sample data. MultiDIC was designed as a ﬂexible library of functions and scripts that users can freely adjust to ﬁt their experimental requirements. The implementation as a toolbox enables users to perform 3D-DIC analyses and to visualize the results using user interfaces (UIs) which do not require interaction with MATLAB syntax. As such, these UIs provide access to the most common functionality as well as usability for 30523 30523 VOLUME 6, 2018 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC the ROI according to selected subset size and spacing, and the corresponding points are detected on all the remaining images using Ncorr [38]. Ncorr is an open-source subset- based 2D-DIC software, described in detail in [38] together with validation results of its metrological performances tested with images from the 2D-DIC-Challenge [18]. E. 3D RECONSTRUCTION In this step, the sets of DLT parameters LCk j and LCl j (j = 1, 2, . . . , 11) associated with the cameras Ck and Cl (k and l represent the indices of the cameras in a speciﬁc stereo-pair), are used to transform each pair of corresponding image points, from their image coordinates xCk p , yCk p and xCl p , yCl p , into the 3D coordinates (X, Y, Z), by rearranging Eq. (5) into the form In practice, this step requires a 3D (non-planar) calibration object having control points whose 3D positions are known with sufﬁcient accuracy. The use of a 3D object with axial- symmetry (a cylinder in the current study) represents the opti- mal solution for a 360◦multi-view system, since images of the calibration object can be acquired from all cameras simul- taneously. The calibration images are then analyzed using MultiDIC to detect the image points (xP, yP) corresponding to the calibration object’s control points, and to sort and match them with their known 3D positions (X, Y, Z). Next, the DLT parameters Lj (j = 1, 2, . . . , 11) and the associated reconstruction errors are calculated and plotted. U = AP, (7) (7) where U =   xCk p −LCk 4 yCk p −LCk 8 xCl p −LCl 4 yCl p −LCl 8  ; P =   X Y Z  ; A =   LCk 1 −LCk 9 xCk p LCk 2 −LCk 10 xCk p LCk 3 −LCk 11 xCk p LCk 5 −LCk 9 yCk p LCk 6 −LCk 10 yCk p LCk 7 −LCk 11 yCk p LCl 1 −LCl 9 xCl p LCl 2 −LCl 10xCl p LCl 3 −LCl 11xCl p LCl 5 −LCl 9 yCl p LCl 6 −LCl 10yCl p LCl 7 −LCl 11yCl p   . (8) B. DISTORTION CORRECTION The purpose of this step is to ﬁnd the parameters for cor- recting the lens distortions and skew affecting the acquired images. This step has to be executed only once per camera and needs not be repeated if a camera pose is changed, as long as the camera’s intrinsic parameters remain unaltered. The MATLAB Camera Calibration Toolbox [39] was uti- lized, which is based on previous works [35], [41]–[43] for evaluating the camera intrinsic parameters, including distor- tion parameters, using the BA method. Speciﬁcally, multiple images of a planar checkerboard pattern are acquired by each camera. Then, the algorithm utilizes BA to minimize the overall re-projection errors of the corner points of the checkerboard pattern. The implemented model allows for the computation of up to six parameters: three for radial distortion, two for tangential distortion, and one for skew, as detailed in Eq. (3) and (6). These parameters are later used before the 3D reconstruction in step 3, to correct the points detected on both the stereo calibration images and the speckle images. Moreover, a triangular mesh is deﬁned on the point grid and saved for the 3D reconstruction step. Speciﬁcally, MultiDIC’s step 2 serves as a wrapper for Ncorr, which allows users to select camera-pairs for analysis and draw the ROI. These data are automatically transferred into Ncorr for further analysis, and the corresponding image points, together with the associated correlation coefﬁcients [38] are returned for deﬁning the triangular mesh, for plotting the results, and for saving the variables necessary for 3D reconstruction. Fig. 3 illustrates how Ncorr was able to obtain high corre- lation accuracy (low values of CLS) over most of the selected ROI with exception of the right border, where the afﬁne func- tion could not accurately describe the subset transformation in this portion of the surface [46]. In this work, a sufﬁciently large number of cameras (i.e. narrow ROI for each camera pair) were chosen to ensure high accuracy correlation, even on complex shapes. A. OVERVIEW OF SOFTWARE ARCHITECTURE AND ALGORITHMS Brieﬂy, Ncorr implements a ﬁrst order shape function to describe the pixel subset transformation [2], the least-square correlation cri- terion CLS as a correlation cost function [38], the Inverse Compositional Gauss-Newton method as a nonlinear opti- mizer [44], and the Reliability-guided method [45] to prop- agate the analysis over the ROI, starting from user-deﬁned seed points. algorithms for computation and visualization of the full-ﬁeld displacement, deformation, strain, and RBM. The methods implemented in each of these steps are detailed in the next sub-sections. C. STEREO CALIBRATION In this step, the mapping between 2D image points and 3D world points is established, using Eq. (5). Since each control point provides two equations, a minimum of six points is required to obtain the set of 11 DLT parameters. Neverthe- less, a greater number of points is preferred, as the effect of experimental errors is reduced by solving an overdetermined system through least-squares minimization. D. 2D-DIC OF STEREO IMAGE PAIRS In this step, which has to be conducted for each camera- pair, corresponding image points are detected using 2D-DIC, on sets of images of a speckled object acquired from two angled views, as illustrated in Fig. 3. First, a reference image is selected, typically the image acquired in the undeformed conﬁguration from either one of the views. Second, a ROI is selected, which corresponds to an area of the object which is visible from both views. Third, a point grid is obtained inside (8) VOLUME 6, 2018 30524 30524 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC FIGURE 3. Point matching of stereo images using 2D-DIC. Portions of a speckled cylindrical object imaged by cameras positioned with an angular displacement of 30◦between them are shown. The corresponding image points detected using DIC inside the ROI are shown on the reference (left) and current (right) images, with colors depicting the values of the correlation coefficient. FIGURE 3. Point matching of stereo images using 2D-DIC. Portions of a speckled cylindrical object imaged by cameras positioned with an angular displacement of 30◦between them are shown. The corresponding image points detected using DIC inside the ROI are shown on the reference (left) and current (right) images, with colors depicting the values of the correlation coefficient. Then, the mean error εM and the root mean square (RMS) error εR are deﬁned as Then, the least-squares solution for P is obtained by Then, the least-squares solution for P is obtained by P = h AT A i−1 AT U. (9) (9) εM = 1 N v u u u t N X n=1 1Xn !2 + N X n=1 1Y n !2 + N X n=1 1Zn !2 ; εR = v u u t 1 N N X n=1 (1Xn)2 + (1Yn)2 + (1Zn)2. (11) Consequently, a 3D point cloud is obtained from each stereo-pair. Since the coordinates of the control points on the calibration are expressed in the global coordinate system for all camera-pairs, all point clouds are automatically recon- structed in the global coordinate system, without the need to perform any additional coordinate transformation procedure. (11) G. POST-PROCESSING In this step, the 3D coordinates of the triangular mesh’s vertices in the reference and current conﬁgurations are used to derive the full-ﬁeld displacement, deformation, and strain. For each triangular element and for each conﬁg- uration, the deformation gradient tensor F is calculated using a variation of the triangular Cosserat point element method [47], [48]. Assuming a homogenous deformation ﬁeld within each element, this method obtains the ﬁnite (nonlinear) deformation ﬁeld with the same spatial resolution as the DIC measurement, and independently from adjacent data points and numerical derivatives. 1. The correlation coefﬁcient CLS, representing the matching quality of each vertex (removing vertices with poorer correlation ﬁrst). 2. The distance between the meshes in the overlap region (removing faces having larger distance to the other mesh ﬁrst). 3. The difference in the local orientation between the meshes in the overlap region (removing faces having larger angu- lar displacement from the other mesh ﬁrst). lar displacement from the other mesh ﬁrst). 4. A combination of 1, 2, and 3, with optional weights. The reference conﬁguration of a given triangular element is denoted by the position vectors {P1, P2, P3} of its three vertices, and the values of these position vectors in a current conﬁguration are denoted by p1, p2, p3 . Moreover, the ref- erence and current conﬁgurations are characterized by the director vectors {D1, D2, D3} and {d1, d2, d3}, respectively, deﬁned as Once the overlap is resolved, as depicted in Fig. 4(b), the meshes are merged by introducing new faces along the gap, connecting original vertices from both meshes, as depicted in Fig. 4(c). The algorithm uses only vertices which exist in the original data. Introduction of new vertices which do not originate from an actual measurement is not desirable, since these vertices cannot be tracked in the deformed con- ﬁgurations and cannot provide reliable displacement and strain measurements. Moreover, keeping all the points and re-meshing the overlapping regions is not preferred because it results in a denser and often lower quality mesh, and might result in a jagged surface in regions where the two surfaces could not accurately merge. F. SURFACE MERGING Moreover, each set of N reconstructed points coordinates [Xn, Yn, Zn] (n = 1, 2 . . . N) on the calibration object, and the associated true coordinates h X(t) n , Y (t) n , Z(t) n i can be used to calculate the reconstruction errors [1Xn, 1Y n, 1Zn] as The tessellation deﬁned in step 2 is used here to form sets of 3D triangular meshes. The meshes obtained from each camera pair are independent, not connected to each other, and may locally overlap. In order to construct a single continuous and merged surface, the overlap between meshes needs to be resolved and adjacent meshes should be stitched together. 1Xn = Xn −X(t) n ; 1Yn=Yn −Y (t) n ; 1Zn = Zn −Z(t) n . (10) VOLUME 6, 2018 30525 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC FIGURE 4. Surface merging example. (a) The green and blue 3D meshes were reconstructed from two adjacent camera-pairs. (b) The overlap regions are identified, and faces are removed from the boundaries until the overlap is resolved and a minimum gap exist between the surfaces. The gap which needs to be filled with new triangles is marked with a magenta line. (c) The gap is filled with new triangular faces by locally applying Delaunay triangulation, using only vertices existing in the original data. FIGURE 4. Surface merging example. (a) The green and blue 3D meshes were reconstructed from two adjacent camera-pairs. (b) The overlap regions are identified, and faces are removed from the boundaries until the overlap is resolved and a minimum gap exist between the surfaces. The gap which needs to be filled with new triangles is marked with a magenta line. (c) The gap is filled with new triangular faces by locally applying Delaunay triangulation, using only vertices existing in the original data. An example schematic for resolving the overlap and for merging two adjacent surfaces is shown in Fig. 4. triangles are formed. Figure 4 demonstrates how overlapping triangular meshes are successfully merged using the original vertices only, despite having different element sizes. To resolve the overlap, an algorithm was developed, whereby redundant faces are iteratively removed from the boundaries of overlapping regions until a user-deﬁned mini- mum gap exists between the meshes. The following heuristics are employed to select which faces to remove: H. EXPERIMENTAL SETUP 3 F = 3 X i=1 di ⊗Di, (14) A 360◦stereo camera rig was designed and fabricated, to hold up to 30 cameras (Raspberry Pi Camera Module V2, featuring a Sony IMX219 8-megapixel 3.674 × 2.760 mm2 CMOS sensor, connected to Raspberry Pi Zero-w) in a 700 mm diameter circle, as shown in Fig. 5. The objects to be imaged were placed in the center of the circle where it could be uni- formly illuminated by circular arrays of ﬂexible LED strips. All cameras were connected through an Ethernet hub and synchronously controlled using a PC. For the experiments described in this paper, twelve cameras were placed coaxially, resulting in twelve stereo-pairs with an angular displacement of 30◦, such that the distance between each pair of contigu- ous cameras was about 181 mm. This stereo-angle is small enough to provide both a sufﬁcient overlapping portion and an acceptable level of distortion between image-pairs, and at the same time large enough to allow for accurate out- of-plane displacement measurements [19], [20]. The system was very low-cost (<$1,000 total, including 12 cameras, cables, LEDs, Ethernet hub, power supplies, and building materials for the rig). A 3D calibration object was prepared by accurately lathing a 250 mm long, 114 mm diameter alu- minum cylinder, to achieve a uniform diameter throughout the cylinder’s length. This cylinder was then wrapped in a white adhesive paper with a printed grid pattern of black square dots (4 mm by 4 mm squares, 12 mm spacing), as shown in Fig. 5(a). (14) where ⊗is the tensor (outer) product. F transforms any material line in the triangular element from the reference to the current conﬁguration, including the director vectors and the principal directions, such that it satisﬁes di = FDi ni = FNi. (15) (15) Next, the right and left Cauchy-Green deformation tensors (C and B, respectively), and the associated Green-Lagrangian and Eulerian-Almansi ﬁnite strain tensors (E and e, respec- tively), are deﬁned by C = FTF; B = FFT E = 1 2 (C −I) ; e = 1 2 I −B−1 , (16) (16) where I is the unity second order tensor. G. POST-PROCESSING Stitching of the gap is performed by locally applying Delaunay triangulation between the ver- tices on the boundaries of the meshes, which maximizes the minimum angle of the triangles and ensures that quality D1 = P2 −P1; D2 = P3 −P1; D3 = D1 × D2 \|D1 × D2\| d1 = p2 −p1; d2 = p3 −p1; d3 = d1 × d2 \|d1 × d2\| D1 × D2 · D3 = 2A, (12) (12) such that D3 and d3 are unit vectors normal to the plane of the triangle in its reference and current conﬁgura- tions, respectively, and A is the triangle’s reference area. Then, the reference reciprocal vectors D1, D2, D3 are 30526 VOLUME 6, 2018 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC deﬁned by D1 = D2 × D3 2A ; D2 = D3 × D1 2A ; D3 = D1 × D2 2A = D3, (13) deﬁned by conﬁguration using the least-squares minimization approach (rigid superimposition) [52], [53]. y D1 = D2 × D3 2A ; D2 = D3 × D1 2A ; D3 = D1 × D2 2A = D3, (13) MultiDIC offers functions for the dynamic visualization of the displacements, stretches and strains (magnitude and direction), surface area change, as well as additional measures such as the correlation coefﬁcient and the color texture from the original images. (13) such that Di·Dj = δi j, where δi j is the Kronecker delta symbol. Consequently, the deformation gradient tensor F is deﬁned by H. EXPERIMENTAL SETUP Moreover, the spec- tral decompositions of these tensors are given by C = λ2 1 (N1 ⊗N1) + λ2 2 (N2 ⊗N2) + λ2 3 (N3 ⊗N3) B = λ2 1 (n1 ⊗n1) + λ2 2 (n2 ⊗n2) + λ2 3 (n3 ⊗n3) E = E1 (N1 ⊗N1) + E2 (N2 ⊗N2) + E3 (N3 ⊗N3) e = e1 (n1 ⊗n1) + e2 (n2 ⊗n2) + e3 (n3 ⊗n3) , (17) where λi are the principal stretches, Ni and ni are the prin- cipal directions in the reference and current conﬁgurations, and Ei and ei are the principal Green-Lagrangian and Euler- Almansi strains, respectively. Since each triangular element is planar, one of the principal stretches must equal to one (e.g. λ3 = 1), and one of the principal strains must equal to zero (e.g. E3 = e3 = 0). Furthermore, their associated directions are normal to the triangle’s surface (N3 = D3 and n3 = d3). It is noted that although strains are more commonly presented, stretches are often preferred in the case of large deformation, since their deﬁnition is unique, while numer- ous strain deﬁnitions exist besides the ones described above (e.g. Seth-Hill class strains [49], [50] and Baˇzant strains [51]). I. METROLOGICAL PERFORMANCE ASSESSMENT To evaluate the errors associated with stereo calibration inde- pendently of the DIC process, an experiment was performed, whereby the 3D positions of the centroids of the black dots on the calibration object were analyzed before and after a 5 mm displacement was applied with a motorized linear stage (Zaber Technologies A-LSQ075B-E01, max 0.5 µm microstep error), as shown at the bottom of the cylinder in Fig. 5(a), and the in Fig. 5(d). The reconstructed 3D points were then superimposed into their reference positions by estimating the optimal RBM between them. The Euclidian distances between corresponding points represent the stereo calibration errors. Furthermore, the dilatation J, which corresponds to the relative surface area change of the triangle, is given by The next set of veriﬁcation experiments involved a 3D speckled test object which was prepared by applying an adhesive paper with a speckle pattern (black 1.4 mm circles printed in a random pattern on white paper) to the aluminum cylinder (see Fig. 3). The cylinder’s surface was imaged using the camera rig and analyzed using MultiDIC. The perfor- mance of the complete 3D-DIC algorithm and experimental J = det (F) = λ1λ2. (18) (18) When the deformation includes large motion, it is often desirable to subtract the average RBM such that the defor- mation can be visualized without large movement of the object. Therefore, the average RBM is estimated for each 30527 30527 VOLUME 6, 2018 VOLUME 6, 2018 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC FIGURE 5. Experimental setup. (a) The 360◦rig holding 12 Raspberry Pi camera units, one of which is shown enlarged in (b). Each pair of adjacent cameras [C1, C2], [C2, C3] . . . [C12, C1], constitute a stereo-pair (P1, P2 . . . P12). The calibration object is placed at the center of the rig and illuminated using flexible LED strips, which are shown enlarged in (c). A motorized translation stage (d) was placed under the calibration object for the validation experiments. FIGURE 5. Experimental setup. (a) The 360◦rig holding 12 Raspberry Pi camera units, one of which is shown enlarged in (b). Each pair of adjacent cameras [C1, C2], [C2, C3] . . . [C12, C1], constitute a stereo-pair (P1, P2 . . . P12). The calibration object is placed at the center of the rig and illuminated using flexible LED strips, which are shown enlarged in (c). I. METROLOGICAL PERFORMANCE ASSESSMENT A motorized translation stage (d) was placed under the calibration object for the validation experiments. system was evaluated by analyzing the accuracy of the recon- structed shape, the merging error between adjacent camera- pairs, the displacement error, and the strain error, as detailed below. the displacements errors were calculated by comparing the displacements measured using 3D-DIC and those measured using the stage. Moreover, the 3D strains, which should the- oretically equal to zero, were analyzed to evaluate the overall accuracy of the strain measurement. The accuracy of the shape measurement was evaluated by comparing the ﬁtted diameter of the reconstructed cylin- der to the diameter measured using a large-capacity caliper (±0.01 mm accuracy). Next, the merging errors between sur- faces reconstructed by different camera-pairs were evaluated by reconstructing a portion of the surface visible by three adjacent cameras (such as the overlapping region illustrated in Fig. 1). The center camera (C2) was deﬁned as the ref- erence camera for the two pairs: [C2, C1] and [C2, C3], such that the same reference point grid was used for both pairs. The 3D point clouds reconstructed by the two camera-pairs should theoretically be identical. Therefore, any distance between them represents a merging error. Finally, to demonstrate the capability of the proposed methods to measure in-vivo shapes and deformations, the sur- face of the lower limb of a human subject (healthy 34 y.o. female) was measured. A custom speckling stamp was made by laser-engraving a rubber sheet with the same speckle pattern described above. A uniform layer of white temporary tattoo ink (ProAiir, ShowOffs Body Art LLC., USA) was applied to the skin surface to enhance contrast and to create a homogenous background. Next, a speckle pattern was applied with black temporary tattoo ink (ProAiir, ShowOffs Body Art LLC., USA) using the stamp. The lower leg of the sub- ject was imaged while the subject performed unconstrained ankle plantarﬂexion (from the most dorsiﬂexed position to the most plantarﬂexed position). The experimental procedure Next, the speckled cylinder was translated using the motor- ized linear stage, in 10 steps of 0.1 mm increment, and 30528 VOLUME 6, 2018 VOLUME 6, 2018 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC FIGURE 6. Stereo calibration error analysis. Two sets of calibration object images (before and after 5 mm displacement) were analyzed. III. RESULTS A. CAMERA INTRINSIC PARAMETERS AND 3D RECONSTRUCTION ERRORS Table 1 reports the mean ± standard deviation (STD) of the intrinsic parameters calculated using the BA method for the twelve cameras. For each camera, 50 images of a ﬂat checkerboard pattern with 15 × 20 black and white 10 × 10 mm2 squares were acquired. The number of signiﬁ- cant digits for each parameter is based on its estimation error, as computed by the BA algorithm. The distortion parameters calculated using the original images were used for correcting the distortion on the detected image points. The corrected points were then used again for estimating the parameters. Therefore, the distortion parameters in the right column of Table 1 represent the values of the residual distortion. The values of the 3D reconstruction errors εM and εR, obtained for all camera-pairs using DLT with and without distortion correction, are reported in Table 2. I. METROLOGICAL PERFORMANCE ASSESSMENT (a) The 3D positions of the control points on the calibration object, as reconstructed in the first set (black hollow circles), and their positions measured in the second (displaced) set superimposed into the first set (plotted with colors depicting the absolute distance between them). (b) The error statistics for all the control points, obtained with and without distortion correction, in the x, y, and z directions, and the error magnitude (Mgn). The boxplots report the median (red line), 25th and 75th quartiles (blue box), and outliers (red crosses). The green dashed line represents zero error, and the magenta dashed line represents the RMS error. FIGURE 6. Stereo calibration error analysis. Two sets of calibration object images (before and after 5 mm displacement) were analyzed. (a) The 3D positions of the control points on the calibration object, as reconstructed in the first set (black hollow circles), and their positions measured in the second (displaced) set superimposed into the first set (plotted with colors depicting the absolute distance between them). (b) The error statistics for all the control points, obtained with and without distortion correction, in the x, y, and z directions, and the error magnitude (Mgn). The boxplots report the median (red line), 25th and 75th quartiles (blue box), and outliers (red crosses). The green dashed line represents zero error, and the magenta dashed line represents the RMS error. TABLE 1. Statistics of the estimated intrinsic parameters. TABLE 1. Statistics of the estimated intrinsic parameters. was approved by the Committee on the Use of Humans as Experimental Subjects of the Massachusetts Institute of Technology (COUHES protocol: 1101004280). All the DIC analyses were performed using 20 pixels subset radius and 10 pixels step size at a ∼10 pixel/mm ratio. All the results represent raw data. No kind of ﬁlter- ing or smoothing procedures were applied. B. STEREO CALIBRATION TEST Fig. 6 reports the stereo calibration errors, represented by the Euclidian distances between corresponding points of the VOLUME 6, 2018 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC FIGURE 7. 3D-DIC results of a rigid speckled cylinder. (a) Merging error depicted as the Euclidian distance between the points reconstructed by two adjacent camera-pairs (such as the overlapping region in Fig. 1); (b) Results from a set of 10 imposed translations (d0) with a 0.1 mm increment. The measured displacements (top), displacement errors (center) and Green-Lagrangian strain magnitude (bottom) are plotted as error bars which represent ± one STD over all data points; (c) The Green-Lagrangian strain magnitude distribution plotted on half of the cylinder, for the 0.5 mm displacement step. FIGURE 7. 3D-DIC results of a rigid speckled cylinder. (a) Merging error depicted as the Euclidian distance between the points reconstructed by two adjacent camera-pairs (such as the overlapping region in Fig. 1); (b) Results from a set of 10 imposed translations (d0) with a 0.1 mm increment. The measured displacements (top), displacement errors (center) and Green-Lagrangian strain magnitude (bottom) are plotted as error bars which represent ± one STD over all data points; (c) The Green-Lagrangian strain magnitude distribution plotted on half of the cylinder, for the 0.5 mm displacement step. TABLE 2. Reconstruction errors for all camera-pairs. with and without distortion correction (∼10−14 mm) , but the RMS of the error magnitudes was smaller when the distortion correction was employed (0.017 mm) than when it was not employed (0.026 mm). C. 3D-DIC TESTS OF A RIGID SPECKLED OBJECT The images in Fig. 8(b-d) represent deformed states of (a). Note that the image distortion with respect to (a) is due to perspective in (b), due to deformation in (c), and due to perspective and deformation in (d). Fig. 9 plots the full surface reconstructed by 12 cameras in the reference (a) and deformed (b and c) conﬁgurations, which corresponds to the same conﬁgurations shown in Fig. 8. The colors on the surface depict the local values of the ﬁrst and second princi- pal stretches, and the black lines represent their directions, with lengths proportional to the stretch magnitude. Since (a) shows the reference conﬁguration, the stretch values equal to one by deﬁnition, and no stretch direction are shown. The images in Fig. 8(b-d) represent deformed states of (a). Note that the image distortion with respect to (a) is due to perspective in (b), due to deformation in (c), and due to perspective and deformation in (d). Fig. 9 plots the full surface reconstructed by 12 cameras in the reference (a) and deformed (b and c) conﬁgurations, which corresponds to the same conﬁgurations shown in Fig. 8. The colors on the surface depict the local values of the ﬁrst and second princi- pal stretches, and the black lines represent their directions, with lengths proportional to the stretch magnitude. Since (a) shows the reference conﬁguration, the stretch values equal to one by deﬁnition, and no stretch direction are shown. C. 3D-DIC TESTS OF A RIGID SPECKLED OBJECT The errors reported in this section are associated with the entire 3D-DIC procedure. The correctness of the shape mea- surement was evaluated by comparing the ﬁtted diameter of the reconstructed cylinder to the 114.00 mm diameter (including the adhesive paper) measured using the caliper. The mean ± STD of the diameter measured using 3D-DIC was 113.93 ± 0.27 and 113.91 ± 0.56 mm, with and without distortion correction, respectively. The merging errors between surfaces reconstructed by two different camera-pairs are shown in Fig. 7(a). The recon- structed points are plotted with colors depicting the mag- nitude of the Euclidean distance between the two sets. The mean ± STD of the errors were 0.06 ± 0.03 mm and 0.08 ± 0.06 mm, with and without distortion correction, respectively. As expected, the distortion correction improved the merging accuracy mostly in the ROI’s upper and lower ends, corresponding to the marginal areas of the images. calibration object measured in two conﬁgurations and rigidly superimposed. Fig. 6(a) illustrates the error magnitudes of individual points, and Fig. 6(b) summarizes the statistics of the 3D components of the errors obtained with and without distortion correction. The mean errors were very small both calibration object measured in two conﬁgurations and rigidly superimposed. Fig. 6(a) illustrates the error magnitudes of individual points, and Fig. 6(b) summarizes the statistics of the 3D components of the errors obtained with and without distortion correction. The mean errors were very small both The displacements and displacement errors are summa- rized in Fig. 7(b) (top and center) as a function of the imposed displacement. Fig. 7(c) shows the strain magni- tude distribution over half of the cylinder, for the 0.5 mm 30530 VOLUME 6, 2018 VOLUME 6, 2018 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC FIGURE 8. In-vivo body segment test – Correlation maps. Sub-figures (a)-(d) show images from adjacent cameras in the reference (a, b) and deformed (c, d) configurations. The ROI is plotted with colors depicting the correlation coefficient, and zoomed-in portions are enlarged in insets. FIGURE 8. In-vivo body segment test – Correlation maps. Sub-figures (a)-(d) show images from adjacent cameras in the reference (a, b) and deformed (c, d) configurations. The ROI is plotted with colors depicting the correlation coefficient, and zoomed-in portions are enlarged in insets. displacement step, while the statistics for all displacement steps are summarized in Fig. 7(b) (bottom). D. 3D-DIC IN-VIVO TEST OF A DEFORMABLE BODY SEGMENT Illustrative results from the 3D-DIC in-vivo measurement are shown in Fig. 8. The full surface of the calf region of the lower leg was imaged ﬁve times while the subject performed ankle plantarﬂexion. Results of the ﬁrst and the last conﬁgu- rations are illustrated in Figs. 8 and 9. Fig. 8 illustrates the 2D-DIC results for one stereo-pair. Fig. 8(a) shows the reference image, on which the point grid was deﬁned. 30531 VOLUME 6, 2018 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC FIGURE 9. In-vivo body segment test – Deformations. The full 360◦surface reconstructed from 12 cameras is shown in its reference (a) and deformed (b, c) configurations. The face colors depict the principal stretch values λ1 (b) and λ2 (c), and the black lines represent the principal directions N1 (b) and N2 (c). Lighting was added to better visualize the surface contours. FIGURE 9. In-vivo body segment test – Deformations. The full 360◦surface reconstructed from 12 cameras is shown in its reference (a) and deformed (b, c) configurations. The face colors depict the principal stretch values λ1 (b) and λ2 (c), and the black lines represent the principal directions N1 (b) and N2 (c). Lighting was added to better visualize the surface contours. The deformation ﬁelds are visibly smooth, despite the facts that it was computed independently on each triangu- lar face and that no smoothing or ﬁltering was applied to the raw data, contrarily to common DIC strain calculation schemes [1], [54]. Animated ﬁgures plotting the dynamic values of the displacement magnitude, the Lagrangian strain magnitude, the principal stretches, the surface area change, and the effect of RBM subtraction, are provided in the online Graphical Abstract video. considering the large size of the calibration object, and the cameras’ large FOV and very low price. Next, the metrological errors associated to the entire multi- view 3D-DIC procedure were evaluated by measuring the shape, displacement and strain error with a speckled cylindri- cal object. The shape measurements revealed errors smaller than 0.01 mm (<0.1%) in obtaining the cylinder’s diameter. The merging errors and displacement errors were also found to be in the order of 0.01 mm, which is comparable to the stereo calibration errors. D. 3D-DIC IN-VIVO TEST OF A DEFORMABLE BODY SEGMENT Furthermore, the strains measured as a result of RBM were in the range of 10−3, which is sufﬁciently accurate in our case, and is acceptable in most biomechanical applications, in which large deformations are involved. Moreover, this strain error is relatively low con- sidering that, contrarily to commonly adopted DIC strain calculation schemes, no kinds of ﬁltering and smoothing were applied to the raw data. IV. DISCUSSION This paper describes the principal algorithms implemented in MultiDIC, a newly developed 3D-DIC MATLAB tool- box for multi-camera systems. The methods and system were designed to overcome some of the limitations involved with the calibration and data merging techniques commonly used in 3D-DIC. One primary advantage of MultiDIC is that it allows for a simple calibration process even when using a large number of cameras, without losing the ability to compensate for lens distortions. Consequently, the new method offers the ﬂexibility of modifying the camera poses during experiments without requiring substantial additional re-calibration time. The MultiDIC toolbox and camera rig were utilized for in- vivo measurements to further illustrate capabilities of the system in clinical settings. It was shown that the dynamic behavior of the entire surface of the shank segment of a human subject can be reconstructed, that a smooth strain ﬁeld can be obtained from the raw data, and that large deforma- tions can be tracked. The results in Fig. 9 and in the online Graphical Abstract illustrate how the subject’s lower limb shape changes due to the contraction of the calf muscles and the plantarﬂexion of the ankle joint. This test served as a proof of principle, and was not intended to provide any physiological or biomechanical ﬁnding. The metrological capabilities of MultiDIC were vali- dated by several experimental tests using a low-cost multi- camera setup. The errors associated with the proposed stereo calibration method were examined independently of the DIC procedure, by analyzing the 3D positions of the calibration object’s control points in two conﬁgurations. The errors when using distortion correction procedure were 0.000 ± 0.017 mm (STD and RMS 35% lower than with- out distortion correction). These errors are relatively low The main limitation in this study is that the metrologi- cal performance tests were performed using very low-cost camera modules. Therefore, the accuracies reported here are 30532 VOLUME 6, 2018 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC adequate only for certain applications. However, the entire procedure is expected to outperform when high quality cam- eras and lenses are used. The use of low-cost hardware was motivated by the large number of cameras required, and was encouraged by previous studies which demonstrated the fea- sibility of using consumer-grade cameras [16], [55]. Future research could determine the performance of MultiDIC using high quality optics. IV. DISCUSSION Another limitation is that only one multi- view setup and calibration object was used in this study. However, the toolbox can be easily adapted to any multi-view setup, as long as a calibration object with a proper geometry is used. [2] B. Pan, K. Qian, H. Xie, and A. Asundi, ‘‘Two-dimensional digital image correlation for in-plane displacement and strain measurement: A review,’’ Meas. Sci. Technol., vol. 20, no. 6, p. 62001, Jun. 2009. [3] O. Faugeras, Three-Dimensional Computer Vision: A Geometric View- point. Cambridge, MA, USA: MIT Press, 1993. [4] F. Hild and S. 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It is noted that additional algorithms for 3D-DIC exist (e.g. [12]–[17], [56]), some of them offer signiﬁcant advances. Moreover, an increasing number of DIC-related open-source tools is seen lately (e.g. [17], [38], [57]–[59]). In order to make these techniques more accessible and the results more reproducible, researchers are encouraged to extend the functionality of the toolbox by implementing addi- tional algorithms as contributors [30], [60]. [8] S. Avril, ‘‘Hyperelasticity of soft tissues and related inverse problems,’’ in Material Parameter Identiﬁcation and Inverse Problems in Soft Tissue Biomechanics. Cham, Switzerland: Springer, 2017, pp. 37–66. Biomechanics. Cham, Switzerland: Springer, 2017, pp. 37–66. [9] A. S. Dickinson, A. C. Taylor, H. Ozturk, and M. Browne, ‘‘Experimental validation of a ﬁnite element model of the proximal femur using digi- tal image correlation and a composite bone model,’’ J. Biomech. Eng., vol. 133, p. 14504, Jan. 2011. [10] R. Maiti et al., ‘‘In vivo measurement of skin surface strain and sub-surface layer deformation induced by natural tissue stretching,’’ J. Mech. Behav. Biomed. Mater., vol. 62, pp. 556–569, Sep. 2016. V. CONCLUSION V. CONCLUSION [16] B. Pan, L. Yu, and D. Wu, ‘‘High-accuracy 2D digital image correlation measurements using low-cost imaging lenses: Implementation of a gener- alized compensation method,’’ Meas. Sci. Technol., vol. 25, no. 2, p. 25001, 2014. This paper presents a newly developed open-source MATLAB toolbox for 3D-DIC, which implements algo- rithms speciﬁcally tailored for multi-camera applications, which are often desirable in biomechanics. Several veriﬁca- tion tests have conﬁrmed that accurate measurements can be achieved using MultiDIC and low-cost hardware. The devel- opment of a user-friendly, high-quality and freely available software, will greatly increase the availability of 3D-DIC to the scientiﬁc community. 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Orteu, and H. Schreier, Image Correlation for Shape, Motion and Deformation Measurements. Boston, MA, USA: Springer, 2009. IV. DISCUSSION Future research by the authors will incorporate the tools established here in biomechanical studies, whereby the measured dynamic shapes of the residual limbs of lower- limb amputees will be used to inform the computational mechanical design of subject-speciﬁc optimized prosthetic sockets [61]. Moreover, the subject-speciﬁc soft-tissue mechanical properties will be evaluated, by analyzing simultaneous force/torque measurements and surface defor- mation measurements of the entire residual limb during in-vivo indentations. Then, the hyper-elastic and visco-elastic properties will be characterized using inverse ﬁnite element analysis [62], [63]. [11] K. Ito et al., ‘‘Dynamic measurement of surface strain distribution on the foot during walking,’’ J. Mech. Behav. Biomed. Mater., vol. 69, pp. 249–256, May 2017. [12] Y. Gao, T. Cheng, Y. Su, X. Xu, Y. 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CONCLUSION He is cur- rently a Research Scientist with the Biomechatron- ics Group and the Center for Extreme Bionics, MIT Media Lab. He is also a Visiting Research Fellow with the Centre for Bioengineering, University of Dublin. His main research interests are biomedical device design, soft tissue biomechanics, image-based modeling, and inverse ﬁnite element analysis. [45] B. Pan, ‘‘Reliability-guided digital image correlation for image deforma- tion measurement,’’ Appl. Opt., vol. 48, no. 8, pp. 1535–1542, Mar. 2009. [46] H. Lu and P. D. Cary, ‘‘Deformation measurements by digital image cor- relation: Implementation of a second-order displacement gradient,’’ Exp. Mech., vol. 40, no. 4, pp. 393–400, Dec. 2000. [47] D. Solav, M. B. Rubin, A. Cereatti, V. Camomilla, and A. Wolf, ‘‘Bone pose estimation in the presence of soft tissue artifact using tri- angular cosserat point elements,’’ Ann. Biomed. Eng., vol. 44, no. 4, pp. 1181–1190, Apr. 2016. [48] D. Solav, H. Meric, M. B. Rubin, D. Pradon, F. Lofaso, and A. Wolf, ‘‘Chest wall kinematics using triangular cosserat point elements in healthy and neuromuscular subjects,’’ Ann. Biomed. Eng., vol. 45, no. 8, pp. 1963–1973, Aug. 2017. 30534 VOLUME 6, 2018 VOLUME 6, 2018 D. Solav et al.: MultiDIC: Open-Source Toolbox for Multi-View 3-D DIC AARON M. JAEGER received the B.S. degree in robotics engineering from the Worcester Poly- technic Institute (WPI) in 2017. In 2015, he was with the Humanoid Robotics Lab, WPI, involved in the DARPA Robotics Challenge. He is currently a member of the Biomechatronics Group, MIT Media Lab. His research interests include biome- chanics, robotic locomotion, exploration, and disaster response. HUGH M. HERR (M’04) is currently a Professor within the Program of Media Arts and Sciences, Massachusetts Institute of Technology (MIT). He also directs the Biomechatronics Group at the MIT Media Lab. His research program seeks to advance technologies that promise to accel- erate the merging of body and machine, includ- ing device architectures that resemble the body’s musculoskeletal design, actuator technologies that behave like muscle, and control methodologies that exploit principles of biological movement. KATIA GENOVESE received the B.S. and M.S. degrees in mechanical engineering from the Polytechnic University of Bari, Italy, in 1998, and the Ph.D. degree in mechanical engineering from Federico II University, Napoli, Italy, in 2002. She has been an Assistant Professor with the School of Engineering, University of Basilicata, Potenza, Italy, since 2002, and an Associate Pro- fessor since 2015. V. CONCLUSION She has authored or co-authored 40 journal papers, book chapters, and over 60 inter- national conference papers. Her research areas concern optical methods for shape and deformation measurement (fringe projection, electronic speckle pattern interferometry, and digital image correlation) and hybrid experimen- tal/numerical methods for material characterization. KATIA GENOVESE received the B.S. and M.S. degrees in mechanical engineering from the Polytechnic University of Bari, Italy, in 1998, and the Ph.D. degree in mechanical engineering from Federico II University, Napoli, Italy, in 2002. She has been an Assistant Professor with the School of Engineering, University of Basilicata, Potenza, Italy, since 2002, and an Associate Pro- fessor since 2015. She has authored or co-authored 40 journal papers, book chapters, and over 60 inter- national conference papers. Her research areas concern optical methods for shape and deformation measurement (fringe projection, electronic speckle pattern interferometry, and digital image correlation) and hybrid experimen- tal/numerical methods for material characterization. 30535 VOLUME 6, 2018
https://openalex.org/W3036480910	https://ieeexplore.ieee.org/ielx7/6287639/8948470/09122523.pdf	English	null	A Wavelet Based Deep Learning Method for Underwater Image Super Resolution Reconstruction	IEEE access	2,020	cc-by	7,382	Received April 28, 2020, accepted June 18, 2020, date of publication June 22, 2020, date of current version July 7, 2020. Received April 28, 2020, accepted June 18, 2020, date of publication June 22, 2020, date of current version July 7, 2020. Received April 28, 2020, accepted June 18, 2020, date of publication June 22, 2020, date of current version July 7, 2020. Digital Object Identifier 10.1109/ACCESS.2020.3004141 YUZHANG CHEN , KANGLI NIU , ZHANGFAN ZENG , AND YONGCAI PAN, (Fellow, IEEE) School of Computer Science and Information Engineering, Hubei University, Wuhan 430062, China Corresponding author: Yuzhang Chen (hubucyz@foxmail.com) YUZHANG CHEN , KANGLI NIU , ZHANGFAN ZENG , AND YONGCAI PAN, (Fellow, IEEE) School of Computer Science and Information Engineering, Hubei University, Wuhan 430062, China Corresponding author: Yuzhang Chen (hubucyz@foxmail.com) This work was supported in part by the General program of Natural Science Foundation of Hubei Province under Grant 2019CFB733, and in part by the Innovation and Entrepreneurship Training Program for College Students in Hubei Province under Grant 201710512051 and Grant S201910512024. ABSTRACT In order to solve the scattering degradation by turbulence and suspended particles in underwater imaging, traditional processing methods including image enhancement, restoration and reconstruction have been continuously researched. But most of them rely on degradation models, and there exist problems of ill- posed. Image super resolution reconstruction based on deep learning has become a hot topic in recent years. In order to further improve the effectiveness and efﬁciency of deep learning based methods, an improved image super-resolution reconstruction algorithm based on deep convolutional neural network is proposed in this paper. The wavelet basis which can effectively simulate the waveform and characteristics of underwater turbulence is selected to replace the neuron ﬁtting function in order to improve the accuracy and efﬁciency of the algorithm. An improved dense block structure (IDB) is introduced into the network which can effectively solve the gradient disappearance problem of deep convolutional neural network and improve the training speed at the same time. The method proposed in this paper has been veriﬁed in laboratory ﬂume, public data set and real water body. The experimental results show that under the same conditions, the proposed algorithm shows improvements on various evaluation parameters compared with DRFN, VDSR and DRCN method. So it can be concluded that the proposed method can effectively improve the quality of deep learning based reconstruction for imaging in natural water. INDEX TERMS Convolutional neural network, super-resolution, signal to noise ratio, underwater image. X TERMS Convolutional neural network, super-resolution, signal to noise ratio, underwater image. INDEX TERMS Convolutional neural network, super-resolution, signal to noise ratio, underwat is work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLUME 8, 2020 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons II. THEORY With the gradual development of underwater imaging devices, turbulence is the bottleneck obstacle to overcome the imaging degradation in natural waters. Turbulence is a complex and irregular state of motion of ﬂuid. Most of the ocean and river are in this random state of motion. Wavelet is a short-term signal whose energy is concen- trated in a short period of time. It can effectively simulate the waveform and characteristics of turbulence and retain the edge information of image. When it is used in neural network, it can make the extraction of image information more accurate and efﬁcient. The two-dimensional discrete wavelet transform is used to transform the discrete image data into coefﬁcients in the wavelet domain. The image data are ﬁltered by selecting a reasonable threshold, and then the desired target image is obtained by inverse wavelet transform. The multi-resolution characteristic of wavelet transform is beneﬁcial to the protection of image edge details and feature extraction. Wavelet bases can be selected ﬂexibly according to different practical problems, and the corresponding pro- cessing effect can be obtained. Therefore, it is very necessary and urgent to ﬁnd a fast and effective method to process underwater image, so as to obtain images with high signal-to-noise ratio and good clarity in real-time underwater imaging. Since the rapid development of super-resolution reconstruction technology based on deep learning in recent years, this paper innovatively introduces it into underwater image restoration to improve the quality of underwater image. In recent years, image super-resolution reconstruction based on deep learning has become a research hotspot. Shen et al. [28]–[30] proposed a super-resolution recon- struction algorithm for MODIS remote sensing images, and made contributions to adaptive norm selection for regular- ized image restoration and super-resolution. Lim et al. [31] proposed EDSR method. The most signiﬁcant improvement is the removal of redundant SRResNet modules so that the size of the model could be expanded to improve the quality of the results. With the same computing resources, EDSR can stack more network layers or extract more features from each layer to get better performance. Wang et al. [32] proposed SFTGAN method, using the image segmentation mask as the prior feature condition of the super-resolution and the prior category information to solve the problem of the unreal super-resolution texture and to restore the real super-resolution texture of the image through the depth space feature transformation. Zhang et al. I. INTRODUCTION mathematical methods such as estimation [5]–[8], fusion [9], color correction [10]–[12], and the combination of depth neu- ral network [13]–[15] to optimize the quality of underwater image restoration and reconstruction. Hou et al. [16]–[20] considered the damage of scattering and optical turbulence to underwater imaging, established a degradation model of underwater imaging, and analyzed the inﬂuence of suspended particles, turbulence, and path scattering on underwater opti- cal imaging. Nootz et al. [21]–[23] established laboratory and ﬁeld underwater turbulence experimental system, and analyzed the inﬂuence of optical turbulence on the resolution of underwater imaging system through ﬁeld measurement. Matt et al. [24], [25] established a turbulent environment experimental platform with variability and reproducibility. The ﬂuid ﬁeld was analyzed by Doppler velocimeter and particle image velocimetry (PIV) system, and the mea- surement results were compensated by computational ﬂuid Imaging detection is an important research topic in marine military, underwater resource development and environmen- tal monitoring. According to previous studies, the serious degradation of underwater image quality is caused by the absorption and scattering of light, suspended particles, turbu- lence distortion and so on, among which turbulence degrada- tion is the most serious problem in natural waters. Optimizing image enhancement algorithms by setting up a degradation model is an effective way to improve the image quality on the basis of maintaining the cost of hardware. Traditional underwater image processing methods include image enhancement and restoration algorithms [1]–[4]. In recent years, scholars have introduced various The associate editor coordinating the review of this manuscript and approving it for publication was Zhan-Li Sun . 117759 Y. Chen et al.: Wavelet Based Deep Learning Method for Underwater Image Super Resolution Reconstruction dynamics model. Farwell et al. [26], [27] studied the intensity and coherent distribution of turbulence in underwater beam propagation based on the power spectrum model of ocean turbulence. of turbulence. Therefore, in this paper, a deep convolution neural network based on wavelet transform is proposed to do the training of the mapping relationship, and an improved dense block structure (IDB) is also proposed to solve the gradient disappearance problem of deep convolutional neural network and improve the training speed at the same time. However, some common problems of above algorithms are as following: (1) The relationship between noise reduc- tion and contrast enhancement cannot be well handled, which leads to inadequate noise elimination or damaged details in reconstructed images. II. THEORY [33] put for- ward RankSRGAN method and optimized SR model for perception index. They introduced ‘‘Ranker’’ to learn the behavior of perceptual indicators through ranking learning. RankSRGAN could combine the advantages of different SR methods to achieve the best performance in perception metrics and restore more realistic texture. I. INTRODUCTION (2) Digital images are mostly two-dimensional or three-dimensional digital matrices, the amount of data is very large, and the iteration time of the algo- rithm is too long to achieve real-time performance. Therefore, the classical method of underwater image processing cannot get high-quality underwater image restoration results quickly and accurately. (3) The problem of low efﬁciency and depen- dence on models limit the application scope and real-time performance. A. WAVELET RECONSTRUCTION FUNCTION Hussain decomposes turbulence signals into Schematic structure of improved dense block (IDB). The output of neural network can be expressed by: The output of neural network can be expressed by: B. IMAGE SUPER RESOLUTION RECONSTRUCTION BASED ON WAVELET DEEP CONVOLUTION NEURAL NETWORK B′(X) = Ac( X i∈Mj xl−1 i × al ij + bl j) (7) (7) (7) The structure of the improved deep intensive convolution neural network designed in this paper is shown in Figure. 1. where xi denotes the values of the pixels in the feature graph, l is the number of the convolution layer, i and j are the position of the pixels, Ac is the activation function for the neural net- work, M is the convolution core, a and b are the weights and constant biases in the convolution core respectively, which are set as scale factor and shift factor of the turbulent coherent structure function in Eq.5 respectively. FIGURE 1. Structure of the wavelet deep convolution neural network. In the training process, the sample image is ﬁrst reduced and then enlarged by interpolation. Activation function is used to assist neuron convolution function to describe the modeling process, which is represented by a and b. And loss function is used to modify these two parameters. In the process of neuron convolution, the convolution nucleus is used to partition, and the ﬁtting function of neuron convo- lution modeling can be expressed by Eq.7. Residual func- tion can be used as loss function, which is guided by the method proposed in VDSR [3]. The image is compensated by zero before each convolution in the convolution neural network proposed in this paper, which ensures that all the feature maps and the ﬁnal output image are consistent in size, and avoids the problem that the image will become smaller and smaller through gradual convolution. Residual structure connects shallow and deep convolution layers by cross-layer in deep network, making the convolution layer ﬁt for the residual of feature map, which greatly reduces the computational complexity of training process, and also reduces the gradient dispersion phenomenon. FIGURE 1. Structure of the wavelet deep convolution neural network. The improved network consists of two main parts: The improved network consists of two main parts: I(X) = A(X) + B(X) (6) (6) where LR is the low-resolution image, HR is the high- resolution image, I(X) is the ﬁnal reconstructed image, X is the captured low resolution image, A(X) is the low-frequency part of the image, which is obtained by bi-cubic interpolation of low-resolution image, B(X) is the high-frequency part of the image, which represents the output image processed by deep convolution neural network. A. WAVELET RECONSTRUCTION FUNCTION Hussain decomposes turbulence signals into f (x, t) = f (x) + fc(x, t) + fr(x, t) (1) (1) where f (x) represents the average time, fc and fr denotes the coherent and the incoherent part of the turbulent structure, among which the energy of turbulence signal is concentrated in coherent structure. Therefore, the coherent structure of turbulence plays an important role in understanding the gen- eration and change of turbulence. 2 The continuous wavelet transform of a signal x(t) ∈L2(R) can be expressed as: Xω(a, b) = 1 √\|a\| Z +∞ −∞ x(t)ψ t −b a dt (2) (2) Its reconstruction (inverse transformation) formula is as follows: Its reconstruction (inverse transformation) formula is as follows: X(t) = Z +∞ 0 Z +∞ −∞ 1 a2 Xω(a, b) 1 √\|a\|ψ t −b a dadb (3) (3) where a is the scale factor that decides the scaling scale of the wavelet. In signal processing, a is used to control the frequency of the wavelet. b is a translation factor which can delay the wavelet signal to the region of interest in the analysis. The wavelet bases for translation and scaling is: However, the common image super-resolution algorithms are ‘‘blind’’ to image processing, which means that the algorithm does not know the defects of the images. Gen- erally, the underwater image is most seriously affected by turbulence. Because of the ﬂexible transformation property of wavelet base, it can simulate the wave form and char- acteristics of turbulence well and reﬂect the randomness ψa,b(t) = 1 √\|a\|ψ t −b a , a, b ∈R, a ̸= 0 (4) (4) 117760 VOLUME 8, 2020 Y. Chen et al.: Wavelet Based Deep Learning Method for Underwater Image Super Resolution Reconstruction FIGURE 2. Schematic structure of improved dense block (IDB). Due to its good autocorrelation performance and low cross- correlation, orthogonal Harr wavelet and Morlet wavelet are used for reconstruction in order to distinguish adjacent har- monics effectively. The coherent structures of turbulence at speciﬁc scales a and b can be obtained by: X(t, a) = Z +∞ −∞ 1 Hψ1ψ2 Wψ1X (t) (b, a) ψ2a,b (t) 1 adb (5) (5) where ψ1 is Morlet wavelet, ψ2 is Harr wavelet, Hψ1ψ2 is a constant related only to the selected wavelet basis. So as long as the speciﬁc scale is determined, the coherent structure of turbulence can be reconstructed. FIGURE 2. TABLE 1. Number and size of data set. (8) During the cross-training process, the training set is divided into 10 parts, each round of training will select 9 as the training set data, the remaining one will be used as the test set to adjust the network parameters. Each round of veriﬁcation set is different, so 10 rounds of cycle constitute cross-training. Through this way, we can effectively select more favorable data sets for network training, so as to prevent the overall effect of the network from being affected by poor performance of some training sets. where N is the number of training samples, Zi is the output high resolution images, Xi is the ith low-resolution input image, r is the residual image information of the standard high-resolution output and low-resolution input. B. IMAGE SUPER RESOLUTION RECONSTRUCTION BASED ON WAVELET DEEP CONVOLUTION NEURAL NETWORK As the low-frequency information carried by the low-resolution image is similar to the low-frequency information of the high-resolution image, it takes a lot of time to carry this part in training. So, we only need to study the high-frequency residual between the high-resolution image and the low-resolution image. Thus the network constructed in this paper can effectively avoid repeated learning of low-frequency part, thus speeding up the convergence of the model. where LR is the low-resolution image, HR is the high- resolution image, I(X) is the ﬁnal reconstructed image, X is the captured low resolution image, A(X) is the low-frequency part of the image, which is obtained by bi-cubic interpolation of low-resolution image, B(X) is the high-frequency part of the image, which represents the output image processed by deep convolution neural network. As the low-frequency information carried by the low-resolution image is similar to the low-frequency information of the high-resolution image, it takes a lot of time to carry this part in training. So, we only need to study the high-frequency residual between the high-resolution image and the low-resolution image. Thus the network constructed in this paper can effectively avoid repeated learning of low-frequency part, thus speeding up the convergence of the model. The middle layer of residual network is improved by intro- ducing an improved dense block structure. The input of each layer of the network is the union of the output of all the layers in front of it. And the feature maps learned by that layer will be passed directly to the back of all layers as input. In this paper, an improved dense block structure (IDB) is proposed by combining residual block with dense block structure. Figure 1 shows the deep convolution neural network applied to learning the high-frequency part of image informa- tion. It consists of four parts, including convolution layer for learning low-level features, which contains two Conv convo- lution layers; improved dense blocks for learning high-level features including 12 IDBs; the fusion layer used to fuse the intensive features of learning; reconstructed blocks for gen- erating high frequency features, including an upper sampling layer and a convolution layer. The input low-resolution image is basically consistent with the low-frequency part of the corresponding high-resolution image, which results that the pixel values of the residual image are mostly very small. In order to learn the high 117761 VOLUME 8, 2020 Y. C. ESTABISHMENT AND TRAINING OF SELF-BUILT DATA SETS In order to ensure the diversity of experimental data, the images for training were captured in the Yangtze River to form self-built data sets. Like the images in the exper- iment, the self-built data set is affected by the underwater turbulence. Therefore, the training of self-built data set can make the network learn the information characteristics of underwater image well. Figure 3 A1 and A2 are the images of test targets 1 and 2 in the water of the Yangtze River, and Figure 3 B1 and B2 are the images of test targets 1 and 2 in the clear water. It can be seen that the image quality in clear water is relatively high, and there is basically no noise, while the image in the Yangtze River water contains more noise, and the resolution is very low. In this paper, a large number of datasets for training are collected to solve the difﬁculty of data acquisition. The aim of data set training is to ﬁnd the least summa- tion of the average Euclidean distances between the original high-resolution image and the reconstructed image: W, b = arg min W,b 1 2N Zi − ⌢Zi 2 2 (9) (9) Peak signal to noise ratio (PSNR) and Structural similar- ity (SSIM) are the two most widely used indicators to measure the quality of image reconstruction. The former quantitatively calculates the errors between the processed results and the original image. Higher PSNR means less distortion, while the closer the SSIM approaches 1, the better the restored results. Therefore, this paper objectively evaluates the advantages and disadvantages of various reconstruction methods by using these two indicators for laboratory experiments. However, in the ﬁeld experiments, there is no reference image, PSNR and SSIM cannot be used. So we select the blur metric (BM) [34], [35], grayscale mean gradient (GMG) and Lapla- cian sum (LS) [34] as the evaluation indexes. The smaller the BM value, the clearer the image, and the higher the GMG and LS values, the higher the image quality. FIGURE 3. Sample images of the self-built data sets. The BM is deﬁned as follows: BM = max(sDvertical, sDhorizontal), sDvertical = m−1,n−1 X i,j=1 Dvertical(i, j), sDhorizontal = m−1,n−1 X i,j=1 Dhorizontal(i, j), i ∈(0, m −1), j ∈(0, n −1), ( Dvertical = \|F(i, j) −F(i −1, j)\| Dhorizontal = \|F(i, j) −F(i, j −1)\| (10) FIGURE 3. B. IMAGE SUPER RESOLUTION RECONSTRUCTION BASED ON WAVELET DEEP CONVOLUTION NEURAL NETWORK Chen et al.: Wavelet Based Deep Learning Method for Underwater Image Super Resolution Reconstruction frequency part end-to-end mapping function F, the idea of residual learning is adopted in the dense convolutional neural network for training high frequency part information. The loss function H(λ) of the network model is constructed as: training, calibration and test images are shown in Table 1. The image blocks collected from these training sets will be used in feature extraction steps, and the convolutional neural network model designed in this paper will be cross-trained. H(λ) = 1 N N X i=1 (Zi −Xi) −F( lim x→∞Xi; λ) 2 = 1 N N X i=1 r −F( lim x→∞Xi; λ) 2 λ = {W1, W2, . . . , Wn, b1, b2, . . . bn} C. ESTABISHMENT AND TRAINING OF SELF-BUILT DATA SETS Sample images of the self-built data sets. In order to improve the applicability of the proposed algo- rithm, a large number of data sets for training were collected. The images before and after degradation of training target were registered to obtain sample training pairs with size of 1064 × 924. The image is ﬁrst converted into Y-channel from YCbCr color space, and then the obtained image is segmented by convolution kernels into 64 × 64 image blocks according to the step size of 10. Then the training set is expanded by rotating and mirroring. The number and size of (10) where Dvertical and Dhorizontal represent different images in the vertical and horizontal directions. F(i, j) is the pixel of 117762 117762 Y. Chen et al.: Wavelet Based Deep Learning Method for Underwater Image Super Resolution Reconstruction TABLE 2. Table of network structure parameters. TABLE 2. Table of network structure parameters. TABLE 2. Table of network structure parameters. coordinate (i, j) on the image plane, and (m, n) is the size of the image. Then the blur metric can be normalized by the range 0 to 1. GMG = 1 (M −1)(N −1) M−1 X i=1 N−1 X j=1 s α2 + β2 2 ( α = f (x, y + 1) −f (x, y) β = f (x + 1, y) −f (x, y) (11) (11) where f (x, y) denotes the point at coordinate (x, y) on image plane, and (M, N) is the size of the image. S = M−1 P i=1 N−1 P j=1 8 × f (x, y) −f (x, y −1) −f (x −1, y)− f (x + 1, y) −f (x, y + 1)− f (x −1, y −1) −f (x −1, y + 1)− f (x + 1, y −1) −f (x + 1, y + 1) (M −2)(N −2) LS = where f (x, y) denotes the point at coordinate (x, y) on the image plane, (M, N) is the size of image. III. FIELD TESTS AND EXPERIMENTAL RESULTS The training platform of algorithm is: the operating system is Ubuntu 14.04 (Canonical Ltd, London, England), the CPU is Core i7–9700K (Quad–core 4.9 GHz) (2200 Mission College Blvd. Santa Clara, CA 95054–1549 USA), and the graphics card is ASUS DUAL RTX2070–O8G–EVO (ASUS, Taipei City, Taiwan). The programming is performed in Anaconda (Austin, Texas, USA). If the computer conﬁguration is low- ered or improved, the algorithm time will increase or decrease accordingly. formed by water pump at different heights. The experimen- tal system uses a circulating pump with a maximum head of 5 meters and a maximum ﬂow of 7.8m3/h to generate ﬂuid power. The laser and sensor are 33 cm from the target plate. In order to reduce the experimental error, the experiment was carried out in dark environment. The three-dimensional structure of the experimental system is shown in Fig. 4. The captured image sequences are processed and com- pared by the proposed algorithm along with MAP-regularized robust reconstruction for underwater imaging detec- tion (ROBUST) [1], Deep Recurrent Fusion Network for Single-Image Super-Resolution With Large Factors (DRFN) [2], Accurate Image Super-Resolution Using Very Deep Convolutional Networks (VDSR) [3], Deeply-Recursive Convolutional Network for Image Super-Resolution (DRCN) [4], the codes of which are obtained from the author’s project code’s open web page. FIGURE 4. Three-dimensional structure of laboratory experiment system. The network structure parameters are shown in Table 2, where ‘‘Kernel size’’ is the size of convolution kernel, ‘‘Padding’’ is the number of layers of each input edge sup- plemented by 0, and ‘‘Bias’’ is whether to add bias. FIGURE 4. Three-dimensional structure of laboratory experiment system. TABLE 3. Experimental results of separation of wavelet and IDB. The experimental results show that in the case of weak turbulence, the image quality can be greatly improved by using wavelet alone. Using IDB alone can reduce running time of the algorithm. When both of them are used, the image quality of underwater image is the best. When the inﬂow velocity reaches 25m / s, it is seen as the strong turbulence environment, and the distortion degree of the image increases greatly. The collected sample image, the result of image restoration and reconstruction, the evalu- ation value and the running time of each algorithm are shown in Figure 7. FIGURE 5. The variances of PSNR during cross-training. It can be seen from the results in the ﬁgure that after cross training, the method in this paper has the best effect on self built data set processing. The image reconstruction results in Figure 7 reveal that the traditional DRFN method has no obvious ringing effect, but its image is as fuzzy as the ROBUST method’s. The results also show that the VDSR method still causes distortion in the strong turbulence state. However, compared with other images, the reconstruction image of the method proposed has a certain degree of fuzziness, it shows the outline of the stripe in the sample image more clearly, which makes the method more practical in the context of strong turbulence. In this paper, peak signal-to-noise ratio (PSNR) and structure similarity index (SSIM) are selected as evaluation indexes. The collected sample image, reconstructed image of each algorithm, the evaluation value of each image and the running time of each algorithm are shown in Figure 6. From the perspective of image reconstruction effect, the ROBUST and DRFN methods eliminate a certain degree of blur, but the processing results are darker, and introduce a larger ringing effect. VDSR improves the ringing effect, while the image distortion is not improved. DRCN improves the resolution of the image, while the color of the image is still dark, and the distortion has not been substantially improved. It can be seen that image restoration and reconstruction algo- rithms have signiﬁcant effect in improving resolution and de blurring, they cannot effectively correct image distortion. Our proposed method can not only eliminate certain image noise, but also signiﬁcantly improve image distortion, which shows the efﬁciency of this algorithm. A. LABORATORY EXPERIMENTS By controlling the velocity of water ﬂow in the water tank, turbulence of different intensity can be obtained. The ﬂow meter can read the velocity in real time, and then calculate the turbulent Reynolds number and turbulence intensity to ensure that the sample image can be obtained in the turbulent environment. When the water velocity of the intake is 5m/s, the target object is photographed 60 times by CCD sensor in 5 seconds, which is considered as the micro turbulence environment. In this paper, the experimental system of underwater turbu- lence is established with 532 nm green semiconductor laser as the light source; high-speed CMOS image sensor is used to collect images. The laser spot size is 10-20 mm and the power is 200 MW. The experimental water tank is made of acrylic plate with high transmittance. More than 90% of the laser source is irradiated on the target plate. Its size is 150cm × 34cm × 33cm (length, height and width). The inlet and outlet are all 40mm round holes, and turbulent ﬂow is 117763 VOLUME 8, 2020 Y. Chen et al.: Wavelet Based Deep Learning Method for Underwater Image Super Resolution Reconstruction In order to compare the effect of adding wavelet basis and IDB to the underwater image processing respectively, we use the above image experiments, and the results are shown in table 3. Self-built data sets are used to cross train each algorithm. In the equation 5, a is set as 0.6745 and b as 0.9875. The vari- ances of PSNR during cross-training are shown in Figure 5. FIGURE 5. The variances of PSNR during cross-training. TABLE 3. Experimental results of separation of wavelet and IDB. TABLE 3. Experimental results of separation of wavelet and IDB. Super-resolution result of our method compared with existing algorithms in strong turbulence environment. time can make the network achieve the best results in under- water image processing. The sample images, reconstruction results, evaluation val- ues and running time of each algorithm from the two datasets are shown in Figure 8. TABLE 3. Experimental results of separation of wavelet and IDB. According to the evaluation results, the PSNR values of VDSR method are larger than those of ROBUST method and DRFN method. Given strong turbulence, the PSNR value of DRCN method is relatively large, while its SSIM value is small, and the SSIM value of the method proposed in this paper is the largest. In comparison to other algorithms, the method proposed still has obvious advantages in processing time. The results show that the method proposed in this paper is superior to other methods in image distortion, which has a greater improvement in image resolution and clarity. In the strong turbulence environment, we separate the wavelet basis and IDB, and the results are shown in Table 4. According to the evaluation results, the SSIM values of DRCN are larger than those of ROBUST method and DRFN method, but only to a small extent. The SSIM value of the image processed by proposed method is the largest, followed by that of the VDSR-processed one. However, the PSNR value of VDSR method is small, which indicates that the distortion gets worse after image processing. TABLE 4. Experimental results of separation of wavelet and IDB. TABLE 4. Experimental results of separation of wavelet and IDB. In terms of run-time, the method proposed in this paper has the shortest time and obvious advantages. Therefore, the method proposed is superior to the other four methods in reconstruction quality and running speed, which is suitable for underwater detection and related appli- cations in low turbulence state. The experimental results show that both wavelet basis and IDB can improve the performance of the network in strong or weak turbulence environment, but using them at the same 117764 VOLUME 8, 2020 Y. Chen et al.: Wavelet Based Deep Learning Method for Underwater Image Super Resolution Reconstruction FIGURE 6. Super-resolution result of our method compared with existing algorithms in micro turbulence environment. FIGURE 6. Super-resolution result of our method compared with existing algorithms in micro turbulence environment. FIGURE 7. Super-resolution result of our method compared with existing algorithms in strong turbulence environment. FIGURE 6. Super-resolution result of our method compared with existing algorithms in micro turbulence environment. FIGURE 6. Super-resolution result of our method compared with existing algorithms in micro turbulence environment. FIGURE 7. Super-resolution result of our method compared with existing algorithms in strong turbulence environment. FIGURE 7. B. PUBLIC DATASETS EXPERIMENTS The experimental results demonstrate that the image reconstruction results of the ROBUST method and the DRFN method are relatively poor, while the VDSR method, the DRCN method and the proposed one show better out- comes. In the milk dataset and the Chlorophyll dataset, the method proposed runs better than other methods, because In order to further verify the effectiveness of the algorithm in this paper, we reconstructed images from the milk dataset with turbidity of 15 and the Chlorophyll dataset with turbidity of 21 from the TURBID Dataset [36], and compared with the other algorithms. 117765 VOLUME 8, 2020 Y. Chen et al.: Wavelet Based Deep Learning Method for Underwater Image Super Resolution Reconstruction FIGURE 8. Public datasets super-resolution result of our method compared with existing algorithms. FIGURE 8. Public datasets super-resolution result of our method compared with existing algorithms. FIGURE 9. Framework of underwater experimental detection system. the datasets are mainly generated for turbidity. According to the paper on datasets, the generation of turbidity mainly affects the scattering. Therefore, the robust light scattering model and the light scattering model considering particle scattering will get better recovery effect. In the turbulent state, neither the ROBUST method nor the DRFN method nor the measurement model depending on the measurement parameters can achieve good results. The method proposed in this paper is applicable to both turbulent and particle scattering conditions, so it is more prac- tical. This method can provide guidance on how to reduce the inﬂuence of turbulent environment on underwater imaging. REFERENCES TABLE 5. Physical properties of experimental system. TABLE 5. Physical properties of experimental system. [1] Y. Chen and K. Yang, ‘‘MAP-regularized robust reconstruction for underwater imaging detection,’’ Optik, vol. 124, no. 20, pp. 4514–4518, Oct. 2013. [2] X. Yang, H. Mei, J. Zhang, K. Xu, B. Yin, Q. Zhang, and X. Wei, ‘‘DRFN: Deep recurrent fusion network for single-image super-resolution with large factors,’’ IEEE Trans. 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(SmartWorld/SCALCOM/UIC/ATC/CBDCom/IOP/SCI), San Francisco, CA, USA, Aug. 2017, pp. 4–8. C. FIELD EXPERIMENTS Experiments on turbulent water environment in the East Lake, Yangtze River and South China Sea were carried out. The sample image is collected by the underwater packaging FIGURE 9. Framework of underwater experimental detection system. 117766 VOLUME 8, 2020 Y. Chen et al.: Wavelet Based Deep Learning Method for Underwater Image Super Resolution Reconstruction FIGURE 10. Super-resolution result of our method compared with existing algorithms in field experiment FIGURE 10. Super-resolution result of our method compared with existing algorithms in field experiment. 117767 Y. Chen et al.: Wavelet Based Deep Learning Method for Underwater Image Super Resolution Reconstruction However, it is undeniable that the training set used in this method is based on underwater images, so its extensibility needs to be studied. Therefore, the next step should focus on studying the applicability of this method in other ﬁelds to improve the universality of this method. imaging system, as shown in Figure 9. The laser and CMOS image sensors working at 465nm are encapsulated in a water-proof water tank, and the image is transmitted to the image processing module by the image sensor. Table 5 shows the physical characteristics of the experimental system. imaging system, as shown in Figure 9. The laser and CMOS image sensors working at 465nm are encapsulated in a water-proof water tank, and the image is transmitted to the image processing module by the image sensor. Table 5 shows the physical characteristics of the experimental system. TABLE 5. Physical properties of experimental system. TABLE 5. Physical properties of experimental system. IV. CONCLUSION Based on the idea of deep learning, an improved deep- intensive convolution neural network is proposed in this paper. The main innovation is that the wavelet basis is intro- duced into the deep learning convolution kernel based on the turbulence structure, and an improved dense block structure is proposed. The experimental results show that, when there is reference image, the value of PSNR and SSIM is effectively improved; when there is no reference image, the values of BM, GMG and LS are also improved. Therefore, it can be concluded that the proposed method can effectively improve the effect of depth neural network in the super resolution reconstruction of imaging in turbulent water. [16] W. Hou, Z. Lee, and A. D. Weidemann, ‘‘Why does the Secchi disk disappear? An imaging perspective,’’ Opt. Express, vol. 15, no. 6, pp. 2791–2802, 2007. [17] W. Hou, D. J. Gray, A. D. Weidemann, and R. A. Arnone, ‘‘Comparison and validation of point spread models for imaging in natural waters,’’ Opt. Express, vol. 16, no. 13, pp. 9958–9965, Jun. 2008. [18] W. Hou, ‘‘A simple underwater imaging model,’’ Opt. Lett., vol. 34, no. 17, pp. 2688–2690, 2009. [19] W. Hou, E. Jarosz, S. Woods, W. Goode, and A. Weidemann, ‘‘Impacts of underwater turbulence on acoustical and optical signals and their linkage,’’ Opt. Express, vol. 21, no. 4, pp. 4367–4375, 2013. [20] W. Hou, S. Woods, E. Jarosz, W. Goode, and A. Weidemann, ‘‘Optical turbulence on underwater image degradation in natural environments,’’ Appl. Opt., vol. 51, no. 14, pp. 2678–2686, 2012. 117768 117768 VOLUME 8, 2020 Y. Chen et al.: Wavelet Based Deep Learning Method for Underwater Image Super Resolution Reconstruction Y. Chen et al.: Wavelet Based Deep Learning Method for Underwater Image Super Resolution Reconstruction [21] G. Nootz, W. Hou, F. R. Dalgleish, and W. T. Rhodes, ‘‘Determination of ﬂow orientation of an optically active turbulent ﬁeld by means of a single beam,’’ Opt. Lett., vol. 38, no. 13, pp. 2185–2187, 2013. YUZHANG CHEN was born in 1984. He received the bachelor’s, master’s, and Ph.D. degrees from the Huazhong University of Science and Tech- nology. He is currently an Associate Professor and a Master Tutor with the School of Computer and Information Engineering, Hubei University. He has been engaged in research on photoelectric imaging detection, photoelectric information per- ception, image processing, and embedded devel- opment for a long time. IV. CONCLUSION His main research contents include laser and LED in water, night vision or underwater scattering medium radiation transmission theory and computer simulation, image acquisition and restoration and reconstruction algorithms, and image processing algo- rithms embedded including the research of android development. [22] G. Nootz, E. Jarosz, F. R. Dalgleish, and W. Hou, ‘‘Quantiﬁcation of optical turbulence in the ocean and its effects on beam propagation,’’ Appl. Opt., vol. 55, no. 31, pp. 8813–8820, 2016. [23] G. Nootz, S. Matt, A. Kanaev, K. P. Judd, and W. Hou, ‘‘Experimental and numerical study of underwater beam propagation in a Rayleigh–Bénard turbulence tank,’’ Appl. Opt., vol. 56, no. 22, pp. 6065–6072, 2017. turbulence tank,’’ Appl. Opt., vol. 56, no. 22, pp. 6065–6072, [24] S. Matt, W. Hou, S. Woods, W. Goode, E. Jarosz, and A. Weidemann, ‘‘A novel platform to study the effect of small-scale turbulent density ﬂuctuations on underwater imaging in the ocean,’’ Methods Oceanogr., vol. 11, pp. 39–58, Dec. 2014. [25] S. Matt, W. Hou, W. Goode, and S. Hellman, ‘‘Introducing SiTTE: A con- trolled laboratory setting to study the impact of turbulent ﬂuctuations on light propagation in the underwater environment,’’ Opt. Express, vol. 25, no. 5, pp. 5662–5683, 2017. KANGLI NIU is currently pursuing the degree in electronic information engineering with the School of Computer and Information Engineering, Hubei University. His main research directions are image processing and computer vision. At present, he has done a lot of research on image recognition and processing in the laboratory, and accumulated a lot of professional knowledge and experimental experience. [26] N. Farwell and O. Korotkova, ‘‘Intensity and coherence properties of light in oceanic turbulence,’’ Opt. Commun., vol. 285, no. 6, pp. 872–875, Mar. 2012. [27] N. H. Farwell and O. Korotkova, ‘‘Multiple phase-screen simulation of oceanic beam propagation,’’ Proc. SPIE, Int. Soc. Opt. Eng., vol. 9224, Oct. 2014, Art. no. 922416. [28] H. Shen, M. K. Ng, P. Li, and L. Zhang, ‘‘Super-resolution reconstruction algorithm to MODIS remote sensing images,’’ Comput. J., vol. 52, no. 1, pp. 90–100, Feb. 2008. [29] H. Shen, L. Zhang, B. Huang, and P. Li, ‘‘A MAP approach for joint motion estimation, segmentation, and super resolution,’’ IEEE Trans. Image Pro- cess., vol. 16, no. 2, pp. 479–490, Feb. 2007. ZHANGFAN ZENG received the B.S., M.S., and Ph.D. IV. CONCLUSION degrees from Wuhan University, China, The University of Manchester, U.K., and the Univer- sity of Birmingham, U.K., in 2006, 2007, and 2013, respectively, all in communication engi- neering. Since December 2015, he has been a full-time Associated Professor with Hubei Uni- versity, China. He has published over 20 research articles and one patent in radar community. His research areas are wireless communication and digital signal processing. [30] H. Shen, L. Peng, L. Yue, Q. Yuan, and L. Zhang, ‘‘Adaptive norm selection for regularized image restoration and super-resolution,’’ IEEE Trans. Cybern., vol. 46, no. 6, pp. 1388–1399, Jun. 2016. [31] B. Lim, S. Son, H. Kim, S. Nah, and K. M. Lee, ‘‘Enhanced deep residual networks for single image super-resolution,’’ in Proc. IEEE Conf. Comput. Vis. Pattern Recognit. Workshops (CVPRW), Jul. 2017, pp. 136–144. [32] X. Wang, K. Yu, C. Dong, and C. Change Loy, ‘‘Recovering realistic texture in image super-resolution by deep spatial feature transform,’’ in Proc. IEEE/CVF Conf. Comput. Vis. Pattern Recognit. (CVPR), Jun. 2018, pp. 606–615. pp [33] W. Zhang, Y. Liu, C. Dong, and Y. Qiao, ‘‘RankSRGAN: Generative adversarial networks with ranker for image super-resolution,’’ in Proc. IEEE/CVF Int. Conf. Comput. Vis. (ICCV), Oct. 2019, pp. 3096–3105. YONGCAI PAN (Fellow, IEEE) is currently a Full Professor and the Head of the Laboratory of Signal Processing and System Analysis, Hubei Univer- sity. His research areas include wireless communi- cation, signal processing, and intelligent systems. He has extensive exposure and experience in industrial projections. [34] Y. Chen and J. Chen, ‘‘Spatial adaptive regularized MAP reconstruc- tion for LD-based night vision,’’ Optik, vol. 125, no. 13, pp. 3162–3165, 2014. [35] Y. Chen, W. Yang, H. Tan, Y. Yang, N. Hao, and K. Yang, ‘‘Image enhancement for LD based imaging in turbid water,’’ Optik, vol. 127, no. 2, pp. 517–521, Jan. 2016. [36] A. Duarte, F. Codevilla, J. D. O. Gaya, and S. S. C. Botelho, ‘‘A dataset to evaluate underwater image restoration methods,’’ in Proc. OCEANS Shanghai, Shanghai, China, Apr. 2016, pp. 1–6. 117769 VOLUME 8, 2020
https://openalex.org/W3154916847	https://baas.aas.org/pub/2021n4i376/download/pdf	English	null	Cracks emerging in NASA’s Discovery Program	null	2,021	cc-by	1,141	Cracks Emerging in NASA’s Discovery Program Cracks Emerging in NASA’s Discovery Program Cracks Emerging in NASA’s Discovery Program John Dove Astronomy and Planetary Research Institute (202) 358-0000 John.aa.dove@gmail.com Discovery is one of NASA’s most successful programs. It has funded ground-breaking missions such as Mars Pathfinder, MESSENGER, Dawn, and Kepler. Unfortunately, overly simplistic narratives and storytelling in recent Discovery proposals is now leading to unrealistic science goals and much lower return on investment. Problems with some of the most recent Discovery missions and some simple remedies are discussed below. InSight (Interior Exploration using Seismic Investigations, Geodesy and Heat Transport): Although sold as “Revealing the Heart of Mars” (Figure 1), with objectives of measuring the martian core radius and the planet’s heat flux, the seismic station has failed to detect anything remotely capable of determining the core radius. The heat flow investigation has been, to date, a failure. The only major discovery so far is that Mars is a “seismically active world” – unsurprising and unworthy of a $1B investment. Psyche: Although sold as a “Journey to a Metal World” (Figure 1), with objectives of determining how an iron world forms, it was known even before selection time that there was a very large chance that the asteroid Psyche is not a metal world. The best estimate for its density is now 3400-4100 kg m-3 [Elkins-Tanton et al. 2020]. These values overlap with ordinary chondrites and the recently visited asteroid 21 Lutetia. The density of iron meteorites is >7800 kg m-3. The mission’s STM and iron-core science objectives are almost irrelevant now. It is uncertain if the science from this body should be a top priority of the United States. Everybody can agree that it would be very interesting to send a suite of instruments to orbit any of the unexplored asteroids. But the point of the Discovery competition is to determine which investigations are the most compelling. If Psyche is now an exploratory survey, instead of a set of measurements to test a specific set of hypotheses tailored for a unique body, the proposed 2015 Mantis asteroid mission to survey nine new worlds would have been more compelling. Mantis does not have a catchy tag line. Discovery 2019 selection in still underway but there are again red flags: Trident: Flying a nearly identical mission profile as Voyager 2, this mission advanced to the next round of competition despite its Category 2 rating. Cracks Emerging in NASA’s Discovery Program By advancing a Category 2 mission, NASA’s message is that achieving the highest quality science is not a main driver for Discovery. Proposing centers have usually worked to perfect the STM, but this will no longer be the top priority. Instead, proposing centers will continue to reach for ever more enticing, easy to digest “story lines” despite their lack of realism. More achievable or compelling science investigations may not even advance to the proposal stage. Could the problems of InSight, Psyche, and Trident have been avoided? InSight: InSight’s objective was extraordinarily ambitious - perform teleseismic science with a single station on a planet with unknown seismicity. There is almost no precedent for performing deep interior seismic investigations with a single station, without making many other assumptions that cannot yet be made for Mars. The mission was relying on extreme luck to be successful. An alternative path forward would have been a lower cost, lower quality wind-protected ground-coupled seismometer flying on another Mars mission(s), such as rovers, to perform preliminary measurements of seismicity, over several months. This could have provided the information needed to design a proper network to actually measure the core radius. Psyche: As with InSight, the most fundamental assumption turned out to be wrong. Estimates of Psyche’s density ranged from 2500-7,000 kg m-3 at the time of proposal submission [Elkins-Tanton et al. 2020 Figure 2]. An alternative path forward would have been to perform follow up measurements to better estimate Psyche’s density. Very high density asteroids could have been found, and then the United States could have actually had a mission to a real metal world. These comments are not meant to speak negatively of the mission teams, as they are composed of some of the world’s top scientists. Recommendations The most basic assumptions of these missions could have been demonstrated to be untenable at the time of proposal. Recommendations #1. Provide significant amounts of funding, $20-50M, to de-risk Step 1 Discovery proposals deemed promising, but are too high-risk to down select. To summarize: -For InSight, a low-cost short-lived seismometer could have been deployed to the surface on a rover mission(s) for preliminary studies at a fraction of the Insight mission cost. France could have still provided the instrument. -For Psyche, fund ground-based observations that would have provided superior error bars on the densities of numerous candidate metal worlds. -For Psyche, fund ground-based observations that would have provided superior error bars on the densities of numerous candidate metal worlds. -For Trident, fund modeling studies that could have been used to alleviate the science risks. This recommendation allows reviewers a chance to decline a proposal while still affirming its importance. This recommendation allows reviewers a chance to decline a proposal while still affirming its importance. #2. Reviewers should be trained to avoid consuming Hollywood story-telling, and to question the most basic assumptions about the mission. Most cutting-edge science is complex, uncertain, and cannot be reduced to a tweet or tag line. On the contrary, if it has been so reduced, one should often be skeptical. #2. Reviewers should be trained to avoid consuming Hollywood story-telling, and to question the most basic assumptions about the mission. Most cutting-edge science is complex, uncertain, and cannot be reduced to a tweet or tag line. On the contrary, if it has been so reduced, one should often be skeptical. Hundreds of scientists obtain their salaries from the Discovery program, including those on the Decadal Survey board, making it difficult to find critics. An honest assessment is important, however, if we want to again fly missions that offer the best possible return to the United States, such as Pathfinder, MESSENGER, Dawn, and Kepler. Figure 1. Artwork for the InSight and Psyche missions. The most basic assumptions of these missions could have been demonstrated to be untenable at the time of proposal. References Observations, Meteorites, and Models: A Preflight Assessment of the Composition and Formation of (16) Psyche, L. T. Elkins-Tanton et al. (2020). Journal of Geophysical Research: Planets, 125, e2019JE006296. https://doi.org/10.1029/2019JE006296 Figure 1. Artwork for the InSight and Psyche missions. The most basic assumptions of these missions could have been demonstrated to be untenable at the time of proposal. Figure 1. Artwork for the InSight and Psyche missions. References Observations, Meteorites, and Models: A Preflight Assessment of the Composition and Formation of (16) Psyche, L. T. Elkins-Tanton et al. (2020). Journal of Geophysical Research: Planets, 125, e2019JE006296. https://doi.org/10.1029/2019JE006296
https://openalex.org/W4380684897	http://anthro-age.pitt.edu/ojs/anthro-age/article/download/475/639	English	null	Book Review: Life Writing in the Posthuman Anthropocene	Anthropology & aging	2,023	cc-by	2,097	Book Review Review of Batzke, Ina, Lea Spinoza Garrido, and Linda M. Hess, eds. Life Writing in the Posthuman Anthropocene. Cham, Switzerland: Palgrave Macmillan. 2022. pp. 268. Price: $139 (Hardcover); $109 (eBook). Book Review Review of Review of Batzke, Ina, Lea Spinoza Garrido, and Linda M. Hess, eds. Life Writing in the Posthuman Anthropocene. Cham, Switzerland: Palgrave Macmillan. 2022. pp. 268. Price: $139 (Hardcover); $109 (eBook). Review of Review of Batzke, Ina, Lea Spinoza Garrido, and Linda M. Hess, eds. Life Writing in the Posthuman Anthropocene. Cham, Switzerland: Palgrave Macmillan. 2022. pp. 268. Price: $139 (Hardcover); $109 (eBook). João Pedro Martinez Pinheiro João Pedro Martinez Pinheiro Pontifical Catholic University of Rio Grande do Sul 1joaopedromp@gmail.com João Pedro Martinez Pinheiro Pontifical Catholic University of Rio Grande do Sul 1joaopedromp@gmail.com Life Writing in the Posthuman Anthropocene illustrates the relevance of posthumanist accounts of the life course in the present-day context of ecological crisis. Past collaborators Ina Batzke and Linda M. Hess (Batzke et al. 2018) are here joined by Lea Espinoza Garrido in presenting an analytically-sophisticated and politically-poignant, yet often entertaining, collection of seven essays and one interview. In addition to their Introduction, these texts show how well the editors’ shared interests in auto/biography, queerness, gender, popular culture, and migration studies synergize with the environmental humanities, a novel dimension in their respective bibliographies. The eight contributions are equally distributed across two sections—“Responsible Relationality” and “Relational Responsibility”—that highlight two general approaches to the following posthumanist concern: how could life writing, the literary epitome of humanism, support more responsible/decentralized human-environmental relations, given that for centuries humanist philosophy helped justify the anthropocentric attitudes that caused the ecological crisis? Katja Sarkowsky frames this concern as a paradox in the opening essay, “Relationality, Autobiographical Voice, and the Posthuman Paradox: Decentering the Human in Leslie Marmon Silko’s Life Writing.” Her case study of Silko’s memoirs, letters, essays, and photobooks uncovers a type of autobiographical storytelling that allows human and nonhuman elements to interface without breaching each other’s ontological boundaries, for example without anthropomorphizing the “other-than-human” (25). To attempt such a breach would only result in an unproductive paradox. Instead, the author argues, this renowned Native-American writer scrutinizes both her material and discursive dependency upon human-nonhuman relations and her own privileged/human-centric position within said relations. João Pedro Martinez Pinheiro Pontifical Catholic University of Rio Grande do Sul 1joaopedromp@gmail.com Anthropology & Aging, Vol 44, No 1 (2023), pp. 118-120 ISSN 2374-2267 (online) DOI 10.5195/aa.2023.475 Anthropology & Aging, Vol 44, No 1 (2023), pp. 118-120 ISSN 2374-2267 (online) DOI 10.5195/aa.2023.475 Book Review \| Pinheiro \| 118 Book Review \| Pinheiro \| 118 Anthropology & Aging Vol 44 No 1 (2023) ISSN 2374-2267 (online) DOI 10.5195/aa.2023.475 http://anthro-age.pitt.edu Book Review For example, when Liptrot’s and her father’s emotional swings are characterized by herself and White as analogous to the archipelago’s extreme weather, this is a nice allegory, but not an instance of more-than-human entanglement. Even the titular outrun, “the rough grazing land on the furthest reaches of a property [...] where domestic and wild animals co-exist” (103), eventually becomes a metaphor for Liptrot’s anxious, though adventurous, personality. When the environment, then, is a mere mirror to one’s soul, is this so different from old nature writing? In “Edges and Extremes in Ecobiography: Amy Liptrot’s The Outrun” Jessica White extols works that, like Liptrot’s, critically update the historically anthropocentric and imperialistic nature writing genre. Echoing a prime posthumanist concern, she asks whether nature is a mere “backdrop [...] to the human world” (115), or are they co-constitutive? For White, Liptrot’s ultimately human struggles with alcoholism and homesickness are nonetheless environmentally contingent. They manifested when she moved to the big city in an attempt to flee the cold, rural isolation of the Scottish archipelago of her youth and the stress caused by her father’s bipolar disorder. Furthermore, Liptrot overcomes her addiction only after reconnecting with her home by farming, ocean swimming, and protecting its endangered bird species. However, as compelling as texts like The Outrun and interpretations like White’s are, “the relationships between a human and their ecosystem” (100) that are presented here often do not exceed the level of metaphor and analogy. For example, when Liptrot’s and her father’s emotional swings are characterized by herself and White as analogous to the archipelago’s extreme weather, this is a nice allegory, but not an instance of more-than-human entanglement. Even the titular outrun, “the rough grazing land on the furthest reaches of a property [...] where domestic and wild animals co-exist” (103), eventually becomes a metaphor for Liptrot’s anxious, though adventurous, personality. When the environment, then, is a mere mirror to one’s soul, is this so different from old nature writing? Finishing the first section, we encounter what is arguably the volume’s most creative piece. “The Sentience of Sea Squirts” is a first-person account whereby poet Clare Brant combines her ocean diving anecdotes with kaleidoscopic philosophical musings. Inspired by “those questions of scale” that accompany “Anthropocene-minded criticism” (124), Brant introduces us to the tiny lives of sea hares, marine snails, and sea squirts. Anthropology & Aging Vol 44 No 1 (2023) ISSN 2374-2267 (online) DOI 10.5195/aa.2023.475 http://anthro-age.pitt.edu Book Review Sarkowsky’s comparative methodology shines particularly bright when she traces the effects of place upon the memoirist’s narrative voice across and within her works: hence there is Silko, the host at the “storied spaces” (40) of her ancestral land in New Mexico, retelling tales from the past, but also Silko, the explorer of her new estate in Arizona, creating stories in the present. The second contribution to this volume is Christina Caupert’s “The Big Picture: Life as Sympoietic Becomings in Rachel Rosenthal’s Performance Art.” Like Sarkowsky’s essay, this one takes the reader on an analytical tour de force through decades-worth of work by a profound female artist. The French- born daughter of Jewish Russians who fled the 1917 Revolution, Rosenthal was, for Caupert, a precocious posthumanist ecofeminist. Caupert reconstructs the philosophical progression of Rosenthal’s one-woman shows from their earlier, feminist iterations (which critiqued individualist, stable, pre-given identities) to their latter-day, posthumanist ones (which often featured untrained Anthropology & Aging Vol 44 No 1 (2023) ISSN 2374-2267 (online) DOI 10.5195/aa.2023.475 http://anthro-age.pitt.edu 119 Book Review \| Pinheiro \| animals and dystopian sci-fi elements). Although illustrations would have been helpful here (supposing that they even exist), Caupert’s vivid descriptions of these “collective sensory, affective, and intellectual journeys” (57) nonetheless convey how topics like interspecies companionship, war, gender norms, and technocratic capitalism collided on Rosenthal’s stage as she openly processed autobiographical (mis)adventures. In “Edges and Extremes in Ecobiography: Amy Liptrot’s The Outrun” Jessica White extols works that, like Liptrot’s, critically update the historically anthropocentric and imperialistic nature writing genre. Echoing a prime posthumanist concern, she asks whether nature is a mere “backdrop [...] to the human world” (115), or are they co-constitutive? For White, Liptrot’s ultimately human struggles with alcoholism and homesickness are nonetheless environmentally contingent. They manifested when she moved to the big city in an attempt to flee the cold, rural isolation of the Scottish archipelago of her youth and the stress caused by her father’s bipolar disorder. Furthermore, Liptrot overcomes her addiction only after reconnecting with her home by farming, ocean swimming, and protecting its endangered bird species. However, as compelling as texts like The Outrun and interpretations like White’s are, “the relationships between a human and their ecosystem” (100) that are presented here often do not exceed the level of metaphor and analogy. Book Review Insofar as it is a radical stance against humanist hierarchies of bodies, groups, and species, posthumanism can be uncomfortable to implement sometimes, as suggested by Bazarbash’s reading of Macdonald’s self-reported display of relational responsibility towards Mabel. In “Writing Life on Mars: Posthuman Imaginaries of Extraterrestrial Colonization and the NASA Mars Rover Missions,” Jens Temmen shows that space colonization politics is not only an extension of European colonialism but it also sidesteps the climate crisis public debate. Powerful states and their ultrawealthy contractors, enamored by the promise of an entire planet’s worth of unmined, unfarmed, and undeveloped land on Mars, have weaponized humanity’s fear of extinction, Temmen argues, by framing the idea of an off-world mass resettlement as a viable solution to environmental collapse. He proceeds to carefully demonstrate how this neoliberal project of privatizing Mars fuels the mediatic processes of animorphization of NASA robots and heroification of billionaire ‘astronauts,’ an analysis which makes this a compelling piece of cultural criticism. The book closes with “(Life) Narrative in the Posthuman Anthropocene: Erin James in Conversation with Birgit Spengler,” a truly effervescent interview of ecocritical, narratological, and feminist proportions. One gets pleasantly lost in the interlocutors’ perspectives on, for example, forest fire science communication and econarratology as an anti-imperialism of the mind. Moreover, James invites scholars to investigate how the environment, broadly conceptualized, is made absent in humanist narratives. Jane Austen’s Mansfield Park is her example here: “the whole plot is made possible by Sir Thomas Bertram’s plantation in Antigua”, “the whole thing is financed by a system of enslavement [...] that largely goes unnarrated” (239-240). Life Writing in the Posthuman Anthropocene is a down-to-earth (quite literally) posthumanist account that substitutes speculative futurism for a much-needed socioecological sensitivity. Students of the aging process would be inspired by the ways in which their object is put into perspective—temporally, philosophically, culturally, and bio-ecologically—by all of the authors discussed above, except perhaps for Brant and Temmen (although the latter does touch upon nonhuman death rituals). The book would also benefit anyone interested in the intersection between environmental and biographical studies; in the search for ‘actually-occurring’ posthumanism; or in the analytical import of theories by figures like Donna Haraway, Rosi Braidotti, and Karen Barad. Book Review “Their lowliness on our scale of recognition makes them easy to overlook. We can’t see the processes by which they filter, feed, repair, reproduce, so we think they do nothing” (129)—and treat them as pests, food, or pharmacopoeia. Her point is that posthumanist life writers should responsibly relate not only to those macroscopic and dry parts of the Earth wherein humans roam, but also to the small and underwater worlds that necessitate attention and care just as much. Scientific biographers, for example, could achieve this by exposing the “human constructedness” (135) of biological discourse, as illustrated in Brant’s brief genealogy of sea squirt taxonomy. The second part of the book is opened by Renata Lucena Dalmaso, for whom “Relational Responsibility,” this section’s title, entails a move away from seeing human-nonhuman relations as merely spatial. Like Brant, Dalmaso invites us to go beyond anthropocentric scales—in this case, of time. Her essay “Humanity, Life Writing, and Deep Time: Postcolonial Contributions” illustrates postcolonial life writing’s power to articulate posthumanist temporalities. The span of celestial and geological formations, deep time is a cornerstone concept in her reading of two books: one by the renowned Indian writer Amitav Ghosh, and another by Brazilian philosopher and Indigenous militant Ailton Krenak. For Dalmaso, these authors’ depictions of their ancestral connection to rivers, forests, and mountains transcend personal, familial, and ethnic narratives. More specifically, they circumvent anthropocentrism by spotlighting nonhuman narrators, like tree rings, sedimentation layers, and the weather. Human agency, a tenet of humanist storytelling, is thereby reframed as but one of the many vectors in a story being told for much longer than anyone’s lifetime. Anthropology & Aging Vol 44 No 1 (2023) ISSN 2374-2267 (online) DOI 10.5195/aa.2023.475 http://anthro-age.pitt.edu 120 Book Review \| Pinheiro The second essay in this section continues the literary criticism format followed by the majority of the contributions. “Helen Macdonald’s H is for Hawk and Critical Posthumanism” by Monir Gholamzadeh Bazarbash is a reminder of the physical, psychological, and social challenges to ethical interspecies relationships. Bazarbash extracts many practical insights from Macdonald’s experience of training Mabel, a goshawk. Evoking “this sense of close connection and radical difference at the same time” (190), her process of understanding (rather than suppressing) Mabel’s wants, needs, and habits involved hunting with and sleeping in the same quarters as Mabel but also coping with her random nightly attacks. Book Review Indeed, although this is not a theoretical book (i.e., one will not find the clarification of basic terms like post-, anti-, trans-, and nonhumanism in it), all of the essays stand as rigorous examples of theory application. Batzke, Ina, Eric C. Erbacher, Linda M. Hess, and Corinna Lenhardt, eds. 2018. Exploring the Fantastic: Genre, Ideology, and Popular Culture. Bielefeld: Transcript. References Batzke, Ina, Eric C. Erbacher, Linda M. Hess, and Corinna Lenhardt, eds. 2018. Exploring the Fantastic: Genre, Ideology, and Popular Culture. Bielefeld: Transcript. Anthropology & Aging Vol 44 No 1 (2023) ISSN 2374-2267 (online) DOI 10.5195/aa.2023.475 http://anthro-age.pitt.edu
https://openalex.org/W2921141022	https://zenodo.org/records/3904385/files/GUILDify_merged_revised.pdf	English	null	GUILDify v2.0: A Tool to Identify Molecular Networks Underlying Human Diseases, Their Comorbidities and Their Druggable Targets	Journal of Molecular Biology/Journal of molecular biology	2,019	cc-by	13,163	© 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ Abstract The genetic basis of complex diseases involves alterations on multiple genes. Unravelling the interplay between these genetic factors is key to the discovery of new biomarkers and treatments. In 2014, we introduced GUILDify, a web server that searches for genes associated to diseases, finds novel disease-genes applying various network-based prioritisation algorithms and proposes candidate drugs. Here, we present GUILDify v2.0, a major update and improvement of the original method, where we have included protein interaction data for seven species and 22 human tissues and incorporated the disease-gene associations from DisGeNET. To infer potential disease relationships associated with multi-morbidities, we introduced a novel feature for estimating the genetic and functional overlap of two diseases using the top-ranking genes and the associated enrichment of biological functions and pathways (as defined by GO and Reactome). The analysis of this overlap helps to identify the mechanistic role of genes and protein-protein interactions in comorbidities. Finally, we provided an R package, guildifyR, to facilitate programmatic access to GUILDify v2.0 (http://sbi.upf.edu/guildify2) GUILDify v2.0: A tool to identify molecular networks underlying human diseases, their comorbidities and their druggable targets Joaquim Aguirre-Plans1, Janet Piñero2, Ferran Sanz2, Laura I. Furlong2, Narcis Fernandez- Fuentes3,4, Baldo Oliva1,* and Emre Guney2,5,* 1- Structural Bioinformatics Group, Research Programme on Biomedical Informatics, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Catalonia 08003, Spain 2- Integrative Biomedical Informatics Group, Research Programme on Biomedical Informatics, Hospital del Mar Medical Research Institute, Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Catalonia 08003, Spain epartment of Biosciences, U Science Tech, Universitat de Vic-Universitat Central de Catalunya Catalonia 08500, Spain 4- Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, SY23 3EB Aberystwyth, United Kingdom 4- Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, SY23 3EB Aberystwyth, United Kingdom 5- Department of Pharmacology and Personalised Medicine, CARIM, FHML, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands 5- Department of Pharmacology and Personalised Medicine, CARIM, FHML, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands Correspondence to Emre Guney: emre.guney@upf.edu Correspondence may also be addressed to Baldo Oliva: baldo.oliva@upf.edu Introduction Complex diseases such as cancer, diabetes, neurodegenerative disorders or cardiovascular diseases are rarely caused by a single genetic perturbation and usually involve polygenic modifications on the underlying interconnected cellular network. Understanding the genetic basis of diseases and the interactions of disease-associated proteins in the protein interaction network (PIN) is essential for the 1 © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ development of new rational therapeutic strategies. Despite recent large-scale genotyping efforts, information on disease-gene associations is still limited, often explaining a small percentage of the phenotypic variance observed among individuals [1]. To address this limitation and infer novel disease-gene associations, various disease-gene prioritisation methods have been suggested, exploiting the “guilt-by-association” principle over certain features of disease-genes such as similarity in sequence and functional annotations, clustering in the linkage interval, or proximity in the PIN [2]. Indeed, albeit the PINs being incomplete [3], the proximity to disease-genes in the PIN has proven extremely useful in prioritising disease-associated genes [4]. Consequently, a number of tools and web servers has been developed to expand the number of disease-associated genes using the interactome [5–9]. development of new rational therapeutic strategies. Despite recent large-scale genotyping efforts, information on disease-gene associations is still limited, often explaining a small percentage of the phenotypic variance observed among individuals [1]. To address this limitation and infer novel disease-gene associations, various disease-gene prioritisation methods have been suggested, exploiting the “guilt-by-association” principle over certain features of disease-genes such as similarity in sequence and functional annotations, clustering in the linkage interval, or proximity in the PIN [2]. Indeed, albeit the PINs being incomplete [3], the proximity to disease-genes in the PIN has proven extremely useful in prioritising disease-associated genes [4]. Consequently, a number of tools and web servers has been developed to expand the number of disease-associated genes using the interactome [5–9]. Previously, we presented GUILDify, a web server that applies the prioritisation algorithms developed in GUILD software to find novel disease-gene associations based on the connectedness of genes in the PIN [10,11]. GUILDify searches for genes starting from user-provided keywords such as the names of diseases or gene symbols in the BIANA knowledge database. It uses the genes associated to the keywords as seeds and the PIN for the selected organism to apply graph theory algorithms to prioritise new disease genes. Introduction Recently, GUILDify has been applied to: (i) find comorbidities across genetic diseases [12]; (ii) construct PINs specific to breast cancer metastasis to lung and brain [13]; (iii) identify candidate genes for body size in sheep [14] and (iv) prioritise preeclampsia pathogenesis [15]. Here, we present a comprehensive upgrade, GUILDify v2.0, where we updated the underlying biological databases in BIANA knowledge database (protein and drug-target interactions, functional and disease annotations) and: (i) facilitated the use of seven species-specific PINs and 22 human tissue-specific PINs; (ii) increased the quality and number of disease-gene associations by incorporating DisGeNET to our datasets; (iii) incorporated the option to search by drug name, allowing the prioritisation of genes based on known drug targets to uncover the neighbourhood of the PIN affected by the drug; (iv) improved the visualisation of the results using cytoscape.js; (v) refined the definition of top-ranking genes based on whether they had similar functional annotations as the seeds, thus providing the biologically most coherent subnetwork relevant to a given disease; (vi) introduced a feature to measure the genetic and functional overlap of the top-ranking genes of two different diseases, supporting the investigation of disease comorbidities; (vii) implemented a new drug repurposing functionality to propose novel indications for a given drug based on the genetic and functional overlap; and (viii) developed an R package to facilitate the programmatic access to the methods implemented in the web server. 1. Identifying genetic and functional similarities across diseases In recent works, we have shown that the genetic and functional similarities of diseases in the PIN can recent works, we have shown that the genetic and functional similarities of diseases in the PIN 2 © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ be used to characterise co- and multi-morbidities across diseases [12] and also to repurpose existing drugs targeting these diseases [16]. Motivated by these findings and to provide systematic insights on disease-disease relationships, GUILDify v2.0 now allows users to identify the overlap between two previously submitted results, i.e. sets of genes linked to two different diseases. Accordingly, given two job IDs corresponding to the prioritisation results of two different diseases, GUILDify v2.0 provides: (i) the overlap between the top-ranking genes of the two diseases; (ii) the overlap between the enriched functions among the top-ranking genes of the two diseases; (iii) the enriched functions among the common top-ranking genes; and (iv) a network visualisation of the interactions between common top- ranking genes. Moreover, GUILDify v2.0 also calculates the Fisher’s exact test to quantify the significance of the overlap between genes and functions and report one-sided P-value (see details in Supplementary Material). GUILDify v2.0 is the first server that permits the use of gene prioritisation results to explore disease-disease relationships with such simplicity and flexibility. 2. Prioritisation of drug targets GUILDify v2.0 now allows to search by a drug in addition to a phenotype and returns a list of drug- target associations integrated from DrugBank [17], DGIdb [18], DrugCentral [19] and ChEMBL [20] (see details in Supplementary Material). This new functionality allows the characterisation of the neighbourhood of the drug in the PIN, i.e. neighbouring proteins to those targeted by the drug, and thus providing insights on the potential mechanism of action of the drug. Moreover, the novel feature of assessing the overlap between two network expansion runs (i.e. two job IDs) can also be applied in multiple scenarios to: (i) identify the similarity between the neighbourhood of two drugs in the PIN, which can be useful to identify drug interactions; (ii) compare the neighbourhood of a disease with the neighbourhood of a drug in the PIN, which can be applied to drug repurposing. Such novel features make GUILDify v2.0 one of the most easy-to-use and flexible web servers to inspect the effect of drugs in the PIN. 3. Screening diseases to identify potential new indications of known drugs Building upon new technical developments mentioned above, GUILDify v2.0 now offers a novel drug repurposing functionality. Given a job ID associated with a drug (or a list of drug targets), this feature automatically calculates the overlap of genes (or functions) between the given drug and a set of pre- calculated diseases. Details on the method and validation of drug repurposing are described in detail at Supplementary Material. 6. Functional-coherency based selection of top-ranking genes One of the main issues when working with disease-gene prioritisation is to select the most relevant (top ranked) genes associated with a given disease. The user can select top 1% or 2% highest scoring genes among all the proteins in the PIN as top ranked genes. In GUILDify v2.0, we also introduced a cutoff based on the functional validation approach described in Ghiassian et al. [5] and provided a new panel visualising the significance of the functional enrichment (P-value) as a function of the number of top-ranking genes included in the validation (implemented in Plotly). In brief, the highest-scoring non-seed proteins are iteratively included in the top-ranking set, provided that they maintain the functional coherency of the existing top-ranking set (see details in Supplementary Material). Note that this approach might be too restrictive for some complex diseases in which the information on known disease-gene associations is limited, failing to represent the functional diversity involved in the disease. 7. Visualisation of the top-ranking subnetwork GUILDify v2.0 uses the JavaScript-based network visualisation library, Cytoscape.js [33], to show the subnetwork of the top-ranking proteins and the drugs targeting these proteins. The user can decide the cutoff to define the top ranked proteins to be visualised (top 1%, top 2% or functionally-coherent as mentioned above). In addition to seeds (green hexagons), top-ranking proteins (yellow circles) and drugs (blue diamonds), the subnetwork includes the proteins that connect the seeds to the largest connected component induced by seeds (named “linkers” and shown as grey circles, see details in Supplementary Material). 4. Tissue and species-specific PINs The analysis of the protein interactions in a tissue-specific context is becoming increasingly relevant to understand genetic diseases and find improved treatments [21]. We have included tissue-specific networks derived from 22 different human tissues (see Supplementary Table S1). To create these networks, we filtered the interactions in the global PIN using RNAseq data from GTEx [22], keeping only the interactions between proteins encoded by genes that are expressed in a given tissue (i.e. considering only transcripts with TPM (transcripts per kilobase million) expression values of 1 or 3 © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ higher (see details in Supplementary Material). We have also included 7 species-specific PINs derived from experimentally determined protein-protein interactions. Although the coverage of interactomic data for some species is low (e.g., 11,943 interactions in rat vs 320,337 interactions in human), these PINs provide a reliable backbone for interactome-based analyses (e.g., in preclinical research) as opposed to PINs generated by predicted interactions based on homology information. higher (see details in Supplementary Material). We have also included 7 species-specific PINs derived from experimentally determined protein-protein interactions. Although the coverage of interactomic data for some species is low (e.g., 11,943 interactions in rat vs 320,337 interactions in human), these PINs provide a reliable backbone for interactome-based analyses (e.g., in preclinical research) as opposed to PINs generated by predicted interactions based on homology information. 5. Disease-gene information from DisGeNET We incorporated DisGeNET, one of the largest repositories of genes and variants associated to human diseases [23]. DisGeNET relies on data from UniProt [24], CTD [25], CLINVAR [26], ORPHANET [27], GWAS Catalog [28], PsyGeNET [29] and HPO [30] and is integrated in BIANA [31]. To investigate the increase in the number of disease-gene associations between versions 1 and 2 of GUILDify, we checked the number of associations for the lowest-level non-obsolete diseases from Disease Ontology [32] that were available in our repositories (2,190 terms). GUILDify v1 contains gene associations for 1,505 diseases and 4,171 genes (2.8 genes per disease), while updated GUILDify v2.0 has gene associations for 2,064 diseases and 11,615 genes (5.6 genes per disease on average). 8. R package 4 4 © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ We have included an R package in order to provide programmatic access to GUILDify v2.0 through R statistical computing environment (https://www.r-project.org/). The package implements methods to query and retrieve results from the web server as an R data frame, allowing users to run multiple queries for more high-throughput and/or systematic analyses. The package and documentation are available online at: http://sbi.upf.edu/guildify2. 1. Input The interface of GUILDify v2.0 is designed to be simple and intuitive. The input varies slightly depending on the desired task: (i) a new search; (ii) retrieving results from a previous run; and (iii) calculating genetic and functional overlap between two previous runs. For a new search, we require two steps: first the selection of seeds (genes associated with a phenotype or drug) and second the selection of parameters to run the prioritisation algorithms. For the selection of seeds the user has to provide: (i) either keyword(s) describing the phenotype/drug of interest or a set of specific gene names separated by a semicolon; (ii) the species of interest (default value: Homo sapiens); (iii) the tissue of interest (default value: All); and (iv) the PIN source (default value: BIANA). If the user provides a keyword (or set of keywords) describing a phenotype or drug, the server searches genes containing the keyword in BIANA knowledge database (i.e. integrating information from many resources), otherwise it uses the list of provided gene names. The server shows the selected seeds, which can still be filtered and selected by the user. Then, for the prioritisation parameters the user can select to run the “disease module detection algorithm” (DIAMOnD, downloaded from https://github.com/dinaghiassian/DIAMOnD) [5] or to use one of the several prioritisation algorithms from the GUILD package (default value: NetScore with default parameters). Finally, to retrieve results, th i d i t i th j b ID f i hil f l l ti ti d f ti l l https://github.com/dinaghiassian/DIAMOnD) [5] or to use one of the several prioritisation algorithms from the GUILD package (default value: NetScore with default parameters). Finally, to retrieve results, the required input is the job ID of a previous run, while for calculating genetic and functional overlap the inputs are two job IDs of previous runs. 1. Exploring the mechanistic links between rheumatoid arthritis and asthma In multiple studies, rheumatoid arthritis and asthma are linked as a potential comorbidity, although the mechanisms underlying this association remain unclear [34]. Using the new functionality of GUILDify v2.0, we can assess the overlap between diseases and thus propose a potential mechanism to explain the association between them. Querying for “rheumatoid arthritis” and “asthma” returns 156 and 96 seeds, respectively coming from DisGeNET, OMIM, and UniProt. There are already 12 seeds in common (Fisher’s exact test, one-sided P-value = 1.4·10-9) and 18 common functions out of the total enriched functions of the seeds (P-value = 9.3·10-23). After running GUILDify v2.0, we select 290 and 181 top ranked genes using functional-coherency based cutoff for rheumatoid arthritis and asthma, respectively. We find that the number of common genes increases to 55 (yielding a P-value = 5.9·10-48), while the number of common functions (biological processes) increases to 31 (P-value = 8.1·10-46). The link between these diseases is significant even when the seeds are removed from the top-ranking genes (see Supplementary Material). Among the shared top-ranking genes, we find Tumor Necrosis Factor (TNF), which has been proposed as a potential drug target for asthma and rheumatoid arthritis, and highlighted as a potential precursor of the comorbidity [12]. We also find HLA-DRB1 and several interleukins (IL18, IL1B, IL3), taking part of the immune response potentially involved in both diseases. Furthermore, the most common enriched functions relate to inflammatory processes such as “inflammatory response”, “positive regulation of interferon-gamma production” and “positive regulation of T-helper 1 cell cytokine production”. These functions appear again if we check the functions enriched by the common genes, along with other functions such as “T-helper 1 type immune response” or “negative regulation of type 2 immune response”, highlighting the involvement of type 1 immune response in both diseases. As negative controls, we repeated the analysis using other disease pairs that are not likely to be comorbid such as “rheumatoid arthritis” - “breast cancer” and “asthma” - “breast cancer”, finding drastically reduced number of genes in the overlap between these disease pairs (see Supplementary Material). The results can be further explored in Figure 1 and in the pre-calculated examples section of the web. Additionally, we compared the functional relevance of the top-ranking genes identified by NetScore with DIAMOnD, based on the analysis in Sharma et al. [35] (see Supplementary Material). 2. Output GUILDify v2.0 outputs the ranking of the nodes in the PIN and the visualisation of the subnetwork involving the top-ranking genes in a cytoscape.js panel. In addition, the output page has: (i) a panel showing the P-values of functional enrichment of the ranked nodes; (ii) two panels with functions enriched among the top-ranking nodes and seeds, respectively; and (iii) one panel with the drugs that target the top-ranking proteins. For the “Overlap between two results” option, the server provides: (i) the list of the common top- ranking genes and the significance of the overlap assessed by a Fisher’s exact test (see details in Supplementary Material); (ii) the network visualisation of the common top-ranking genes including the “linkers” (see above); (iii) the list of enriched functions of the common genes; iv) the list of common enriched functions of both results and the significance of the overlap; and v) the drugs targeting the proteins of the common PIN. Using this functionality, the users can identify the overlap between any two queries such as between two diseases, two drugs or a disease and a drug. Although we do not 5 © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ provide the overlap between interactions of top-ranking proteins in a separate table, these interactions can be investigated in the network visualisation panel. 1. Exploring the mechanistic links between rheumatoid arthritis and asthma We checked the enrichment of top-ranking genes among the pathways containing the seed genes of asthma and rheumatoid arthritis, showing that both methods significantly recover the pathways in each disease. Furthermore, NetScore identified more genes that belonged to the pathways shared between asthma and rheumatoid arthritis compared to DIAMOnD. Datasets GUILDify v2.0 uses BIANA [31] for the integration of biological interaction databases with information on drugs, genes, proteins, functions, pathways and diseases. To create the tissue-specific PINs, we use the RNAseq data from GTEx V7 [22]. Phenotype-gene associations are extracted from DisGeNET, OMIM, Uniprot, and Gene Ontology. Drug-target associations are taken from DrugBank [17], DGIdb [18], DrugCentral [19] and ChEMBL [20]. See Supplementary Material for details on the datasets. Prioritisation algorithms GUILDify v2.0 uses four different network-based prioritisation algorithms: NetShort, NetZcore, NetScore and DIAMOnD. For details on these algorithms see references [5,10,11] and the Supplementary Material. 2. Study of the mechanism of non-small cell lung carcinoma drugs Indeed, the characterisation of the neighbourhood in the PIN that is affected by drugs opens a wide range of possibilities for drug repurposing research. 2. Study of the mechanism of non-small cell lung carcinoma drugs Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Typically induced by exposure to toxic substances, the NSCLC pathology has been specially associated with a mutation in 6 6 © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ the Epidermal Growth Factor Receptor (EGFR) [36]. In a recent study, 9 drugs were proposed to treat this disease [37], 6 of them having drug-target interactions reported: Afatinib, Ceritinib, Crizotinib, Erlotinib, Gefitinib and Palbociclib. Given that we can now identify potentially new relationships between drugs and diseases using drugs as queries, we investigate whether the neighbourhood of the targets of these drugs in the PIN significantly overlaps with the neighbourhood of the genes associated with NSCLC. We used GUILDify v2.0 to define this neighbourhood. We observe that the genetic overlap is always significant, except for one of the drugs (Palbociclib, see Table 1). We confirm the significance by applying the same approach to breast cancer, showing that Ceritinib, Crizotinib and Palbociclib produce a significant genetic overlap, although the number of common genes in each case is substantially lower than it is in NSCLC (see Table 1). These results are consistent with the fact that Palbociclib is primarily indicated for breast cancer and it has been recently repurposed for NSCLC [38]. The small but significant overlap of Ceritinib and Crizotinib suggests that these two drugs might also be considered as potential repurposing candidates. We note that using the top-ranking nodes increases the significance of the genetic overlap (with lower P-values) compared to the overlap using only seeds (genes associated with a pathophenotype and direct targets of drugs). The significant overlap between the top ranked genes identified using these drugs and the top ranked genes for NSCLC (but not for the top ranked genes for breast cancer) suggests that GUILDify v2.0 can help understanding how drugs exert their action on certain diseases. Indeed, the characterisation of the neighbourhood in the PIN that is affected by drugs opens a wide range of possibilities for drug repurposing research. The significant overlap between the top ranked genes identified using these drugs and the top ranked genes for NSCLC (but not for the top ranked genes for breast cancer) suggests that GUILDify v2.0 can help understanding how drugs exert their action on certain diseases. Overlap and functional enrichment analysis We use one-sided Fisher’s exact test to calculate the overlap between two sets of genes or functions and use Benjamini-Hochberg multiple hypothesis testing procedure (where applicable). The functions 7 7 © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ enriched among seeds and top-ranking nodes as well as common functions between two diseases are calculated as explained in a previous work [12] (see details in Supplementary Material). Conflicts of Interest Statement None declared. Acknowledgements J.A.P. and E.G. would like to acknowledge the technical support from GRIB IT team, in particular A. Gonzalez Pauner and M. A. Sánchez Gómez. The authors received support from: ISCIII-FEDER (PI13/00082, CP10/00524, CPII16/00026); IMI-JU under grants agreements no. 116030 (TransQST) and no. 777365 (eTRANSAFE), resources of which are composed of financial contribution from the EU-FP7 (FP7/2007- 2013) and EFPIA companies in kind contribution; the EU H2020 Programme 2014-2020 under grant agreements no. 634143 (MedBioinformatics) and no. 676559 (Elixir-Excelerate); the Spanish Ministry of Economy (MINECO) [BIO2017-85329-R] [RYC-2015-17519]; “Unidad de Excelencia María de Maeztu”, funded by the Spanish Ministry of Economy [ref: MDM-2014-0370]. The Research Programme on Biomedical Informatics (GRIB) is a member of the Spanish National Bioinformatics Institute (INB), PRB2-ISCIII and is supported by grant PT13/0001/0023, of the PE I+D+i 2013-2016, funded by ISCIII and FEDER. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license © 2019. 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Farré, L. Fernández- [29] 9 TABLE AND FIGURES LEGENDS Figure 1. GUILDify v2.0 example study on the comorbidity between asthma and rheumatoid arthritis. First, we run the prioritisations of the two diseases by searching (1) and selecting (2) the genes. After obtaining the ranking of proteins from the prioritisation (3), we use both job IDs to check their overlap (4) and inspect the genetic and functional relationships between them (see details at htt // bi f d / ildif 2 i th l l t d l ti http://sbi.upf.edu/guildify2 in the pre-calculated examples section. http://sbi.upf.edu/guildify2 in the pre-calculated examples section. Table 1. Results of the genetic and functional overlap between the subnetwork of genes associated with “non small cell lung carcinoma” and “breast cancer” (top ranking genes and seeds) and the subnetwork of genes associated with the targets of drugs Afatinib, Ceritinib, Crizotinib, Erlotinib, Gefitinib and Palbociclib (drug targets and top-ranking genes obtained with GUILDify v2.0). P-values shown have been corrected using the Benjamini-Hochberg correction for multiple tests. Results with non-significant P-value are highlighted in red. 10 Search a phenotype asthma Search 1 3 Ranking 2 “rheumatoid arthritis” Select genes asthma rheumatoid _arthritis Search GUILDify! GUILDify! Introduce 2 job IDs asthma rheumatoid_arthritis Check overlap 4 q ADA q ADAM33 q ADCY2 q ABCB1 q ABCC2 q ABCC3 CCR3 – 1.00 CCL2 – 0.93 IL4 – 0.89 IL10 – 1.00 MMP10 – 0.84 IL6R – 0.83 Search a phenotype asthma Search 1 3 Ranking Genetic overlap: Functional overlap: 2 Visualisation: “rheumatoid arthritis” 235 126 55 51 41 31 Select genes TNF HLA-DRB1 IL1B, IL18, MMP10, CCL2, VEGFA, CAT… asthma rheumatoid _arthritis Search GUILDify! GUILDify! Introduce 2 job IDs asthma rheumatoid_arthritis Check overlap 4 q ADA q ADAM33 q ADCY2 q ABCB1 q ABCC2 q ABCC3 CCR3 – 1.00 CCL2 – 0.93 IL4 – 0.89 IL10 – 1.00 MMP10 – 0.84 IL6R – 0.83 Functions of common genes: chemokine activity positive regulation of T-helper 1 cell cytokine production T-helper 1 type immune response negative regulation of type 2 immune response … inflammatory response pos. regulation of interferon-gamma pos. regulation of T-helper 1 cell cytokine … Ranking asthma rheumatoid _arthritis Genetic overlap: Functional overlap: Visualisation: 235 126 55 51 41 31 TNF HLA-DRB1 IL1B, IL18, MMP10, CCL2, VEGFA, CAT… Functions of common genes: chemokine activity positive regulation of T-helper 1 cell cytokine production T-helper 1 type immune response negative regulation of type 2 immune response … inflammatory response pos. TABLE AND FIGURES LEGENDS regulation of interferon-gamma pos. regulation of T-helper 1 cell cytokine … Genetic overlap: Functional overlap: Visualisation: 235 126 55 51 41 31 TNF HLA-DRB1 IL1B, IL18, MMP10, CCL2, VEGFA, CAT… Functions of common genes: chemokine activity positive regulation of T-helper 1 cell cytokine production T-helper 1 type immune response negative regulation of type 2 immune response … inflammatory response pos. regulation of interferon-gamma pos. regulation of T-helper 1 cell cytokine … inflammatory response pos. regulation of interferon-gamma pos. regulation of T-helper 1 cell cytokine … Functional overlap: chemokine activity positive regulation of T-helper 1 cell cytokine production T-helper 1 type immune response negative regulation of type 2 immune response … Functions of common genes: “non-small cell lung carcinoma” “breast cancer” Genetic overlap Functional overlap Genetic overlap Functional overlap Top Seeds Top Seeds Top Seeds Top Seeds Nº P- val. Nº P-val. Nº P-val. Nº P-val. Nº P- val. Nº P-val. Nº P-val. Nº P-val. Afatinib 9 7.80 E-06 2 1.90E- 03 0 1 6 1.70E- 05 3 1.40 E-01 0 1 0 1 1 7.50E- 01 Ceritinib 18 4.20 E-15 4 6.60E- 05 5 1.70E- 07 9 1.80E- 06 6 1.20 E-02 0 1 0 1 0 1 Crizotinib 13 1.20 E-09 4 7.00E- 05 3 1.10E- 04 8 7.70E- 06 5 2.00 E-02 0 1 0 1 0 1 Erlotinib 16 6.30 E-13 4 6.60E- 05 5 1.20E- 06 10 2.60E- 07 3 1.40 E-01 0 1 0 1 0 1 Gefitinib 10 1.10 E-06 3 1.20E- 04 1 3.60E- 02 11 7.20E- 10 3 1.40 E-01 0 1 0 1 0 1 Palbociclib 3 1.40 E-01 0 1 0 1 2 8.90E- 02 5 2.00 E-02 1 4.70E -01 0 1 1 7.50E- 01 Supplementary material for: GUILDify v2.0: A tool to identify molecular networks underlying human diseases, their comorbidities and their druggable targets Joaquim Aguirre-Plans, Janet Piñero, Ferran Sanz, Laura I. Furlong, Narcis Fernandez- Fuentes, Baldo Oliva* and Emre Guney* 1. Datasets 2 2. Prioritisation algorithms 3 3. Functional enrichment analysis 3 4. Description of BIANA integration pipeline 3 5. Description of tissue-specific PIN generation pipeline 6 6. Functional-based selection of top-ranking genes 8 7. Significance of the overlap between genes/functions 9 8. Results of the case study of asthma and arthritis rheumatoid and the negative controls with breast cancer 11 9. Comparison of the case study results of asthma and rheumatoid arthritis with DIAMOnD 13 10. 1.2. Tissue-specific protein-protein interaction networks To create the tissue-specific PINs, we retrieved the RNA-sequencing gene TPMs from GTEx V7 [10]. We use the samples from subjects for which the reason for death was traumatic injury (point 1 in Hardy Scale) and we discard the tissues with less than 5 samples (a total of 675 samples from 40 tissues). For each gene, we calculate the median expression of all samples of a tissue. We unify tissues into a unique “main” tissue (i.e. “Adipose – Subcutaneous” and “Adipose – Visceral Omentum” belong to the main tissue “Adipose”) by considering the highest median expression of all samples [11]. We note that using this approach, the final tissue profiles could be biased towards the subtypes of tissue that have a higher number of samples. This is a limitation, as there may be subtypes of tissue that are more represented than others in the final expression of the tissue (Supplementary Table S1). GUILDify v2.0 includes a total of 22 tissues (see details in Supplementary Material). 1.1. Protein-protein interaction networks GUILDify v2.0 relies on a knowledge database called BIANA [1], which integrates biological interaction databases together with information on genes and proteins and its associated functions, diseases and phenotypes. Currently, we have up-to-date information of protein-protein interactions from IntAct [2], BioGRID [3] and DIP [4]. As an additional option, we provide the user with 5 other PIN sources: HIPPIE (high confidence score threshold >= 0.7) [5], InBio_Map (score threshold >= 0.15) [6], ConsensusPathDB [7], I2D [8], and STRING (score >= 0.7) [9]. TABLE AND FIGURES LEGENDS Screening diseases to identify potential new indications of known drugs 13 11. Visualisation of the top-ranking subnetwork 16 References 16 1. Datasets 2 2. Prioritisation algorithms 3 3. Functional enrichment analysis 3 4. Description of BIANA integration pipeline 3 5. Description of tissue-specific PIN generation pipeline 6 6. Functional-based selection of top-ranking genes 8 7. Significance of the overlap between genes/functions 9 8. Results of the case study of asthma and arthritis rheumatoid and the negative controls with breast cancer 11 9. Comparison of the case study results of asthma and rheumatoid arthritis with DIAMOnD 13 10. Screening diseases to identify potential new indications of known drugs 13 11. Visualisation of the top-ranking subnetwork 16 References 16 1 1.3. Phenotype-gene associations Phenotype-gene associations are extracted from DisGeNET, OMIM, Uniprot and Gene Ontology extracting information from relevant sections. In the case of DisGeNET, we parse disease-gene associations from curated sources: UniProt [12], CTD [13], ORPHANET [14], PsyGeNET [15] and HPO [16]. In OMIM [17], we retrieve disease-gene associations from the OMIM’s Synopsis of the Human Gene Map. For Uniprot [30], we collect the protein information of the categories “Description”, “Function”, “Keyword” and “Disease”. Finally, in the case of the Gene Ontology (GO) [18,19], we parse the functional annotations of genes. 3. Functional enrichment analysis We calculate the enriched functions of seeds and top-ranking nodes and calculate the significance of common functions between two diseases (or phenotypes) as in a previous work on comorbidities [28]. Briefly, functions are defined by GO biological processes, GO molecular functions and Reactome pathways. In the case of GO, we only use high confident annotations (codes of evidence EXP, IDA, IMP, IGI, IEP, ISS, ISA, ISM or ISO). We calculate the significance of the enrichment using a one-sided Fisher's exact test (the alternative hypothesis is that the overlap would be greater than observed overlap). Then, we correct the P-value by either applying the Benjamini-Hochberg correction for multiple tests and keeping the functions for which the adjusted P-value < 0.05. We also offer the user the possibility to use Bonferroni correction at the results page. 1.4. Drug-target integration Drug-target interactions are retrieved from DrugBank [20], DGIdb [21], DrugCentral [22] and ChEMBL [23], and integrated following the procedure in Piñero et al. [24]. In DrugBank, we only select the therapeutic targets (excluding enzymes, transporters and carriers). In the case of data from DrugCentral, we retrieve the targets in the “Tclin” category. From DGIdb we select the targets from “Chembl”, “GuideToPharmacology”, “Tdg Clinical Trial”, “FDA”, “TEND” and “TTD”. Finally in ChEMBL, we collect targets with a DrugBank identifier cross-reference. 2 2. Prioritisation algorithms GUILDify v2.0 uses four different network-based prioritisation algorithms: NetShort, NetZcore, NetScore and DIAMOnD. For details on these algorithms see references [25–27]. In brief, NetShort (10-20 minutes of computation time) incorporates “phenotypic-relevance” of the path between a node and the nodes of a given phenotype by considering the number of edges to phenotype-associated nodes (seeds). NetZcore (5-10 minutes of computation time) iteratively assesses the relevance of a node for a given phenotype by averaging the normalised scores of the neighbours. NetScore (5-10 minutes of computation time) is based on the propagation of information through the nodes of the network by considering multiple shortest paths from the source of information to the target. NetCombo (10-20 minutes of computation time) combines NetScore, NetShort and NetZcore by calculating the mean of the normalised score of each prioritisation method. DIAMOnD [27] (5 minutes of computation time) determines the “connectivity significance” of all the proteins of the network, iteratively ranking and selecting the nodes with highest scores. 4. Description of BIANA integration pipeline We used BIANA [1] to compile different types of biological data in an integrated database and to create the protein-protein interaction networks (PIN). The information in BIANA is updated annually to keep the resources underlying the web server up-to-date. The details of data retrieval, integration, unification and network generation pipeline are as follows: We use five sources of protein-protein interaction data: o UniProt swissprot: retrieved from o UniProt swissprot: retrieved from ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/ (Release of 28-Mar-2018). ftp://ftp.uniprot.org/pub/databases/uniprot/current_release/knowledgebase/complete/ (Release of 28-Mar-2018). o Taxonomy: downloaded from ftp://ftp.ncbi.nih.gov/pub/taxonomy (Release of 19-Apr- 2018). o Taxonomy: downloaded from ftp://ftp.ncbi.nih.gov/pub/taxonomy (Release of 19-Apr- 2018). o Gene Ontology: downloaded from http://www.geneontology.org/ontology/ (Release of 19-Apr-2018). o Gene Ontology: downloaded from http://www.geneontology.org/ontology/ (Release of 19-Apr-2018). o NCBI Gene: downloaded from ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/gene2ensembl.gz (Release of 28-Nov-2017). o NCBI Gene: downloaded from ftp://ftp.ncbi.nlm.nih.gov/gene/DATA/gene2ensembl.gz (Release of 28-Nov-2017). o DisGeNET: downloaded from http://www.disgenet.org/web/DisGeNET/menu/downloads (Version 5.0). o DisGeNET: downloaded from http://www.disgenet.org/web/DisGeNET/menu/downloads (Version 5.0). o DrugBank: downloaded from https://www.drugbank.ca/releases/latest (Version 5.1.0). o DrugBank: downloaded from https://www.drugbank.ca/releases/latest (Version 5.1.0). o DrugBank: downloaded from https://www.drugbank.ca/releases/latest (Version 5.1.0). o DrugCentral: downloaded from http://drugcentral.org/download (Release of 29-Aug- o DrugBank: downloaded from https://www.drugbank.ca/releases/latest (Version 5.1.0). o DrugCentral: downloaded from http://drugcentral.org/download (Release of 29-Aug- 2017). g p g ( ) o DrugCentral: downloaded from http://drugcentral.org/download (Release of 29-Aug- 2017). o DrugCentral: downloaded from http://drugcentral.org/download (Release of 29-Aug- 2017). o DGIdb: downloaded from http://dgidb.org/downloads (Version 3.0.2). o DGIdb: downloaded from http://dgidb.org/downloads (Version 3.0.2). o ChEMBL: (Version ChEMBL_24) downloaded from: o ChEMBL: (Version ChEMBL_24) downloaded from: o ChEMBL: (Version ChEMBL_24) downloaded from: o https://www.ebi.ac.uk/chembl/drug/targets > Downloads > Download all txt o https://www.ebi.ac.uk/chembl/drugstore > Downloads > Download all txt o https://www.ebi.ac.uk/chembl/drug/indications > Downloads > Download all txt o https://www.ebi.ac.uk/chembl/target/browser > Select All > Fetch selected targets > Please select… > Download All (tab-delimited) 1. Download the data We use five sources of protein-protein interaction data: We use five sources of protein-protein interaction data: 3 o IntAct: retrieved from https://www.proteinatlas.org/download/normal_tissue.tsv.zip (Release of 22-Mar-2018). o IntAct: retrieved from https://www.proteinatlas.org/download/normal_tissue.tsv.zip (Release of 22-Mar-2018). o BioGRID: downloaded from https://downloads.thebiogrid.org/BioGRID (Version 3.4.159). o BioGRID: downloaded from https://downloads.thebiogrid.org/BioGRID (Version 3.4.159). o DIP: downloaded from http://dip.doe-mbi.ucla.edu/dip/Download.cgi (Release of 05- Feb-2017). o DIP: downloaded from http://dip.doe-mbi.ucla.edu/dip/Download.cgi (Release of 05- Feb-2017). o iRefIndex: downloaded from http://irefindex.org/download/irefindex/data/archive/release_14.0/psi_mitab/MITAB2.6/ (Version 15.0). o iRefIndex: downloaded from http://irefindex.org/download/irefindex/data/archive/release_14.0/psi_mitab/MITAB2.6/ (Version 15.0). o HIPPIE: downloaded from http://cbdm-01.zdv.uni- o HIPPIE: downloaded from http://cbdm-01.zdv.uni- mainz.de/~mschaefer/hippie/download.php (Version 2.1). And we also incorporate additional databases to complement interactomics data: And we also incorporate additional databases to complement interactomics data: And we also incorporate additional databases to complement interactomics data: o UniProt swissprot: retrieved from 2. Parse and unify the data y We parse all the external databases according to the manual of BIANA (available at http://sbi.imim.es/web/BIANA.php). Once we have all the databases incorporated in BIANA knowledge database, we find the equivalent entries across databases to unify the data. This means that two entities coming from different databases (or in some cases from the same database) can be unified in a unique entity provided that they satisfy certain equivalence criteria. 4 If they do so, they will be given the same unique ID called BIANA ID. The rules to unify data (equivalence criteria) are the following: o Same Entrez Gene ID (applied to all databases) o Same Taxonomy ID AND protein sequence (applied to all databases) o Same UniProt entry (only applied to ConsensusPathDB and Uniprot databases) o Same UniProt accession (applied to InBio_Map, I2D, HitPredict and Uniprot) o Same UniProt accession (applied to DrugBank, DrugCentral, ChEMBL and Uniprot databases, to unify the drug targets with the rest of proteins) o Same DrugBank ID (applied to DrugBank, DCDB and DrugCentral to unify drugs) o Same PubChem Compound (applied to DrugBank and DCDB to unify drugs) o Same ChEMBL ID (applied to DrugBank, DGIdb and ChEMBL to unify drugs) o Same Entrez Gene ID (applied to all databases) o Same Entrez Gene ID (applied to all databases) o Same Taxonomy ID AND protein sequence (applied to all databases) o Same UniProt entry (only applied to ConsensusPathDB and Uniprot databases) o Same UniProt accession (applied to InBio_Map, I2D, HitPredict and Uniprot) o Same UniProt accession (applied to DrugBank, DrugCentral, ChEMBL and Uniprot databases, to unify the drug targets with the rest of proteins) o Same DrugBank ID (applied to DrugBank, DCDB and DrugCentral to unify drugs) o Same PubChem Compound (applied to DrugBank and DCDB to unify drugs) o Same ChEMBL ID (applied to DrugBank, DGIdb and ChEMBL to unify drugs) 3. Generate protein-protein interaction networks (PIN) To generate PINs, we retrieve the protein-protein interactions from BIANA knowledge database that have the same Taxonomy ID (reported in the same organism) for human, mouse, rat, yeast, worm, fly and plant. To generate PINs, we retrieve the protein-protein interactions from BIANA knowledge database that have the same Taxonomy ID (reported in the same organism) for human, mouse, rat, yeast, worm, fly and plant. Once we have the PIN, we filter the interactions depending on the detection method used to characterise each interaction. Recent studies highlighted that several protein interaction detection techniques tended to provide a higher number of interactions for more studied proteins in the interactome [29,30]. Thus, for human, we only include interactions coming from the following detection methods that we consider to be less biased: o Two hybrid (ID: 18). o Cross-linking study (ID: 30). o Protein array (ID: 89). o Two hybrid array (ID: 397). o Two hybrid pooling approach (ID: 398). o Biochemical (ID: 401). o Biochemical (ID: 401). o Enzymatic study (ID: 415). o Two hybrid prey pooling approach (ID: 1112). o Proximity labelling technology (ID: 1313). o Validated two hybrid (ID: 1356). For mouse, we included all the methods listed above and the following ones: For mouse, we included all the methods listed above and the following ones: o Affinity chromatography technology (ID: 4) o Anti tag coimmunoprecipitation (ID: 7) o Coimmunoprecipitation (ID: 19) o Cosedimentation in solution (ID: 28) o Pull down (ID: 96) o Affinity chromatography technology (ID: 4) o Anti tag coimmunoprecipitation (ID: 7) o Coimmunoprecipitation (ID: 19) o Cosedimentation in solution (ID: 28) o Pull down (ID: 96) 5 o X-ray crystallography (ID: 375) o X-ray crystallography (ID: 375) o Chromatin immunoprecipitation assay (ID: 810) o Tandem affinity purification (ID: 676) 1. Download the data 1. Download the data We used the RNA-Seq data from GTEx Portal version 7, downloaded from https://gtexportal.org/home/datasets. 1. Download the data We used the RNA-Seq data from GTEx Portal version 7, downloaded from https://gtexportal.org/home/datasets. 2. Process the data o Process the subjects file (GTEx_v7_Annotations_SubjectPhenotypesDS.txt) and get the subjects with cause of death by traumatic injury (DTHHRDY=1). We end up with 29 subjects. o Process the samples file (GTEx_v7_Annotations_SampleAttributesDS.txt) and get the samples coming from the subjects filtered in the previous step. We end up with 699 samples. o Count the tissues present in the samples and the number of samples for each tissue. Remove the tissues that have less than 5 samples. We end up having 40 tissues and 675 samples. o Read the TPM file (GTEx_Analysis_2016-01-15_v7_RNASeQCv1.1.8_gene_tpm.gct.gz) and get the TPM values of the 675 samples mentioned in the previous step. o For each gene in the TPM file, calculate the median of TPM values across all the samples in each tissue. o If several tissues belong to a more general tissue (i.e. “Adipose – Subcutaneous” and “Adipose – Visceral Omentum” belong to the main tissue “Adipose”), we unify them by considering the highest median expression value among these tissues. After unifying the tissues, we end up having 22 main tissues. The tissues unified are listed in Supplementary Table S1. 5. Description of tissue-specific PIN generation pipeline 1. Download the data We used the RNA-Seq data from GTEx Portal version 7, downloaded from https://gtexportal.org/home/datasets. 3. Filter the PIN The last step is to filter the interactions of the PIN based on the tissue annotation of the proteins. For each pair of interacting proteins, first, we check if we have information of the proteins in the GTEx file that we have processed. If so, we get the TPM values for the tissue of interest and the two proteins, and if in both cases the TPM values are higher or equal than 1, we maintain the interaction in the network. If not, we remove the interaction. 6 Supplementary Table S1. Tissues considered for the creation of tissue-specific PIN. In the left we show the 40 initial tissues and in the right the 22 final tissues after the unification of some of the tissues into a broader one. We included the number of samples considered for each tissue. Supplementary Table S1. Tissues considered for the creation of tissue-specific PIN. In the left we show the 40 initial tissues and in the right the 22 final tissues after the unification of some of the tissues into a broader one. We included the number of samples considered for each tissue. GTEx tissues Unified tissues Name Num. samples Name Num. 3. Filter the PIN samples Adipose – Subcutaneous 18 Adipose 28 Adipose – Visceral (Omentum) 10 Adrenal Gland 5 Adrenal Gland 5 Artery – Aorta 16 Artery 52 Artery – Coronary 9 Artery – Tibial 27 Brain – Amygdala 10 Brain 144 Brain – Anterior cingulate cortex (BA24) 12 Brain – Caudate (basal ganglia) 13 Brain – Cerebellar Hemisphere 13 Brain – Cerebellum 16 Brain – Cortex 14 Brain – Frontal Cortex (BA9) 8 Brain – Hippocampus 13 Brain – Hypothalamus 11 Brain – Nucleus accumbens (basal ganglia) 12 Brain – Putamen (basal ganglia) 12 Brain – Spinal cord (cervical c- 1) 10 Breast – Mammary Tissue 16 Breast – Mammary Tissue 16 Cells – Transformed fibroblasts 20 Cells – Transformed fibroblasts 20 Colon – Sigmoid 8 Colon – Sigmoid 8 Esophagus – Gastroesophageal Junction 8 Esophagus 36 Esophagus – Mucosa 15 Esophagus – Muscularis 13 Heart – Atrial Appendage 14 Heart 39 Heart – Left Ventricle 25 Liver 11 Liver 11 Lung 25 Lung 25 Muscle – Skeletal 31 Muscle – Skeletal 31 Nerve – Tibial 26 Nerve – Tibial 26 7 7 Ovary 5 Ovary 5 Pituitary 14 Pituitary 14 Prostate 10 Prostate 10 Skin – Not Sun Exposed (Suprapubic) 16 Skin 44 Skin – Sun Exposed (Lower leg) 28 Testis 15 Testis 15 Thyroid 25 Thyroid 25 Uterus 5 Uterus 5 Vagina 6 Vagina 6 Whole Blood 110 Whole Blood 110 6. Functional-based selection of top-ranking genes The functional-based selection of top-ranking genes is a procedure that we followed to identify if the set of ranking genes is functionally similar to the set of initial seed genes. The procedure was first implemented in Ghiassian et al. [27]. First, we find the enriched Gene Ontology (GO) terms in the seeds by calculating the functional enrichment following the procedure in Rubio-Perez, et al [28]. We only use high confidence annotations from Biological Processes or Molecular Functions associated with the evidence codes EXP, IDA, IMP, IGI, IEP, ISS, ISA, ISM or ISO. From the enriched GO terms, we identify the ones that are significantly enriched using a one-sided Fisher's exact test of significance where the alternative hypothesis is that the overlap would be greater than observed overlap. We correct the significance applying either a Bonferroni or a Benjamini-Hochberg correction for multiple tests and selecting a P-value < 0.05 (the case studies use Benjamini-Hochberg). For each candidate gene in the top-ranking genes, we search if it is annotated within any of the significant GO terms of the seeds. The genes annotated are considered true positives. For each candidate gene in the ranking, we define a sliding window with a size corresponding to the number of seeds. For instance, if there are 66 seeds, the interval for top ranking node i will be [i-66/2, i+66/2]. We calculate the number of true positives among the proteins in the sliding window. We calculate the statistical significance in the sliding window by using a Fisher’s exact test. In the end, we obtain a plot that goes from the first position of the sliding window (ranking = # of seeds / 2 + 1) to the final position (500 - # of seeds/2). In each position, we show the result of the Fisher’s test calculation for the positions of the sliding window. We consider as enriched positions all the positions until the last sliding window giving a P-value < 0.05. 8 Supplementary Figure S1. Functional-based selection plot in the case of the example GUILDify v2.0 run using 96 seeds for asthma keyword and the NetScore algorithm with default parameters. Supplementary Figure S1. Functional-based selection plot in the case of the example GUILDify v2.0 run using 96 seeds for asthma keyword and the NetScore algorithm with default parameters. In the figure S1, we observe an example of the functional-based selection plot for asthma using 96 seeds. 6. Functional-based selection of top-ranking genes Therefore, the plot starts at position 49 and ends at position 452. The last sliding window with a P-value < 0.05 is at position 133, and ranges from position 85 to 181. We consider as enriched positions all the top-ranking genes until the 181st position, including the 96 initial seeds and 85 additional non-seed genes. 7. Significance of the overlap between genes/functions To calculate the significance of the overlap between top-ranking genes and their functions, we use a one-sided (the alternative hypothesis is that the odds ratio based on the overlap is greater than the observed odds ratio) Fisher’s exact test with the contingency table given in Supplementary Table S2. Supplementary Table S2. Contingency table used to calculate the significance of the overlap between top-ranking genes and their functions. Top 2 Non-top 2 Top 1 Nº common Nº top 1 – Nº common Non-top 1 Nº top 2 – Nº common Nº total – Nº top 1 – Nº top 2 – Nº common Top 2 Non-top 2 Top 1 Nº common Nº top 1 – Nº common Non-top 1 Nº top 2 – Nº common Nº total – Nº top 1 – Nº top 2 – Nº common Supplementary Table S3 is an example of the contingency table for the genetic overlap between asthma and rheumatoid arthritis, where we obtain an Odds Ratio of 23.542 and a P-value of 5.910-48. 9 Supplementary Table S3. Contingency table used in Fisher's exact text for the genetic overlap between asthma and rheumatoid arthritis. Top genes 2 Non-top genes 2 Top genes 1 55 290 – 55 = 235 Non-top genes 1 181 – 55 = 126 13,090 – 181 – 290 + 55 = 12,674 The contingency table for the functional overlap of the same example, where we obtain an Odds Ratio of 155.334 and a P-value of 1.310-58 is below (Supplementary Table S4). Supplementary Table S4. Contingency table used in Fisher's exact text for the functional overla between asthma and rheumatoid arthritis Top functions 2 Non-top functions 2 Top functions 1 38 84 – 38 = 46 Non-top functions 1 94 – 38 = 56 10,670 – 94 – 84 + 38 = 10,530 Supplementary Table S5. Job IDs to access to the case studies in the web server and parameters used. 7. Significance of the overlap between genes/functions Keyword Parameters Job ID asthma Seeds: all genes Organism: Homo sapiens Tissue: All Network: BIANA Method: NetScore (nRepetition=3, nIteration=2) asthma “rheumatoid arthritis” Seeds: all genes Organism: Homo sapiens Tissue: All Network: BIANA Method: NetScore (nRepetition=3, nIteration=2) rheumatoid_arthritis “non small cell lung carcinoma” Seeds: all genes Organism: Homo sapiens Tissue: All Network: BIANA Method: NetScore (nRepetition=3, nIteration=2) non_small_cell_lung_carcinoma “breast cancer” Seeds: all genes Organism: Homo sapiens Tissue: All Network: BIANA Method: NetScore (nRepetition=3, nIteration=2) breast_cancer 10 P 10 “breast cancer” Seeds: DisGeNET and OMIM Organism: Homo sapiens Tissue: All Network: BIANA Method: NetScore (nRepetition=3, nIteration=2) breast_cancer_omim_disgenet afatinib Seeds: all genes Organism: Homo sapiens Tissue: All Network: BIANA Method: NetScore (nRepetition=3, nIteration=2) afatinib ceritinib Seeds: all genes Organism: Homo sapiens Tissue: All Network: BIANA Method: NetScore (nRepetition=3, nIteration=2) afatinib crizotinib Seeds: all genes Organism: Homo sapiens Tissue: All Network: BIANA Method: NetScore (nRepetition=3, nIteration=2) crizotinib erlotinib Seeds: all genes Organism: Homo sapiens Tissue: All Network: BIANA Method: NetScore (nRepetition=3, nIteration=2) erlotinib gefitinib Seeds: all genes Organism: Homo sapiens Tissue: All Network: BIANA Method: NetScore (nRepetition=3, nIteration=2) gefitinib palbociclib Seeds: all genes Organism: Homo sapiens Tissue: All Network: BIANA Method: NetScore (nRepetition=3, nIteration=2) palbociclib 8. Results of the case study of asthma and arthritis rheumatoid and the negative controls with breast cancer We query “asthma” in GUILDify v2.0, obtaining 96 seeds. After the running GUILD and selecting the functionally-coherent top genes, we obtain 181 genes conforming the neighbourhood of asthma. We do the same for “rheumatoid arthritis”, retrieving 158 seeds and creating a neighbourhood of 290 functionally-coherent top genes. Between the top ranking genes of the two phenotypes there are 55 common genes (Fisher’s exact test, one-sided P-value = 5.9·10-48) which 11 is more significant than the 12 common genes between the seeds (P-value = 1.4·10-9). When removing the seeds from the top ranking genes of the two phenotypes, we find an overlap of 43 genes which is even more significant than before (P-value = 3.7·10-65). If we focus on the functional overlap, we find 38 common enriched functions from the top ranking genes (P-value = 1.3·10-58), 18 common enriched functions from the seeds (P-value = 1.7·10-24), and 24 common functions removing the seed-functions from the top-functions (P-value = 3.5·10- 47). To have a negative control, we query “breast cancer” and retrieve 119 seeds. We select the 182 functionally-coherent top genes and compare the neighbourhood with the phenotypes of asthma and rheumatoid arthritis. In the case of asthma and breast cancer, we observe a significant overlap between 8 genes (P-value = 3.8·10-3), but the functional overlap is not significant (only 1 common function). In the case of rheumatoid arthritis and breast cancer the overlap is not significant. It is important to remark that 101 of the 119 breast cancer seeds are from Uniprot and may not be as much reliable. We repeated the same analysis selecting only 28 seeds from OMIM and DisGeNET, and in general the results of the overlap are not significant neither for asthma nor rheumatoid arthritis. The results can be explored with more detail in Supplementary Table S6. Supplementary Table S6. Results of the genetic and functional overlap between the subnetwork of genes associated to asthma and rheumatoid arthritis, asthma and breast cancer, and rheumatoid arthritis and breast cancer. Breast cancer has been calculated either using DisGeNET and OMIM seeds (D+O) or using all the seeds (all). P-values have been corrected using the Benjamini-Hochberg correction for multiple tests. Results with non-significant P-value are highlighted in red. Genetic overlap Functional overlap Top Top without seeds Seeds Top Top without seeds Seeds Nº P-val. Nº P-val. Nº P-val. 8. Results of the case study of asthma and arthritis rheumatoid and the negative controls with breast cancer Nº P-value Nº P-value Nº P-value Asthma – Rheumatoid arthritis 55 2.90E-47 43 1.80E- 64 12 7.00E- 09 31 4.00E- 45 18 5.50E- 34 18 4.60E-22 Asthma – Breast cancer (all) 8 9.50E-03 5 1.30E- 04 3 9.50E- 02 1 2.10E- 01 1 1.40E- 02 0 1 Rheumatoid arthritis – Breast cancer (all) 4 7.20E-01 2 2.30E- 01 2 5.20E- 01 0 1 0 1 0 1 Asthma – Breast cancer (D+O) 2 2.30E-01 0 1 2 4.50E- 02 0 1 0 1 0 1 Rheumatoid arthritis – Breast cancer (D+O) 0 1 0 1 0 1 0 1 0 1 1 1 12 9. Comparison of the case study results of asthma and rheumatoid arthritis with DIAMOnD We have compared the functional enrichment of top-ranking genes identified by NetScore and DIAMOnD in asthma and rheumatoid arthritis. We based the analysis in Sharma et al., where the authors present a comparison of DIAMOnD and several other prioritisation algorithms, showing that DIAMOnD outperforms existing algorithms in predicting asthma related genes [31]. Following the procedure described in the original article, for each seed-gene, we determine the set of MSIgDB pathways [32] associated with the gene. For each pathway, we analyse its enrichment among the set of seed-genes using Fisher’s exact test. The p-values are corrected using the Benjamini-Hochberg correction procedure for multiple tests. We choose the pathways with a significance level of p-value<0.01 as being associated with the set of seed-genes (enriched pathways). We calculate a Fisher’s exact test between the top-ranking genes (based on functional-coherency) of the algorithm under analysis and the genes of the enriched pathways. The p-value of the Fisher’s exact test gives the enrichment of the top-ranking genes. The enrichment of the pathways using top-ranking genes from NetScore and DIAMOnD as well as using the original set of seed-genes are shown in Supplementary Figure S2. When measuring the overlap between the two diseases, NetScore outperforms DIAMOnD, finding more genes involved in the pathways enriched in both diseases. Supplementary Figure S2. Bar plot showing the enrichment of the top-ranking genes in terms of -log p-value. Supplementary Figure S2. Bar plot showing the enrichment of the top-ranking genes in terms of -log p-value. 2. Set of pre-calculated drugs The list of drugs was obtained by retrieving all drugs with targets stored in BIANA knowledge database. Out of this list, we selected those drugs with at least 10 targets (retrieved from at least one of the following databases in BIANA: DrugBank, ChEMBL, DGIdb, DrugCentral), obtaining a final list of 362 drugs. We run the prioritisation using the R package and default parameters (BIANA network, NetScore algorithm). 10. Screening diseases to identify potential new indications of known drug UILDify v2.0 introduces a “Drug Repurposing” functionality that can be accessed from the home 13 page of the web server. This functionality takes a job ID as input, i.e. results for a drug (or a disease) and screens across a set of pre-calculated diseases (or drugs) for the significance of the overlap of genes and functions between the given job ID and the set of pre-calculated diseases (or drugs). The generation of the pre-calculated sets and the validation of the drug repurposing approach using these sets are explained below. 3. Set of drug-disease indications We tested the quality of the predictions of indications of drugs using as benchmark the indications of Hetionet [33]. Accordingly, we used 161 drugs and 64 diseases that appeared in both Hetionet and the lists of pre-calculated drugs and diseases, producing a final set of 329 drug-disease pairs with known indications. 1. Set of pre-calculated diseases We created a list of diseases using the UMLS concept unique identifiers from DisGeNET. Specifically, we obtained all diseases with gene associations reported by curated sources (UniProt, ORPHANET, PsyGeNET and HPO). We did not include CTD because it provides several clinically ambiguous phenotypes such as “liver cirrhosis, experimental”. From this list, we selected those diseases associated to at least 10 gene from DisGeNET, obtaining a final list of 757 diseases. We ran the prioritisation using the guildifyR package and the default parameters (BIANA network, and NetScore algorithm). 4. Finding the indication among the top-ranked results We plotted a histogram of the correct indications among the top ranked (see Supplementary Figure S3). This showed that 30% of the correct indications were already among the top 10 indications selected (and 50% of correct indications appeared among the top 25 predicted indications). We calculated how many disease indications could be guessed depending on the number of top- ranked indications for a drug. We transformed this calculation in True Positive Rate (TPR) and False Positive Rate (FPR), calculated as: 𝑇𝑃𝑅= # 𝑡𝑟𝑢𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑠 # 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑠 = # 𝑔𝑢𝑒𝑠𝑠𝑒𝑑 𝑖𝑛𝑑𝑖𝑐𝑎𝑡𝑖𝑜𝑛𝑠 # 𝑡𝑜𝑡𝑎𝑙 𝑖𝑛𝑑𝑖𝑐𝑎𝑡𝑖𝑜𝑛𝑠 14 Using the TPR and FPR we plotted the Receiver Operating Characteristic (ROC) curve (see Supplementary Figure S3). Using the top-scoring 1% of genes and functions, we obtained an Area Under the Curve (AUC) of 0.59 for genetic overlap and 0.61 for functional overlap. Supplementary Figure S3. Cumulative distribution of the ranking positions achieved by the indications using the drug repurposing feature of GUILDify v2.0. Supplementary Figure S3. Cumulative distribution of the ranking positions achieved by the indications using the drug repurposing feature of GUILDify v2.0. Supplementary Figure S4. Receiver Operating Characteristic (ROC) curve. The values of the Area Under the Curve (AUC) are indicated in the legend. Supplementary Figure S4. Receiver Operating Characteristic (ROC) curve. The values of the Area Under the Curve (AUC) are indicated in the legend. 15 11. Visualisation of the top-ranking subnetwork In the results page, we provide a visualisation panel to inspect in detail the interactions between the top-ranking nodes. Initially, we provide the user with an image of the subnetwork created with Matplotlib Python library [34]. The users have the option to click to “activate interactive visualization”, where the top-ranking nodes and interactions are displayed in a visualisation panel using the JavaScript-based network visualisation library, Cytoscape.js [35]. In addition to seeds (green hexagons), top-ranking proteins (yellow circles) and drugs (blue diamonds), the subnetwork includes the proteins that connect the seeds to the largest connected component induced by seeds (named “linkers” and shown as grey circles). 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https://openalex.org/W2565406467	https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-016-3326-6	English	null	TEA: the epigenome platform for Arabidopsis methylome study	BMC genomics	2,016	cc-by	7,118	© The Author(s). 2016 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. * Correspondence: paoyang@gate.sinica.edu.tw; cylin@iis.sinica.edu.tw †Equal contributors 4Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 2Institute of Information Science, Academia Sinica, Taipei, Taiwan Full list of author information is available at the end of the article Abstract Background: Bisulfite sequencing (BS-seq) has become a standard technology to profile genome-wide DNA methylation at single-base resolution. It allows researchers to conduct genome-wise cytosine methylation analyses on issues about genomic imprinting, transcriptional regulation, cellular development and differentiation. One single data from a BS-Seq experiment is resolved into many features according to the sequence contexts, making methylome data analysis and data visualization a complex task. Results: We developed a streamlined platform, TEA, for analyzing and visualizing data from whole-genome BS-Seq (WGBS) experiments conducted in the model plant Arabidopsis thaliana. To capture the essence of the genome methylation level and to meet the efficiency for running online, we introduce a straightforward method for measuring genome methylation in each sequence context by gene. The method is scripted in Java to process BS- Seq mapping results. Through a simple data uploading process, the TEA server deploys a web-based platform for deep analysis by linking data to an updated Arabidopsis annotation database and toolkits. Conclusions: TEA is an intuitive and efficient online platform for analyzing the Arabidopsis genomic DNA methylation landscape. It provides several ways to help users exploit WGBS data. TEA is freely accessible for academic users at: http://tea.iis.sinica.edu.tw. Sheng-Yao Su1,2,3†, Shu-Hwa Chen2†, I-Hsuan Lu2, Yih-Shien Chiang4, Yu-Bin Wang2, Pao-Yang Chen4* and Chung-Yen Lin2,5,6* From 15th International Conference On Bioinformatics (INCOB 2016) Queenstown, Singapore. 21-23 September 2016 From 15th International Conference On Bioinformatics (INCOB 2016) Queenstown, Singapore. 21-23 September 2016 Background an epigenetic marker, i.e., to alter genome activity with- out changing the sequence context [5–7]. 5-methylcytosine (5 mC) is the best-characterized methylation type in genomic DNA. It can be classified into three different sequence contexts by the neighboring bases, CG, CHG, and CHH (H = A, C, or T). In plants, CG methylation is maintained by DNA methyltransferase 1 (MET1), the plant homolog of DNMT1 [8–12]. The plant-specific DNA methyltransferase chromomethylase 3 (CMT3) activity promotes CHG methylation, which is linked to the histone H3 lysine 9 (H3K9) dimethylation condition [13–15]. Asymmetric CHH methylation is me- diated by two DNA methyltransferases: chromomethylase 2 (CMT2), and domains rearranged methyltransferase 2 (DRM2) [16]. CMT2 mainly functions at the pericentro- meric and long transposable element region, and DRM2 mediates CHH methylation through the RNA-directed DNA methylation (RdDM) pathway [17, 18]. The fact that an epigenetic marker, i.e., to alter genome activity with- out changing the sequence context [5–7]. an epigenetic marker, i.e., to alter genome activity with out changing the sequence context [5–7]. 5-methylcytosine (5 mC) is the best-characterized methylation type in genomic DNA. It can be classified into three different sequence contexts by the neighboring bases, CG, CHG, and CHH (H = A, C, or T). In plants, CG methylation is maintained by DNA methyltransferase 1 (MET1), the plant homolog of DNMT1 [8–12]. The plant-specific DNA methyltransferase chromomethylase 3 (CMT3) activity promotes CHG methylation, which is linked to the histone H3 lysine 9 (H3K9) dimethylation condition [13–15]. Asymmetric CHH methylation is me- diated by two DNA methyltransferases: chromomethylase 2 (CMT2), and domains rearranged methyltransferase 2 (DRM2) [16]. CMT2 mainly functions at the pericentro- meric and long transposable element region, and DRM2 mediates CHH methylation through the RNA-directed DNA methylation (RdDM) pathway [17, 18]. The fact that Genomic DNA methylation is a long observed phenomenon. It was first described in prokaryotes and was found as a defense system of the genome against foreign DNA invasion (e.g., R-M system, [1]). In eukaryotes, it is known to play roles in regulating gene activity [2–4]. DNA methylation can be a stable change, such as gen- omic imprinting in diploid organisms to label the par- ental origin of the genome constituents in the zygote or to regulate gene dosage in sexual dimorphic chromo- somes. The Author(s) BMC Genomics 2016, 17(Suppl 13):1027 DOI 10.1186/s12864-016-3326-6 The Author(s) BMC Genomics 2016, 17(Suppl 13):1027 DOI 10.1186/s12864-016-3326-6 The measurement of methylation level The methylation landscape in TEA is based on the gene, in which DNA methylation levels in the promoter and gene body are estimated from the WGBS data. The DNA methylation level for individual cytosines is esti- mated as Equation (1.1): the DNA methylation level for individual cytosine i ¼ Ci ¼ # read C # readC þ readT ð1:1Þ ( ) g g y Arabidopsis thaliana is a popular model plant. It is a selfing (or self-fertilization) species with a small and completely sequenced genome from the standard strain Col-0. The genome of A. thaliana is comprised of five nuclear chromosomes plus mitochondrial and chloro- plast DNA. The present genome build, TAIR10, is 135 Mb. Arabidopsis has been used in a wide range of studies which are collected in a centralized resource [29]. One special interest in studying plant genomes is the relationship between DNA methylation and trans- posable element silencing. In plants, most DNA methy- lation occurs at transposable elements and other repetitive DNA sequences. The DNA methylation path- way is described as a powerful tool for flowering plants to silence these high amounts of transposon parasites in their genomes [30]. Recently, a comprehensive survey on the Arabidopsis methylome published a whole series of BS-Seq data from a panel of gene silencing mutants at single-nucleotide resolution [31]. Reanalyzing the openly accessible methylome data can serve as a good starting point for bench scientists to conceive of new studies; however, it is often hampered by a ceiling on data- handling skills. Various features according to the se- quence contexts and locations need to be resolved and compared. The workflow for a BS-Seq data analysis process is more complicated than a transcriptome workflow. ð1:1Þ Calculating the average promoter or gene body methy- lation levels within the promoter or gene body is then the average Ci within the range by Equation (1.2): Average DNA methylation level in promoter or gene body ¼ X i∈X ci X i∈X 1 ð1:2Þ X b d ð1:2Þ X = promoter or gene body We count the number of reads mapped to each C with a minimum threshold of four, making an average methy- lation percentage of at least five C sites of each sequence context type, and give six measurements (i.e., pmt-CG, pmt-CHG, pmt-CHH, gene_CG, gene_CHG, gen- e_CHH) of a gene. Background Recent studies reveal that genomic DNA methy- lation can be changed dynamically; working together with histone codes, DNA methylation is recognized as * Correspondence: paoyang@gate.sinica.edu.tw; cylin@iis.sinica.edu.tw †Equal contributors 4Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 2Institute of Information Science, Academia Sinica, Taipei, Taiwan Full list of author information is available at the end of the article The Author(s) BMC Genomics 2016, 17(Suppl 13):1027 Page 142 of 193 Page 142 of 193 for performing analysis online, we introduce a straight- forward method for measuring genome methylation level in each sequence context by gene. This method is imple- mented in an in-house program (EpiMolas.jar) to process BS-Seq mapping results from CGmap by BS- Seeker2 [32] and CX_report by Bismark [33] into a small, tab-delimited data file, mtable. The mtable upload process triggers a website deployment by linking data files to gene annotation databases (TAIR10, KEGG, GO etc.) and to versatile data analysis and display modules in TEA. Using this user-friendly interface, the summary of gene and promoter methylation levels among experi- ment conditions are retrieved and analyzed easily. methylation of cytosine in different sequence contexts is maintained/modified by different enzymes and pathways implies complex regulation of genomic methylation status and attracts researchers interested in various subjects. j Bisulfite sequencing (BS-Seq) has become a standard technology to profile genome- wide DNA methylation at single-base resolution. Briefly, genomic DNA is treated with sodium bisulfite before high-throughput next generation sequencing (NGS). Bisulfite modification converts non-methylated cytosines to uracils (read as T in the sequencing reaction) while methylated cytosines remain unchanged. The frequency of C/C + T at each C is calculated from reads mapped to the position to give the cytosine methylation measurement at each C in the genome. Tools have been developed to deal with whole- genome BS-Seq (WGBS) data, such as BiSeq [19], BSmooth [20], DMAP [21], methPipe [22], methylKit [23], methylPipe [24], methylSig [25], MOABS [26], rad- Meth [27], and WBSA [28]. Most of these analysis tools focus on identifying differentially methylated regions (DMRs) and defining differentially methylated genes (DMGs) as genes with DMRs in or near the gene body. The measurement of methylation level Whole Genome Bisulfite sequencing is actually considered the gold standard approach for profiling genome-wide DNA methylation [34], and has become the standard profiling method in major epige- nome consortiums, such as NIH Roadmap [35], EN- CODE [36], Blueprint [37], and IHEC [38]. The major sources of error originate from DNA sequencing errors and incomplete bisulfite conversion. The former is now well controlled, with the advancement of NGS, and is generally <0.01%. The latter is usually >99.5% in a stable lab practice. When a region such as a promoter or gene body is analyzed, TEA requires at least five cytosines to In this paper, we present an intuitive data analysis plat- form for Arabidopsis whole-genome bisulfite sequencing (WGBS) data, TEA (The Epigenomic platform for Arabi- dopsis, http://tea.iis.sinica.edu.tw). To crop the essence of genome methylation status and to meet the efficiency Page 143 of 193 The Author(s) BMC Genomics 2016, 17(Suppl 13):1027 Page 143 of 193 be included in order that any potential bias from indi- vidual cytosines can be minimized. Institute of Information Science, Academia Sinica, Taiwan. The TEA annotation database includes genome structure and gene annotation for A. thaliana. It based on Ensembl (Ensembl Plants release 32) and function-rich annotations like GO terms from Gene Ontology (Aug. 2016) and KEGG Pathways (Apr. 2016). Data retrieving, integration and real-time calculation in the analysis process are implemented in scripts written in Java (OpenJDK 7), Python (version 2.7), and PHP (version 5.1). Graphical visualization used HTML Canvas and SVG library to provide a high level of data interactivity. Biodalliance gen- ome browser (version 0.13.7) [39] and Circos software package (version 0.69) [40] were integrated for browsing the genome structure and for accessing a gene list of chromosome coordinates, respectively. The methylation level is thus a normalized score from 0 (all observed sites are unmethylated) to 1 (all observed sites are methylated), or “NaN” for genes that do not have sufficient reads/sites to calculate the index. The overall calculation process is shown in Fig. 1. The method is scripted in Java to process BS-Seq map- ping results, e.g., .CGmap from BS SEEKER2 [32] or CXre- port.txt from Bismark [33]. This program EpiMolas.jar (download from link in http://tea.iis.sinica.edu.tw/tea/mta ble.html) also requires the Arabidopsis gene annotation file (gtf) to calculate the methylation measurement file mtable. mtable is a tab-delimited pure text file, indexed by gene identifiers and six measurements of methylation levels. Heat map for 2D presentation in color p p The heat map plot is implemented in java with jquery (version 2.1.4) and d3.js libraries (version 3.5.17, https:// d3js.org/). Tree topologies of the dataset grouping and the methylation level of the subjects (promoter or gene body for a selected C context) are calculated by the SIN- GLE_LINKAGE in EUCLIDIAN_ DISTANCE method. We added tooltip functions to enhance the legibility as well as to access source data intuitively. For example, we introduced CanvasRenderingContext2D.drawImage with d3 to zoom and pan the heat map on the gene name The measurement of methylation level It is the upload format for the TEA server. Processes for execut- ing EpiMolas.jar to generate mtable are described in TEA online help. Venn diagram for all possible logical relationships A working platform in TEA starts with a data uploading process. Users should make their mtable first (described previously). A step-by-step process guides users to de- fine and check the overall data-to-TEA database map- ping conditions in real time. The data deployment will take a few minutes to complete. Measurements on pro- moter regions of RNA genes (rRNAs, pre-tRNA, snRNAs, snoRNAs, miRNAs, and other RNA genes) and transposable genes (TE) are not included on the TEA- derived website. The whole process of TEA is illustrated in Fig. 2. Data-uploading details can be viewed through the demo tutorial on “New Submission”. Unregistered users can upload the data to TEA and run “have a trial” to get the analysis in a dynamic URL for 1-month access. g g For showing and counting all possible logical relation- ships between a finite collection of different sets, we im- plemented a Venn plot generator to render a diagram. It is a pure java script developed by our team to generate SVG without dependency on jquery or other libraries. This plot is also an interactive visualization tool to assist in subset selection without sophisticated Boolean oper- ation. The implemented Venn plot function can generate comparison results for up to four selected sets. All of the possible relationships among different gene sets can be saved as a new gene list to module “Gene List Analysis” for deciphering deeper biological meanings. System implementation TEA (The Epigenomic platform for Arabidopsis) is con- structed using LAPP system architecture (Ubuntu 14.04, Apache 2.04, PostgreSQL 9.1, and PHP 5.1) with bootstrap 3 CSS framework (http://getbootstrap.com/) to provide an intuitive user experience. The whole system runs in a vir- tual machine (CPUs of 2.27 GHz, four cores, 16 GB RAM and 500 GB storage) on the cloud infrastructure of the Fig. 1 Flowchart of methylation level extraction in EpiMolas.jar. EpiMolas.jar accepts output from several types of BS-seq mapping reports and generates six methylation profiles in three contexts, CG/CHG/CHH, and two locations, promoter/gene body, based on a gene annotated file Fig. 1 Flowchart of methylation level extraction in EpiMolas.jar. EpiMolas.jar accepts output from several types of BS-seq mapping reports and generates six methylation profiles in three contexts, CG/CHG/CHH, and two locations, promoter/gene body, based on a gene annotated file The Author(s) BMC Genomics 2016, 17(Suppl 13):1027 Page 144 of 193 The Author(s) BMC Genomics 2016, 17(Suppl 13):1027 converted to methylation data files (.mtable) using EpiMolas.jar as described previously. (the Y axis on a heat map) or on the dataset label (the X axis on a heat map). Users can select a subset of genes from the heat map by mouse clicking on the gene name and saving the list for the module “Gene List Analysis.” (the Y axis on a heat map) or on the dataset label (the X axis on a heat map). Users can select a subset of genes from the heat map by mouse clicking on the gene name and saving the list for the module “Gene List Analysis.” Data modules TEA provides several ways to help users exploit their uploaded high-throughput data (Fig. 3). The data mod- ules include the word search function on the annotation table, the gene ID list search function, the quantitative analysis functions on the differences (subtraction) or a threshold (cutoff) of a methylation measurement, and the canonical view in pathways. The gene list from all of these data retrieval steps can be kept as a gene list for later uses. Gene Ontology terms and KEGG pathway enrichment calculation A summary of data mapping condition and usable measurements for each dataset and each methylation index are logged in the data website “home”. User should check this summary first. Unexpected values, such as a low gene id mapping rate or a low percentage of analyzable genes/promoters, are warning signs of problems in the data, for example, improper files were used in the data preprocessing steps or a low read throughput were used for resolving the methylome. Functional enrichment analysis pipelines are built in TEA for detecting overrepresented GO terms and KEGG entities in the given gene list compared with a random sampling from a whole-genome background. Custom- ized P-value is an applicable cutoff for displaying enriched GO terms or KEGG pathways. The gene set enrichment score is measured using the hypergeometric test in Equation (2): The basic data display view in TEA is the gene-centric page containing gene information, the methylation pro- file on CG, CHG, and CHH contexts of the gene (gene body) and the promoter region (−2500 to +500), an em- bedded genome browser, and gene function annotation (GO terms and KEGG pathways). The neighboring gene function allows users to browse the genes located up to a 5-Mb flanking range. p xjN; m; n ð Þ ¼ m x N−m n−x N n ð2Þ ð2Þ where x is the number of genes in the test set in a par- ticular annotation class (e.g., a given GO term), m is the gene number of the test set, N is the number of total genes being annotated in the whole sample space (i.e., the genome), n is the number of the genome in the given annotation class. Demo datasets The demo datasets are from Zhong et al. [41], which in- cludes five previously published WGBS datasets, GSM881756, GSM1193638, GSM981015, GSM981017, and GSM981040 [31, 42]. All of these bisulfite sequen- cing reactions were carried out in 50-mer single-end for- mat using an Illumina HiSeq2000. Raw read data files were downloaded from SRA/NCBI and were prepro- cessed with Cutadapt [43] to remove TruSeq adaptors. Cleaned reads were mapped to TAIR10 genome using BS Seeker2 [32] and Bismark [33]. The output files (*.CGmap /BS Seeker2 and CX_report.txt) were then Find Genes by Text In the module Full Text Search, users can find genes by Gene ID, gene symbol, gene description, and KEGG pathway description from the integrated annotation table in TEA. Further constraints on the The Author(s) BMC Genomics 2016, 17(Suppl 13):1027 Page 145 of 193 Fig. 2 The workflow of TEA from raw reads to data visualization and deep analysis. a Align BS-seq read to the reference genome. b Generate mtable from CGmap, the output of BS Seeker2, or CX_report, the output of Bismark. c The main portal of TEA for data submission. d Data visualization with Venn diagram, boxplot, histogram, heatmap, and Circos plot Fig. 2 The workflow of TEA from raw reads to data visualization and deep analysis. a Align BS-seq read to the reference genome. b Generate mtable from CGmap, the output of BS Seeker2, or CX_report, the output of Bismark. c The main portal of TEA for data submission. d Data visualization with Venn diagram, boxplot, histogram, heatmap, and Circos plot on pathway maps. Items in a given map can be saved as a gene list for further analysis. gene biotypes and chromosome location can be applied to refine the search simultaneously. In the module Import Genelist query, users can paste or upload a file of a list in TAIR10/ ensembl gene IDs or gene symbols and obtain the matches. In the module KEGG GlobalView, users can browse genes g y Find Genes by Values Module DMGs (differentially methylated genes) is a pairwise comparison workflow for two data pools (experimental conditions) to which a single or Fig. 3 Six data modules in TEA. The full text search module is to search for genes of interest in the keyword search from the context of gene ID, gene symbol, gene description, and KEGG description. Demo datasets With the aid of the data displaying approach “show Venn diagram”, overlapping of elements (i.e., genes) in different gene lists is easily solved. Further manipulation on Boolean algebra, such as union, intersection, or other combinations, can be carried out on the interactive diagram to generate a new gene list. Demo datasets Through a customized and flexible parameter setting, genes that fulfill the criteria are selected and can be further constrained on particular gene biotypes and chromosome locations. Module mC Threshold is used for selecting genes above or below a cutoff in at least one or among all of the selected datasets. These two modules provide different ways to access methylation landscapes in six different sequence contexts. trimmed and mapped the reads to the Arabidopsis refer- ence genome and produced the mtable file for each data- set. The five mtable files were uploaded to TEA and joined to TEA annotation database to build a data analysis website (http://tea.iis.sinica.edu.tw/tair10_demo_new/). p Firstly, we examined the summary of the five datasets. The percentage on gene id mapping and the usable mea- surements were acceptable. The methylation measure- ment data distribution showed a higher level of CG methylation in both gene body and promoter regions, agreeing with the general idea that CG is the major methylation type in the A. thaliana genome. Moreover, mutant drm2 had the most obvious effect (decreased methylation level) on the overall CHH methylation sta- tus in both gene body and promoter regions, whereas the drm3 mutant had the mildest effect. We further ex- amined the gene body CHH methylation level using a criterion of gene body CHH methylation level difference ≥0.15 from the control group (Col-1 + Col-2). Gene lists from the three comparisons performed in the “DMGs” module were analyzed in the module “GeneList Analysis” using the Venn diagram and heatmap plot approaches with 2D clustering (Fig. 4). The control-to- drm3 (Ctl_drm3) DMGs number were the smallest set. The heatmap-2D clustering result indicated that the profile of DMGs selected by gene body CHH level were alike in drm2 and nrpe1 (polV). Gene List Analysis Gene List Analysis Gene list items are derived from text search, id search, or a value comparison process and are kept for users’ needs. With the aid of the data displaying approach “show Venn diagram”, overlapping of elements (i.e., genes) in different gene lists is easily solved. Further manipulation on Boolean algebra, such as union, intersection, or other combinations, can be carried out on the interactive diagram to generate a new gene list. Gene List Analysis Gene list items are derived from text search, id search, or a value comparison process and are kept for users’ needs. Discussion The report of a BS-Seq mapping tool is often a table to count mappings on each C base in the reference gen- ome, numerating the “C” and “C + T” events with or without the sequence context notation. Although the mapping info has been degenerated a lot from the ori- ginal alignment result, the file size of the mapping report is still hard to handle in a web-based analysis tool. The measurement introduced in this study is a further reduc- tion on the methylation landscape from the single-base level to the gene level. Each measurement is an average of five observations (a particular C context) and each observation is based on at least four events (mapped reads). A deviation of 0.1 in the measurement reflects a change in the methylation state of 10% of the Cs in the observation. Although we cannot tell whether the changes are concentrated in a few sites or more dis- persed among the observed sites, using this straightfor- ward approach, we reanalyzed a published dataset and observed the same trends in the methylation landscapes caused by gene silencing. Demo datasets The DMGs module is to select differentially methylated genes (DMGs) on the basis of Fig. 3 Six data modules in TEA. The full text search module is to search for genes of interest in the keyword search from the context of gene ID, gene symbol, gene description, and KEGG description. The DMGs module is to select differentially methylated genes (DMGs) on the basis of customized criteria. The mc Threshold module is to select DMGs by a cutoff value. The Import Genelist module is to upload lists of genes of interest. The KEGG Global View module is to display which genes are involved in each category of KEGG pathways. The gene list analysis module is to view the gene set from different analytic approaches including Venn diagram, heatmap, Circos plot, GO terms, and KEGG pathway enrichment analysis Fig. 3 Six data modules in TEA. The full text search module is to search for genes of interest in the keyword search from the context of gene ID, gene symbol, gene description, and KEGG description. The DMGs module is to select differentially methylated genes (DMGs) on the basis of customized criteria. The mc Threshold module is to select DMGs by a cutoff value. The Import Genelist module is to upload lists of genes of interest. The KEGG Global View module is to display which genes are involved in each category of KEGG pathways. The gene list analysis module is to view the gene set from different analytic approaches including Venn diagram, heatmap, Circos plot, GO terms, and KEGG pathway enrichment analysis Fig. 3 Six data modules in TEA. The full text search module is to search for genes of interest in the keyword search from the context of gene ID, gene symbol, gene description, and KEGG description. The DMGs module is to select differentially methylated genes (DMGs) on the basis of customized criteria. The mc Threshold module is to select DMGs by a cutoff value. The Import Genelist module is to upload lists of genes of interest. The KEGG Global View module is to display which genes are involved in each category of KEGG pathways. The gene list analysis module is to view the gene set from different analytic approaches including Venn diagram, heatmap, Circos plot, GO terms, and KEGG pathway enrichment analysis The Author(s) BMC Genomics 2016, 17(Suppl 13):1027 Page 146 of 193 multiple datasets can be assigned. Data displaying approaches We designed several ways to deepen the view of a gene list including “Show gene list”, “Plot on the location of genes on chromosome map” “Calculate GO term enrich- ment”, “Calculate KEGG pathway enrichment”, and “Draw heatmap with 2D clustering”. As mentioned in previous paragraphs, genes can be selected for different reasons via different data modules. Using “show gene lists”, the selected genes can be saved as a GeneList item for later uses. The approach “Show Venn Diagram” is only available in module GeneList Analysis to help on sub-list selection. Demo case: BS-Seq data reanalysis To help users become familiar with TEA, we provide a demo dataset from Zhong et al. (2015) entitled “DO- MAINS REARRANGED METHYLTRANSFERASE3 con- trols DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis” [41]. Briefly, this study focused on elucidating the function of DRM3 (do- main rearranged methyltransferase 3) in the RdDM path- way, which contains a catalytically inactive enzyme domain, but is required for de novo DNA methylation in vivo. The major conclusions from this study are: 1. DRM3 has moderate effects on global DNA methylation and 2. DRM3 interacts with Pol V, implying regulatory roles of Pol V- involved RNA-directed DNA methylation. Five BS-Seq libraries were prepared from 3-week old leaf for detecting genome-wide methylation status under four conditions: two biological replicates in the control group and one dataset for each of the three gene silencing mutants (drm2, drm3, and nrpe1/Pol V catalytic domain). We first downloaded the raw read files from SRA/ NCBI, DMRs identified from different studies are not easily inferred as equivalent because the methods applied are not guaranteed to be compatible to each other. There- fore, a reanalysis of published datasets is necessary for reusing data. It is cost-effective to perform data mining Page 147 of 193 The Author(s) BMC Genomics 2016, 17(Suppl 13):1027 Fig. 4 A reanalysis of the case study dataset. a Using the criterion of differentially methylated (Δ = 0.15) on the methylation index CHH-gene, DMGs were selected from drm2, drm3, and nrpe1 (polV) mutants in comparison with the control group. b The left panel is a Venn diagram to show the overlap of genes among the three DMG sets. A resizable 2D heat map plot of the union set indicates the profile pattern (similarity) among the five datasets Fig. 4 A reanalysis of the case study dataset. a Using the criterion of differentially methylated (Δ = 0.15) on the methylation index CHH-gene, DMGs were selected from drm2, drm3, and nrpe1 (polV) mutants in comparison with the control group. b The left panel is a Venn diagram to show the overlap of genes among the three DMG sets. A resizable 2D heat map plot of the union set indicates the profile pattern (similarity) among the five datasets the input methylation data from the single-base level to a gene-level methylation profile. It provides several ways to help users exploit and discover their uploaded high- throughput data. Declarations h l h This article has been published as part of BMC Genomics Volume 17 Supplement 13, 2016: 15th International Conference On Bioinformatics (INCOB 2016). The full contents of the supplement are available online at https:// bmcgenet.biomedcentral.com/articles/supplements/volume-17-supplement-13. Acknowledgements Th h f d g The research was funded by Ministry of Science and Technology (MOST), Taiwan, for financially supporting this research through MOST 103-2311-B- 001 -033 -MY3 and Innovative Translational Agricultural Research Program (ITAR) from Academia Sinica, Taiwan to CYL, MOST 101-2321-B-001-043-MY2 and MOST 102-2811-B-001-046 to SHC, MOST-103-2313-B-001-003-MY3 and a grant from Academia Sinica to PYC. Demo case: BS-Seq data reanalysis It can also facilitate data sharing among cooperators. An unregistered usage is available for creating a working platform with a dynamic URL for one-month access. TEA is freely available for academic users. We welcome researchers to ask for cooperation, keeping the data on a password-controlled access web- site or an open-access website. before conducting a new experiment. In a more practical scenario, researchers can try to find a compatible dataset from some metadata depository, such as SRA/NCBI, TAIR, or MPSS [44] and conduct the analysis together with their experimental data to increase the power of the data. An interesting aspect of reanalyzing openly accessed data is to dig out some novel findings not men- tioned in the article, because either the authors were not interested in them, or the findings were not covered in the article’s scope. In summary, we present TEA, a streamlined WGBS analysis platform with versatile analysis and display modules. It provides a straightforward methodology to explore the methylation status in different sequence con- texts. The openly accessible TEA provides a dynamic URL for one-month access. Further extension of this work will include more model organisms in the platform and more sophisticated and robust models to select DMGs. 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Conclusions g Publication charges for this article have been partly funded by MOST-103- 2313-B-001-003-MY3 to PYC and partly supported by a grant from Academia Sinica to PYC. TEA is a user-friendly platform for WGBS analysis. It is fast and efficient to select the DMGs because it shrinks Page 148 of 193 Page 148 of 193 The Author(s) BMC Genomics 2016, 17(Suppl 13):1027 The Author(s) BMC Genomics 2016, 17(Suppl 13):1027 The Author(s) BMC Genomics 2016, 17(Suppl 13):1027 41. Zhong X, Hale CJ, Nguyen M, Ausin I, Groth M, Hetzel J, Vashisht AA, Henderson IR, Wohlschlegel JA, Jacobsen SE. Domains rearranged methyltransferase3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis. Proc Natl Acad Sci U S A. 2015;112(3):911–6. 42. Greenberg MV, Deleris A, Hale CJ, Liu A, Feng S, Jacobsen SE. Interplay between active chromatin marks and RNA-directed DNA methylation in Arabidopsis thaliana. PLoS Genet. 2013;9(11):e1003946. 43. Martin M. 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W4319316975.txt	https://drpress.org/ojs/index.php/fbem/article/download/4897/4747	en		Is Bitcoin A Blessing or A Curse?	Frontiers in business, economics and management	2,023	cc-by	1,745	Frontiers in Business, Economics and Management ISSN: 2766-824X \| Vol. 7, No. 2, 2023 Is Bitcoin A Blessing or A Curse? Yawen Lu1, * 1HD Ningbo School, CO 315100, China author: Yawen Lu (Email: 925210597@qq.com) *Corresponding Abstract: In this article, several perspectives of bitcoin are briefly stated. First, there is an introduction for bitcoin, such as the concepts and the process of its launch, in order to have a general idea. Then, I borrow a few main ideas from Adam Smith, which help to support that bitcoin is a blessing. Based on six behavioral motivations from Adam Smith, I tried to define the property of bitcoin and find out the development of bitcoin in the future. In the other hand, bitcoin is limited, which can not satisfy all the consumers and do not have a supervision department. Keywords: Bitcoin, Blessing, Adam Smith, Liberalism, Marxist. question that deserves our investigation and debates. 1. Introduction 2. Organization Bitcoin as an electronic currency was first introduced in 2008 when the world financial crisis broke out. Since then, this virtual form of currency has become a central topic across the world. Some people are optimistic and love to see the wide range of bitcoin used in different aspects of life, while others doubt its future prospect. Meanwhile, economists worldwide take a close look at the evolution of bitcoin but find its future remains unpredictable. Like it or not, bitcoin emerges in the age of quantification, financialization and digitalization. Traditionally, official currencies, backed by central governments and banks, are used as people believe they are a reliable store of value. Bitcoin is different in that it builds its trust on algorithms and encryption. Unlike the paper money backed up by gold depositories and credit of states, bitcoin is a decentralized payment system which is not governed by any central governments or banks. This system is based on the application of the blockchain technology in which “peers can connect and make financial exchanges within a network without the need for mediation with a central node, being it a bank, financial institution, or government)” (Baldwin 2). The system is a distributed peer-to-peer network, and all nodes therein are equal peers. Moreover, bitcoin depends on unique arithmetic. Solving algorithms to create new blocks—this can be seen as the mining for coins” (baidu baike). Despite its advantage as an alternative payment method, the total number of Bitcoin is limited. The currency system has only no more than 10.5 million in four years, and the total number thereafter will be permanently limited to 21million” (baidu baike). This may lead to fierce scramble for the blockchain. Nevertheless, ever since bitcoin was conceived, cyber-libertarians who hold the doctrine of free will in the internet firmly has been arguing that bitcoin would challenge the existing payment systems and even monetary systems. The emergence of bitcoin indeed shows the fact that a network society is coming into our horizon and perhaps the old economic system will come to the end. At this point we are faced with two completely different judgements about Bitcoin. The currency is either regarded as a revolutionary progress that will dramatically change our economic form or a privilege, or “a game of the few” that is only accessible for social upper class or simply a group of financial elites. Is Bitcoin a blessing or a curse is then a 2.1. A Liberalist View of Bitcoin The emergence of bitcoin ignites economic, social and ethical debate. Adam Smith (3), the founding father of the liberalist school, advocates the end of feudalism and mercantilist regulation by state monopolies and, instead, the establishment of ‘laissez-faire’ government”. He proposes six behavioral motivations, such as sympathy and desire of freedom. People are motivated because they are free to make choices according to their private interests in the market. In the Wealth of Nations, he claims that it would be better if people get out of the government control and follow the rules of nature in their economic activities. Clearly, bitcoin represents the core concept of economic liberalism, which is that “desires are to be accommodated not proscribed” (Berry 15). It meets the need of self-interests. Adam Smith demonstrates an ideal society where economic decisions are made between buyers and sellers out of appetite and desire. The same game can be applied to individual's choice of money. People are blessed to choose bitcoin because it promises to bring future economy with benefit and convenience. With this liberalist understanding of Bitcoin comes the notion of decentralization. As an alternative to official currencies, Bitcoin is then believed to be able to challenge existing financial authorities and give people back the rights to bargaining, financing, and setting prices. Some even claim that Bitcoin is “a socialist solution to a capitalist problem” (Baldwin 3). While it does seem too naive to entrust Bitcoin with the task of changing the entire capitalist system, its decentralizing potential is not unrealistic. To imagine this, we simply have to look at how electronic payment has in recent years dramatically changed our lives. In China, for example, whose memories of poverty and famine are still not very far away, it is nowadays extremely difficult to see people carrying purses or wallets while shopping, regardless of their age, social class, or income level. This makes us wonder, if the development of information technology can revolutionize our payment, why it is not possible for it to bring changes to our currencies, especially when Bitcoin and other virtual currencies are already invented? We can even compare the future of Bitcoin to smart phone. Nowadays, everyone has to have a smart phone, because we need to use it to pay and go 223 the same way as the bubble of the housing industry did. With Bitcoin in hands, one might one day become extremely rich, but it is likely that he will fall into destitute on the next morning. The lesson we learn from fetishism in the past three hundred years of capitalist expansion is that the glory of goods is never truly tangible. When a commodity is out there in the market, we feel anxious because we cannot resist its temptation. But when we finally buy it home, making it our own property, its glory soon vanishes, which makes us even more anxious, so that something else has to be bought as a compensation. Buying thus goes on forever, constantly infusing energy to capitalism, whereas the buyer eventually gets nothing. It this is the case, Bitcoin is a commodity that creates for the people nothing but desires for endless consumption. out as a traffic permit. When everyone cannot live a normal life without it, then Bitcoin is no doubt a blessing. However, in the past few years, people have turned their attention to another subject of debate: is the bitcoin system sustainable? The concern derives from the fact that the bitcoin doesn’t have an entity, it doesn’t have any exact value as a product. If buyers do not have this consensus, bitcoin is physically nothing. The question is what makes people to choose to use bitcoin. 2.2. Marxist View of Bitcoin Fetishism can be defined as a belief in the magical power or a worship of religion. Because the bitcoin doesn’t have any physical form, it doesn’t have any exact value as a product. We can consider that its existent value was given by the people who brought it or invest it. If buyers do not have consensus, bitcoin is physically nothing. The background of generation had a far-reaching influence in people’s subjective judgment. It is easy to form a situation, called follow the suit. This looks like people believe in religion. Because more and more people believed in the same religion, the religion became true. Most people who brought the bitcoin didn’t have a deep understand and blindly followed others. This is the same reason why there are so much people buying bitcoin. However, in the daily life, people will find the differences between the reality and their imagine. People will have a sense of unevenness. Finally, the trend of the situation may lose control. Also, bitcoin has an obvious exclusiveness which means the bitcoin does not lift a restriction to everyone in the world. If not all of the people participate in buying bitcoin, the currency may be glorified by the people who join in. In this case, the people who have no knowledge bitcoin may be ignore from the beginning. Besides, bitcoin may create an assumption. Although decentralization is a main character of bitcoin which seems governments lost their control of bitcoin, it’s still possible that the bitcoin was controlled by a powerful consortium. For example, Sanxing which is a famous smartphone producer intervened the trend of bitcoin. If this assumption is tenable, bitcoin may become an advertisement to propaganda of the consortium. If the consortium enhanced the thresholds of entry the bitcoin and negotiated each other, the bitcoin will be a course. Because it became a tool to help capitalism to gain money. All of these points to an opinion that all our fantasies revolving around Bitcoin may one day suddenly collapse in 3. Conclusion When we talking about whether the bitcoin is a blessing or not, we need to redefine the type of the bitcoin. If we define the bitcoin as a kind of investment, it will become a tool which the consortium gains the wealth. If we define it as a kind of new currency, it is likely that it will improve our lives. Bitcoin may replace the function of the existing currency so that more and more people will use it. This can avoid the problem of fetishism that always excludes the majority from its games. Above all, the final standard of our judgement can be grounded on the “Greatest Happiness Principle” proposed by Jeremy Bentham, which is the idea that a policy is good when it can create “the greatest happiness of the greatest number”. In the same way, we can say that Bitcoin will be a blessing when it can bring the greatest change for the greatest number of us. References [1] Baldwin Jon. “In digital we trust: Bitcoin discourse, digital currencies, and decentralized network fetishism.” Palgrave Communication 4.14 (2018): 1-10. Print. [2] Berry Christopher J. “Adam Smith’s moral economy” The Kyoto Economic Review 79.1 (2010): 2-15. Print. [3] “Liberalism.” Editorial. Baidu Baike. Web. 3 Jun. 2022. [4] “Jeremy Bentham.” Stanford Encyclopedia of Philosophy. Stanford University, 8 Dec. 2021. Web. 26 Jun. 2022. 224
https://openalex.org/W2303927938	https://europepmc.org/articles/pmc4808141?pdf=render	English	null	Missed opportunities of inclusion in a cohort of HIV‐infected children to initiate antiretroviral treatment before the age of two in West Africa, 2011 to 2013	Journal of the International AIDS Society	2,016	cc-by	10,715	Abstract Abstract Introduction: The World Health Organization (WHO) 2010 guidelines recommended to treat all HIV-infected children less than two years of age. We described the inclusion process and its correlates of HIV-infected children initiated on early antiretroviral therapy (EART) at less than two years of age in Abidjan, Coˆte d’Ivoire, and Ouagadougou, Burkina Faso. Methods: All children with HIV-1 infection confirmed with a DNA PCR test of a blood sample, aged less than two years, living at a distance less than two hours from the centres and whose parents (or mother if she was the only legal guardian or the legal caregiver if parents were not alive) agreed to participate in the MONOD ANRS 12206 project were included in a cohort to receive EART based on lopinavir/r. We used logistic regression to identify correlates of inclusion. Results: Among the 217 children screened and referred to the MONOD centres, 161 (74%) were included and initiated on EART. The main reasons of non-inclusion were fear of father’s refusal (48%), mortality (24%), false-positive HIV infection test (16%) and other ineligibility reasons (12%). Having previously disclosed the child’s and mother’s HIV status to the father (adjusted odds ratio (aOR): 3.20; 95% confidence interval (95% CI): 1.55 to 6.69) and being older than 12 months (aOR: 2.05; 95% CI: 1.02 to 4.12) were correlates of EART initiation. At EART initiation, the median age was 13.5 months, 70% had reached WHO Stage 3/4 and 57% had a severe immune deficiency. Conclusions: Fear of stigmatization by the father and early competing mortality were the major reasons for missed opportunities of EART initiation. There is an urgent need to involve fathers in the care of their HIV-exposed children and to promote early infant diagnosis to improve their future access to EART and survival. Keywords: children; West Africa; HIV; acceptability; early antiretroviral therapy; linkage to care; access to care. two months of age in reducing infant mortality in HIV- infected infants compared with those treated according to the World Health Organization (WHO) 2006 recommenda- tions: 4% versus 16% respectively [6]. Since then, the WHO has recommended the initiation of EART in all HIV-infected children before the age of 12 months, regardless of their disease progression [7], extended up to 24 months of age in 2010 [8]. Received 19 July 2015; Revised 22 January 2016; Accepted 22 February 2016; Published 23 March 2016 Copyright: – 2016 Dahourou DL et al; licensee International AIDS Society. This is an Open Access article distributed under the terms of the Creative Commons Attribution 3.0 Unported (CC BY 3.0) License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Consequently, a systematic early infant diagnosis of HIV was recommended for all HIV-exposed children from the age of six weeks [8] in order to initiate EART in those infected by HIV. Missed opportunities of inclusion in a cohort of HIV-infected children to initiate antiretroviral treatment before the age of two in West Africa, 2011 to 2013 De´sire´ L Dahourou§,1,2,3, Madeleine Amorissani-Folquet4,5, Malik Coulibaly1,2, Divine Avit-Edi4, Nicolas Meda1,3,6, Marguerite Timite-Konan7, Vic Arendt8, Diarra Ye6,9, Clarisse Amani-Bosse4, Roger Salamon2, Philippe Lepage10, Vale´riane Leroy11 for the Monod Anrs 12206 Study Group §Corresponding author: De´sire´ L Dahourou, MONOD Project, ANRS 12206, Centre de Recherche Internationale pour la Sante´, Ouagadougou, Burkina Faso, PO Box 09 BP 168, Ouagadougou, Burkina Faso. Tel: 226 78833924. (ddahourou@gmail.com) Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 Missed opportunities of inclusion in a cohort of HIV-infected children to initiate antiretroviral treatment before the age of two in West Africa, 2011 to 2013 Introduction two months of age in reducing infant mortality in HIV- infected infants compared with those treated according to the World Health Organization (WHO) 2006 recommenda- tions: 4% versus 16% respectively [6]. Since then, the WHO has recommended the initiation of EART in all HIV-infected children before the age of 12 months, regardless of their disease progression [7], extended up to 24 months of age in 2010 [8]. Consequently, a systematic early infant diagnosis of HIV was recommended for all HIV-exposed children from the age of six weeks [8] in order to initiate EART in those infected by HIV. The paediatric epidemic of HIV remains a major concern in the world. In 2012, three million children were living with HIV in- fection worldwide, 91% of whom were in sub-Saharan Africa [1]. Despite a decrease in HIV prevalence among pregnant women over the last decade, the epidemic is still widespread in Burkina Faso (1.7% prevalence in 2011 [2]) and in Coˆte d’Ivoire (4.5% prevalence in 2009 [3]). Although the coverage of prevention of mother-to-child transmission (PMTCT) programmes has improved in both countries, it still remains insufficient. In 2011, 67% and 29% of HIV-positive women received the PMTCT intervention in Burkina Faso [2] and Coˆte d’Ivoire [4], respectively, with a growing HIV paediatric epidemic. In the absence of any intervention, the mortality of HIV-infected children remains high, reaching 50% at the age of two [5]. However, access to EART for HIV-infected children is complex in the African context as operational issues further complicate the situation. Overall, access to antiretroviral therapy (ART) has significantly improved, but remains insufficient, with only 34% of eligible children in sub-Saharan Africa having access to treatment in 2012, compared to 64% of adults [1]. First, access to early infant diagnosis under the In 2008, the antiretroviral CHER trial demonstrated the efficacy of starting early antiretroviral therapy (EART) before 1 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 greater than 750/mm3; creatinine five times below the normal threshold; aspartate aminotransferase (AST) and alanine-amino-transferase (ALT) five times below the normal threshold and whose parents (or mother if she was the only legal guardian, or the legal caregiver if parents were not alive) consented to participation in the MONOD ANRS 12206 project, were initiated on EART. Data collection ’ d Patients’ data were collected prospectively using standar- dized questionnaires at their first contact, pre-inclusion and inclusion visits. The socio-demographic data of mothers and children and PMTCT exposure data were collected at the first contact visit. After obtaining the parents’ written informed consent to participate in the project, all socio-demographic data, data related to the father’s HIV status, DNA PCR to confirm the child’s HIV status, haematology, biochemistry, CD4 percentage and viral load data of the children were collected at the pre-inclusion visit (four weeks before inclusion). At the inclusion visit, children’s anthropometric and clinical data were collected. Clinical stage and immuno- deficiency were defined according to the WHO recommenda- tions issued in 2006 [16]. Study design We studied the inclusion process of children screened to be enrolled in the MONOD ANRS 12206 project until their confirmed inclusion with EART initiation. Eligible children were initially included in a 12-month therapeutic prospective cohort with a twice-daily EART combination (zidovudine (AZT) or abacavir (ABC))lamivudine (3TC)lopinavir/ritonavir (LPV/r) in addition to cotrimoxazole prophylaxis and ther- apeutic education (teaching the caregiver how to comply with the daily dosages and hours of treatment). Baseline categorical data are presented as frequencies (percentage), whereas continuous variables are presented using the mean and standard deviation (SD) for normally distributed continuous data or using median and interquar- tile ranges (IQR) for non-normally distributed continuous data. We compared categorical data using the chi-square test or Fisher’s exact test, and we used Student’s t-test or Wilcoxon rank-sum test to compare continuous data. Statistical analysis d ib d h Statistical analysis We described the pre-ART inclusion process of children initially diagnosed as HIV infected and the causes of non- inclusion. We described the socio-demographic characteris- tics of the children at their first contact and pre-inclusion visits according to their inclusion in the EART therapeutic cohort. For children with a confirmed HIV infection status, we analyzed the correlates of inclusion in the therapeutic cohort, using a multivariate logistic regression including all uncorre- lated variables associated in the univariate analysis with pB0.25. Then, we conducted a stepwise descending ad- justed analysis. Introduction Children diagnosed with tuberculosis at pre-inclusion were included in a separate cohort and received an efavirenz (EFV)-based therapy be- cause of the drug interaction between LPV/r and tuberculosis treatment [15]. age of 18 months requires sophisticated PCR techniques due to the persistence of maternal antibodies in the child’s blood. These techniques are not available everywhere [9]. Second among the barriers, the lack of service delivery and low acceptability by healthcare workers and the community are frequent [1012]. Consequently, providing a continuum of care for HIV-exposed children remains challenging. Those HIV-infected children who have a chance to access ART are frequently treated late, at an advanced age (median age greater than five years) and at an advanced stage of the disease [13,14]. age of 18 months requires sophisticated PCR techniques due to the persistence of maternal antibodies in the child’s blood. These techniques are not available everywhere [9]. Second among the barriers, the lack of service delivery and low acceptability by healthcare workers and the community are frequent [1012]. Consequently, providing a continuum of care for HIV-exposed children remains challenging. Those HIV-infected children who have a chance to access ART are frequently treated late, at an advanced age (median age greater than five years) and at an advanced stage of the disease [13,14]. Since the promulgation of the WHO 2010 recommenda- tions, no study has assessed the missed opportunities of the pre-EART cascade beyond HIV diagnosis and the acceptability of EART proposed in HIV-infected children less than two years old in West Africa. We studied the inclusion process and the family acceptability of EART as part of the pre-ART cascade and its correlates among HIV-infected children less than two years old in Abidjan and Ouagadougou through the descrip- tion of the inclusion process in the therapeutic cohort of the MONOD ANRS 12206 project. Methods Settings g The MONOD ANRS 12206 project was funded by the French National Agency for Research on AIDS and Viral Hepatitis (ANRS), the European and Developing Countries Trials Partnership (EDCTP) and the Luxembourg Institute of Health. This was a multicentre project launched in two West African countries (Burkina Faso and Coˆte d’Ivoire) targeted at EART implementation before the age of two as recommended by the WHO in 2010. This project was conducted in the two paediatric wards of the Yalgado Oue´draogo and Charles de Gaulle University Hospitals in Ouagadougou, Burkina Faso, and in two public paediatric HIV programme sites (FSU Abobo-Avocatier, CEPREF-Yopougon) as well as in the two paediatric wards of the Yopougon and Cocody University Hospitals in Abidjan, Coˆte d’Ivoire. Cohort profile From May 2011 to February 2013, 217 children had a first contact visit, and of these 161 were included in the MONOD cohort (156 in the initial cohort and 5 in the tuberculosis cohort). The cumulative rate of inclusion was 74%. The main reasons for non-inclusion (N56) were linked to the refusal of EART for the child by the father or father’s denial of child’s HIV status (27/56; 48%), early mortality (13/56; 24%), child found to be HIV negative on retested sample (9/56; 16%) and ineligibility for geographical reasons or due to severe disorders of baseline blood haematology/chemistry (7/56; 12%). Thus, among the 208 HIV-infected children confirmed eligible for treatment, 77% were initiated on EART (95% confidence interval (95% CI): 71% to 83%) (Figure 1). Correlates of inclusion were investigated in a multivariate analysis (Table 2). Children who were accompanied by both parents at the first contact visit were 10 times more likely to be included and initiated on EART. In the final adjusted analysis, we found that children were more likely to be included when they were older than 12 months at the first contact visit (adjusted odds ratio (aOR): 2.05; 95% CI: 1.02 to 4.12) and when the father was informed about the HIV status of both the mother and the child (aOR: 3.20; 95% CI: 1.55 to 6.69) (Table 2). Participants Weight-for-height, weight-for-age and height-for-age were used to assess children’s nutritional status. Each outcome was standardized (z-score) for age and gender using the WHO growth standards [17]. We defined underweight at baseline as weight-for-age z-score (WAZ) B2 SD and stunting as height-for-age z-score (HAZ) B2 SD. All HIV-1-infected children screened by HIV DNA PCR on dried blood spot samples under the umbrella of the national screening programme in urban centres, less than two years old and ART-naive except for PMTCT prophylaxis of HIV were referred with their parents (or the legal caregiver if parents were not alive) to one of the MONOD healthcare facilities in Abidjan and Ouagadougou to be pre-included. Those children with HIV infection confirmed by DNA PCR on blood sample, with haemoglobin levels greater than 7 g/dl; neutrophils All statistical analyses were performed with SAS 9.3 (SAS Institute Inc., Cary, NC, USA) except for anthropometric indicators, which were analyzed with ENA/Epi Info 3.5.1. 2 2 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 Ethical considerations median age at first contact. However, median age at first HIV diagnosis was significantly different between those included and non-included children (p0.03): children included were three months older at first HIV diagnosis than those not included. At the first contact visit, children were most often accompanied by both parents (56.7%); this proportion was significantly higher in children included (pB0.01). The mothers of the children included were more often single mothers compared to those of children not included (p0.02). Overall, 62% of fathers had already been informed of the HIV status of their own child and of the mother; this proportion was significantly higher in children included (pB0.01). Both parents (or the mother if she was the only legal guardian, or the legal caregiver if parents were not alive) of each child enrolled gave their written informed consent to participate in the MONOD ANRS 12206 project, which was approved by both national ethics committees of Burkina Faso and Coˆte d’Ivoire. The consent had to be signed no later than the pre-inclusion visit. Characteristics of eligible children according to their inclusion in the EART therapeutic cohort (N208) The baseline characteristics of the 161 children included in the MONOD cohort are described in Table 3 according to their study site. The median age at ART initiation of children enrolled was 13.7 months (IQR: 7.7 to 18.4). This age was significantly different between the two study sites (p0.03): children in Ouagadougou were two months older than those in Abidjan. The children were female (54%) and lived with both parents (67%) regardless of the study sites. Overall, the Table 1 provides the baseline characteristics of the children according to their inclusion in the MONOD cohort. Overall, the median age at first HIV diagnosis and at the first contact visit were 8.4 months (IQR: 2.9 to 15.6) and 11.7 months (IQR: 6.0 to 17.3), respectively; 54% were girls. According to inclusion in the MONOD cohort, there was no difference in First contacts: N = 217 (Abidjan: n = 135, Ouagadougou: n = 82) Pre-inclusions: N = 183 (Abidjan: n = 114, Ouagadougou: n = 69) Inclusions: N = 161 (Abidjan: n = 103, Ouagadougou: n = 58) • 4 deaths • 21 refusal linked to the father • 4 fathers not found • 1 trisomic child • 1 transfer out to initiate immediate ART • 3 not eligible (1 HIV not confirmed, 2 place of residence not compatible with the follow-up) • 9 deaths • 2 parents refusal • 3 not eligible (1 age > 24 months, 2 blood hematology/chemistry disorders) • 8 HIV-negative on retesting N = 34 N = 22 Tuberculosis cohort: N = 5 (Abidjan: n = 4, Ouagadougou: n = 1) Initial cohort: N = 156 (Abidjan: n = 99, Ouagadougou: n = 57) Figure 1. Cohort profile ofthe ANRS 12206 MONOD study, Abidjan, Ouagadougou, May 2011 to February 2013. Characteristics of eligible children according to their inclusion in the EART therapeutic cohort (N208) First contacts: N = 217 (Abidjan: n = 135, Ouagadougou: n = 82) 4 deaths 21 refusal linked to the father 4 fathers not found 1 trisomic child 1 transfer out to initiate immediate ART 3 not eligible (1 HIV not confirmed, 2 place of residence not compatible ART • 3 not eligible (1 HIV not confirmed, 2 place of residence not compatible with the follow-up) N = 22 Inclusions: N = 161 (Abidjan: n = 103, Ouagadougou: n = 58) Initial cohort: N = 156 (Abidjan: n = 99, Ouagadougou: n = 57) Tuberculosis cohort: N = 5 (Abidjan: n = 4, Ouagadougou: n = 1) Figure 1. Cohort profile ofthe ANRS 12206 MONOD study, Abidjan, Ouagadougou, May 2011 to February 2013. Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 Table 1. Characteristics of eligible children according to their inclusion in the EART therapeutic cohort (N208) Characteristics of eligible children according to their inclusion in the ANRS 12206 MONOD study, Abidjan, Ouagadougou, May 2011 to February 2013 Total n208 Not included n47 Included n161 Characteristics n (%) n (%) n (%) p Median child’s age (in months) at first contact (IQR) 11.9 (6.4 to 17.3) 9.5 (5.7 to 17.0) 12.9 (6.6 to 17.3) 0.17* Child’s age at first contact (in months) 0.08 B12 105 (50.5) 29 (61.7) 76 (47.2) ]12 103 (49.5) 18 (38.3) 85 (52.8) Median child’s age (in months) at first HIV diagnosis (IQR) 8.4 (2.9 to 15.6) 5.9 (2.4 to 13.0) 9.0 (3.4 to 16.0) 0.03* Age at first HIV diagnosis (in months) 0.07 B2 29 (14.1) 10 (22.2) 19 (11.8) ]2 177 (85.9) 35 (77.8) 142 (88.2) Gender 0.87 Male 95 (45.7) 21 (44.7) 74 (46.0) Female 113 (54.3) 26 (55.3) 87 (54.0) Vital status of the father B0.01 Alive 146 (70.2) 11 (23.4) 135 (83.8) Deceased 10 (4.8) 1 (2.1) 9 (5.6) Unknown/missing 52 (25.0) 35 (74.5) 17 (10.6) Person accompanying child at the first contact B0.01 Mother alone 80 (38.5) 38 (80.8) 42 (26.1) Father alone 3 (1.4) 0 (0.0) 3 (1.9) Both parents 118 (56.7) 8 (17.0) 110 (68.4) Legal guardian 2 (1.0) 0 (0.0) 2 (1.2) Other 5 (2.4) 1 (2.2) 4 (2.4) Mother still in touch with the child’s father 0.02 Yes 175 (84.1) 46 (97.9) 129 (80.1) No 19 (9.1) 0 (0.0) 19 (11.8) Deceased 10 (4.8) 1 (1.8) 9 (5.6) Unknown 4 (2.0) 0 (2.1) 4 (2.5) Father informed of the mother’s HIV status (N198*) B0.01 Yes 124 (62.6) 20 (43.5) 104 (68.4) No 66 (33.3) 25 (54.3) 41 (27.0) Unknown 8 (4.1) 1 (2.2) 7 (4.6) Father informed of his child’s HIV status (N198) B0.01* Yes 123 (62.1) 20 (43.5) 103 (67.8) No 74 (37.4) 26 (56.5) 48 (31.5) Unknown 1 (0.51) 0 (2.1) 1 (0.7) IQR: interquartile range; p-value from Wilcoxon rank-sum test; p-value from Fisher’s exact test; *excluding data for children whose fathers were deceased. teristics of eligible children according to their inclusion in the ANRS 12206 MONOD study, Abidjan, Ouagadougou, ruary 2013 IQR: interquartile range; p-value from Wilcoxon rank-sum test; p-value from Fisher’s exact test; excluding data for children whose fathers were deceased. 14% of the children. Characteristics of eligible children according to their inclusion in the EART therapeutic cohort (N208) The majority of the children were malnourished and started EART at an advanced clinical and immunological stage. These figures show a significant delay in Ouagadougou compared to Abidjan. In addition, we reported a limited access to PMTCT interventions, with more than half of the children included not having received any PMTCT interven- tion, despite this being recommended in these settings. of CD4 cells was 20% (IQR: 13.5 to 26.2) and 85% of children had moderate to severe immune deficiency at baseline. Children from Ouagadougou had a significantly lower CD4 percentage compared to those in Abidjan (pB0.01). Children included in Ouagadougou were also significantly more malnourished than those in Abidjan when considering all anthropometric indicators, with average weight-for-age, height-for-age and weight-for-height z-scores being signifi- cantly lower (pB0.01). The median haemoglobin level was 9.0 grams per decilitre (IQR: 8.2 to 9.9) and 80% of children had a haemoglobin level B10 grams per decilitre, with a proportion significantly higher in Ouagadougou than in Abidjan (pB0.01). The average viral load was 6 log10 (SD: 1.1), children from Ouagadougou having a significantly higher viral load compared to those in Abidjan (pB0.01) (Table 4). of CD4 cells was 20% (IQR: 13.5 to 26.2) and 85% of children had moderate to severe immune deficiency at baseline. Children from Ouagadougou had a significantly lower CD4 percentage compared to those in Abidjan (pB0.01). Children included in Ouagadougou were also significantly more malnourished than those in Abidjan when considering all anthropometric indicators, with average weight-for-age, height-for-age and weight-for-height z-scores being signifi- cantly lower (pB0.01). The median haemoglobin level was 9.0 grams per decilitre (IQR: 8.2 to 9.9) and 80% of children had a haemoglobin level B10 grams per decilitre, with a proportion significantly higher in Ouagadougou than in Abidjan (pB0.01). The average viral load was 6 log10 (SD: 1.1), children from Ouagadougou having a significantly higher viral load compared to those in Abidjan (pB0.01) (Table 4). p g g Of the 217 children referred to the MONOD study, 9% were found to be falsely HIV positive after the confirmatory test usually required before the initiation of EART. Indeed, the positive predictive value of this test depends on the pre- valence of the disease in the tested population. Characteristics of eligible children according to their inclusion in the EART therapeutic cohort (N208) Only 64% of the fathers reported having taken an HIV test, but this proportion was significantly higher in Abidjan than in Ouagadougou: 68% versus 57%, respec- tively (p0.02). Among the fathers who underwent screen- ing for HIV infection, 40% reported being HIV positive, of whom 51% were on ART. main caregivers were mothers (83%). However, more fathers in Ouagadougou (26%) than in Abidjan (1%) were the main caregivers (p0.01). In Abidjan, parents were significantly more likely to have access to tap water, electricity and refrigerators at home compared to those in Ouagadougou. Over half (51%) of the mothers did not receive any prenatal PMTCT intervention, and only 35% had ongoing ART at the time of inclusion of their child. Over half (51%) of the children included had not received any postnatal prophylaxis for PMTCT. Self-reported father’s HIV test was unknown for At the initiation of ART, 62% of the children had already reached a WHO clinical Stage 3 or 4: this proportion was significantly higher in Ouagadougou than in Abidjan, 88% versus 48%, respectively (pB0.01). The median percentage 4 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 Table 2. Factors associated with inclusion in the ANRS 12206 MONOD study, Abidjan, Ouagadougou, May 2011 to February 2013 (N208) Table 2. Factors associated with inclusion in the ANRS 12206 MONOD study, Abidjan, Ouagadougou, May 2011 to February 2013 (N208) Unadjusted bivariate analysis Adjusted multivariate analysis Characteristics OR 95% CI OR 95% CI Child’s age at first contact (months) B12 Reference Reference ]12 1.80 0.92 to 3.50 2.05 1.02 to 4.12 Child’s age at first HIV diagnosis (months) B2 Reference ]2 2.13 0.91 to 4.99 Gender Male Reference Female 0.95 0.49 to 1.82 Person accompanying child at the first contact Mother or legal guardian Reference Both parents or other 10.24 4.59 to 22.84 Father informed of HIV status of the mother and/or his child No/unknown Reference Reference Mother and child 3.04 1.50 to 6.19 3.20 1.55 to 6.59 Mother only 5.18 1.10 to 24.34 6.21 1.29 to 29.78 Child only 3.96 0.82 to 19.04 4.08 0.83 to 19.89 OR: odds ratio; CI: confidence interval. OR: odds ratio; CI: confidence interval. of age), were referred at a median age of 12 months and subsequently initiated ART at an advanced age (median 14 months). Characteristics of eligible children according to their inclusion in the EART therapeutic cohort (N208) As a result, in low HIV prevalence settings as in our context, the rate of false positives remains of concern and fully justifies the need for a systematic confirmatory test recommended by WHO [18]. According to an HIV transmission rate of 5%, the rate of false- positive DBS could reach 10.3% [19]. This finding confirms the need to verify the first HIV positive test result in the setting of intermediate HIV prevalence, but further highlights the need for lasting stringent quality control and confirmatory testing. In African settings, the long delay between the collection of the initial blood sample and the availability of the results after a confirmatory test complicates the disclosure of the initial positive result to the family, as well as the monitoring of the children over the cascade of care [20]. The problem of false- positive results can also undermine community trust in the reliability of early infant diagnosis and lead to possible diagnosis and EART delays. It also exposes HIV-uninfected children to the risk of antiretroviral side effects if they are not identified. Discussion Our study reported, for the first time, pre-EART attrition during the first 24 months of life in West African settings of intermediate HIV prevalence and in the context of very low coverage of early infant diagnosis (29% in Ouagadougou; 16% in Abidjan). The inclusion rate of children referred after a first positive DBS was 74%. The reasons for non-inclusion in the MONOD cohort included refusal of EART due to fear of stigmatization by the father, early competing infant mortality and false-positive HIV test on DBS. Good communication between parents, especially when the mother’s and the child’s HIV status are disclosed to the father, and being older than 12 months were two factors associated with a greater acceptability of EART in this research context. As a result of the low early infant diagnosis coverage in this context, children were diagnosed at a late age (median eight months 5 5 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org f y , http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 Table 3. Discussion Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 Table 3 (Continued ) Total n161 Abidjan n103 Ouagadougou n58 Characteristics n (%) n (%) n (%) p No 59 (57.3) 41 (58.6) 18 (54.5) Unknown 3 (2.9) 0 (0.0) 3 (9.1) Father on ART (N41) 0.87 Yes 21 (51.2) 14 (48.3) 7 (58.3) No 18 (43.9) 13 (44.8) 5 (41.7) Unknown 2 (4.9) 2 (6.9) 0 (0.0) ART: antiretroviral therapy; EART: early ART; PMTCT: prevention of mother-to child transmission; IQR: interquartile range; p-value from Wilcoxon rank-sum test; p-value from Fisher’s exact test; *self-reported. ART: antiretroviral therapy; EART: early ART; PMTCT: prevention of mother-to child transmission; IQR: interquartile range; p-value from Wilcoxon rank-sum test; p-value from Fisher’s exact test; self-reported. We show that EART is feasible in our context, reaching 77% of the children with a confirmed HIV diagnosis. However, despite the referral network set up to capture children eligible for EART through the MONOD project, the initiation of antiretroviral treatment was considerably delayed compared to the CHER strategy, especially in Burkina Faso, cumulating in delays in the pre-EART cascade (HIV diagnosis, HIV confirma- tion, EART initiation). The MONOD cohort aimed at initiating ART as soon as possible after early infant diagnosis (from 12 weeks). However, our findings showed that more than half of the children started EARTafter their first birthday, with high competing mortality before the initiation of EART. Children in Burkina Faso were older at the initiation of EART compared to those in Coˆte d’Ivoire. This difference between the study sites can be explained by the fact that there is a closer link between PMTCT services and postnatal services in Abidjan as compared to Ouagadougou. One of the healthcare centres of the MONOD project in Abidjan included a PMTCT project. Another likely explanation is the fact that the majority of children in Ouagadougou were recruited from the two university hospitals while they presented HIV-related symp- toms, which led the paediatricians to propose an HIV diagnostic test for them. This late HIV infection testing at a symptomatic stage of illness has already been described elsewhere in Ethiopia [21]. Other studies have indicated that paediatric ART is often started too late, when children are already advanced in their disease and are immunodeficient [2227]. Discussion This also occurred in the field reality of the MONOD settings and most likely explained the early high mortality rate, highlighting the importance of starting ART as early as possible. opportunities to diagnose children in hospital and the low acceptability of early screening by the community and healthcare workers [11,12,2932]. In our study, the cumulative rate of EART initiation before the age of two years reached 77%. Five studies conducted in Africa and Asia have reported proportions of ART initiation ranging between 40% and 99% [33]. This variability can be partly explained by the fact that the studies were performed at different timing regarding the subsequent recommenda- tions for antiretroviral treatment initiation. In addition, these studies have included older children with a median age ranging between 2.2 and 6.5 years [33]. In our study, we report that variables related to the quality of communication within the parental couple regarding maternal and child HIV status disclosure proved to be important factors to favour an EART initiation. EART initiation also depends on several socio- economic factors such as the cost of transportation, avail- ability of food aid, time constraint, perception of child health, stigma, beliefs and paternal support [34]. In our study, the cost of transportation was not considered as an obstacle as we provided transportation fees to the caregivers. The non- disclosure of the maternal and the child’s HIV status to the father was clearly a missed opportunity to initiate EART. A child’s HIV positive status usually indicates maternal infection and the resultant anxiety, fear of blame, social and healthcare discrimination as well as marital abandon- ment may negatively influence the mother’s acceptance of the HIV status of the child [35,36]. Braitstein and al. showed that 30% of children’s caregivers in Kenya reported that the child was lost-to-follow up because they did not want to share the child’s HIV status with their partner [37]. We also found that being older than 12 months at the first contact visit increased the probability of initiation of EART. This result was likely due to the delay in HIV diagnosis, with testing often performed late, but also reflects the perception of the child’s health by the parents. Indeed, parents agreed more readily to an HIV test and to initiation of therapy in children who were symptomatic [34]. Discussion Baseline socio-demographic characteristics of the 161 HIV-infected children included in the ANRS 12206 MONOD study according to site (Abidjan, Ouagadougou, May 2011 to February 2013) Total n161 Abidjan n103 Ouagadougou n58 Characteristics n (%) n (%) n (%) p Median child’s age (in months) at ART initiation (IQR) 13.6 (7.7 to 18.4) 12.9 (6.7 to 18.3) 14.6 (10.1 to 19.5) 0.03 Child’s age at EART initiation (in months) B12 70 (43.5) 50 (48.5) 20 (34.5) 0.08 ]12 91 (56.5) 53 (51.5) 38 (65.5) Gender 0.38 Male 74 (46.0) 50 (48.5) 24 (41.4) Female 87 (54.0) 53 (51.5) 34 (58.1) Main caregiver B0.01 Mother 133 (82.6) 93 (90.3) 40 (69.0) Father 16 (9.9) 1 (1.0) 15 (25.8) Other 12 (7.5) 9 (8.7) 3 (5.2) Type of housing 0.02 Individual housing 83 (51.5) 46 (44.7) 37 (63.8) Shared housing 78 (48.5) 57 (55.3) 21 (36.2) Tap water at home B0.01 Yes 108 (67.1) 87 (84.5) 21 (36.2) No 53 (32.9) 16 (15.5) 37 (63.8) Electricity at home B0.01 Yes 120 (74.5) 98 (95.2) 22 (37.9) No 41 (25.5) 5 (4.8) 36 (62.1) Fridge at home 0.02 Yes 36 (22.4) 29 (28.2) 7 (12.1) No 125 (77.6) 74 (71.8) 51 (87.9) Orphan 0.17 Both parents alive 146 (90.7) 91 (88.4) 55 (94.8) Mother deceased 6 (3.7) 6 (5.8) 0 (0.0) Father deceased 9 (5.6) 6 (5.8) 3 (5.2) Family structure 0.65 Two parents 109 (67.3) 71 (68.9) 38 (65.5) Single parent/missing 52 (32.1) 32 (31.1) 20 (34.5) Mother on ART 0.06 Yes 56 (34.8) 42 (40.8) 14 (24.1) No 94 (58.4) 53 (51.4) 41 (70.7) Unknown 11 (6.8) 8 (7.8) 3 (5.2) Maternal PMTCT prophylaxis 0.79 Yes 66 (41.0) 41 (39.8) 25 (43.1) No 83 (51.5) 55 (53.4) 28 (48.3) Unknown 12 (7.5) 7 (6.8) 5 (8.6) Postnatal PMTCT prophylaxis 0.93 Yes 66 (41.0) 43 (41.7) 23 (39.7) No 83 (51.5) 52 (50.5) 31 (53.4) Unknown 12 (7.5) 8 (7.8) 4 (6.9) Father tested for HIV 0.03 Yes 103 (64.0) 70 (68.0) 33 (56.9) No 35 (21.7) 24 (23.3) 11 (19.0) Unknown 23 (14.3) 9 (8.7) 14 (24.1) Father HIV positive* (N103) 0.06 Yes 41 (39.8) 29 (41.4) 12 (36.4) 1 to February 2013) Total n161 Abidjan n103 Ouagadougou n58 n (%) n (%) n (%) p 13.6 (7.7 to 18.4) 12.9 (6.7 to 18.3) 14.6 (10.1 to 19.5) 0.03 70 (43.5) 50 (48.5) 20 (34.5) 0.08 91 (56.5) 53 (51.5) 38 (65.5) 0.38 74 (46.0) 50 (48.5) 24 (41.4) 87 (54.0) 53 (51.5) 34 (58.1) B0.01 133 (82.6) 93 (90.3) 40 (69.0) 16 (9.9) 1 (1.0) 15 (25.8) 12 (7.5) 9 (8.7) 3 (5.2) 0.02 83 (51.5) 46 (44.7) 37 (63.8) 78 (48.5) 57 (55.3) 21 (36.2) B0.01 108 (67.1) 87 (84.5) 21 (36.2) 53 (32.9) 16 (15.5) 37 (63.8) B0.01 120 (74.5) 98 (95.2) 22 (37.9) 41 (25.5) 5 (4.8) 36 (62.1) 0.02 36 (22.4) 29 (28.2) 7 (12.1) 125 (77.6) 74 (71.8) 51 (87.9) 0.17 146 (90.7) 91 (88.4) 55 (94.8) 6 (3.7) 6 (5.8) 0 (0.0) 9 (5.6) 6 (5.8) 3 (5.2) 0.65 109 (67.3) 71 (68.9) 38 (65.5) 52 (32.1) 32 (31.1) 20 (34.5) 0.06 56 (34.8) 42 (40.8) 14 (24.1) 94 (58.4) 53 (51.4) 41 (70.7) 11 (6.8) 8 (7.8) 3 (5.2) 0.79 66 (41.0) 41 (39.8) 25 (43.1) 83 (51.5) 55 (53.4) 28 (48.3) 12 (7.5) 7 (6.8) 5 (8.6) 0.93 66 (41.0) 43 (41.7) 23 (39.7) 83 (51.5) 52 (50.5) 31 (53.4) 12 (7.5) 8 (7.8) 4 (6.9) 0.03 103 (64.0) 70 (68.0) 33 (56.9) 35 (21.7) 24 (23.3) 11 (19.0) 23 (14.3) 9 (8.7) 14 (24.1) 0.06 41 (39.8) 29 (41.4) 12 (36.4) Dahourou DL et al. Discussion Over one-third of children included in our study were already severely malnourished, with a higher proportion in Ouagadougou than in Abidjan. The WHO clinical stage and immunodeficiency The delay in the initiation of EART is essentially due to the difficulty of early infant diagnosis, as already observed in other studies, in low income countries [11,27,28]. This delay in access to early diagnosis can be explained by the lack of service delivery, especially because of the insufficient avail- ability of virological diagnostics, the lack of reagents, the time required to perform tests, the lack of support services for the samples’ transportation between healthcare sites and labo- ratories, the lack of integration of testing within existing programmes such as immunization programmes, the missed 7 7 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/1 Table 4. Baseline clinical, anthropometric, haematological, immunological and virological characteristics of the 161 HIV-infected children included in the ANRS 12206 MONOD study according to site (Abidjan, Ouagadougou, May 2011 to February 2013) Table 4. Baseline clinical, anthropometric, haematological, immunological and virological characteristics of the 161 HIV-infected children included in the ANRS 12206 MONOD study according to site (Abidjan, Ouagadougou, May 2011 to February 2013) status were also more advanced, consistently reflecting the recruitment bias in the hospital settings in Ouagadougou compared to Abidjan. Our study also highlighted the limited access to PMTCT HIV prevalence, with more than 50% of children not having received any PMTCT prophylaxis. This situation is partly due to the missed opportunities of prenatal HIV testing. PMTCT interventions are proposed in a context where stigma Table 4. Discussion Baseline clinical, anthropometric, haematological, immunological and virological characteristics of the 161 HIV-infected children included in the ANRS 12206 MONOD study according to site (Abidjan, Ouagadougou, May 2011 to February 2013) Total N161 Abidjan n103 Ouagadougou n58 Characteristics n (%) n (%) n (%) p WHO stage at ART initiation B0.01 1 48 (29.8) 41 (39.8) 7 (12.1) 2 13 (8.1) 13 (12.6) 0 (0.0) 3 63 (39.1) 34 (33.0) 29 (50.0) 4 37 (30.0) 15 (14.6) 22 (37.9) Weight-for-age z-score Mean (SD) 2.4 (1.7) 2.0 (1.6) 3.1 (1.5) B0.01 ]2 75 (46.6) 61 (59.2) 14 (24.1) B0.01 2, 3 28 (17.4) 13 (12.6) 15 (25.9) B3 58 (36.0) 29 (28.2) 29 (50.0) Height-for-age z-score Mean (SD) 2.3 (1.8) 1.9 (1.9) 2.8 (1.6) B0.01 ]2 78 (48.5) 61 (59.2) 17 (29.3) B0.01 2, 3 34 (21.1) 17 (16.5) 17 (29.3) B3 49 (30.4) 25 (24.3) 24 (41.4) Weight-for-height z-score Mean (SD) 1.6 (1.5) 1.3 (1.5) 2.2 (1.5) B0.01 ]2 103 (64.0) 77 (74.8) 26 (44.9) B0.01 2, 3 30 (18.6) 15 (14.6) 15 (25.8) B3 28 (17.4) 11 (10.6) 17 (29.3) Haemoglobin at initiation (g/dl) Median (IQR) 9.0 (8.2 to 9.9) 9.2 (8.5 to 10.0) 8.4 (7.8 to 9.5) B0.01* ]10 32 (19.9) 25 (24.2) 7 (12.1) 0.03** 9 to 10 48 (29.8) 34 (33.0) 14 (24.1) 7 to 9 79 (49.1) 42 (40.8) 37 (63.8) Missing 2 (1.2) 2 (2.0) 0 (0.0) Median CD4 percentage at ART initiation (IQR) 19.5 (13.5 to 26.2) 21.5 (15.6 to 28.8) 16.6 (11.0 to 23.5) B0.01* Immune status at ART initiation 0.58 Normal 21 (13.0) 14 (13.6) 7 (12.1) Moderate 20 (12.5) 16 (15.5) 4 (6.9) Advanced 25 (15.5) 15 (14.6) 10 (17.2) Severe 92 (57.1) 56 (54.4) 36 (62.1) Missing 3 (1.9) 2 (1.9) 1 (1.7) Mean viral load at ART initiation (log10) 6.1 (1.1) 5.9 (1.1) 6.5 (0.9) B0.01 Viral load class at ART initiation 0.02 56 log 64 (39.7) 48 (46.6) 16 (27.6) 6 log 95 (59.0) 53 (51.4) 42 (72.4) Missing 2 (1.3) 2 (2.0) 0 (0.0) SD: standard deviation; IQR: Interquartile range; ART: antiretroviral therapy; WHO: World Health Organization. Immune status: Age B11 months: normal CD4% 35%, moderate CD4% 535% and ]30%, advanced CD4% 525% and ]29%, severe CD4% B25%; Age ]11 months: normal CD4% 30%, moderate CD4% 525% and ]30%, advanced CD4% 520% and ]24%, severe CD4% B20%. SD: standard deviation; IQR: Interquartile range; ART: antiretroviral therapy; WHO: World Health Organization. Immune status: Age B11 months: normal CD4% 35%, moderate CD4% 535% and ]30%, advanced CD4% 525% and ]29%, severe CD4% B25%; Age ]11 months: normal CD4% 30%, moderate CD4% 525% and ]30%, advanced CD4% 520% and ]24%, severe CD4% B20%. p-value from Wilcoxon rank-sum test; p-value from Fisher’s exact test; *WHO 2006 growth standards. Discussion p-value from Wilcoxon rank-sum test; p-value from Fisher’s exact test; WHO 2006 growth standards. Haemoglobin at initiation (g/dl) SD: standard deviation; IQR: Interquartile range; ART: antiretroviral therapy; WHO: World Health Organization. Immune status: Age B11 months: normal CD4% 35%, moderate CD4% 535% and ]30%, advanced CD4% 525% and ]29%, severe CD4% B25%; Age ]11 months: normal CD4% 30%, moderate CD4% 525% and ]30%, advanced CD4% 520% and ]24%, severe CD4% B20%. p-value from Wilcoxon rank-sum test; p-value from Fisher’s exact test; *WHO 2006 growth standards. HIV prevalence, with more than 50% of children not having received any PMTCT prophylaxis. This situation is partly due to the missed opportunities of prenatal HIV testing. PMTCT interventions are proposed in a context where stigma remains high and where a significant number of pregnant status were also more advanced, consistently reflecting the recruitment bias in the hospital settings in Ouagadougou compared to Abidjan. Our study also highlighted the limited access to PMTCT intervention in West African settings with an intermediate 8 8 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 couples about the disclosure of the child’s HIV status and finally by fighting against stigma at the community level and among healthcare workers. These efforts are needed to provide early HIV care to infants and to improve their survival as efficiently as shown in the CHER trial [6]. More recently, in 2015, universal treatment of all HIV-infected patients was recom- mended by WHO [42]. However children less than two years old are those with the most advanced stage of the disease, requiring more rapid initiation of ART compared to older ones [43]. In the view of the currently enormous unmet need for paediatric ART provision compared to adults and the 2015 guidelines [42], it is necessary to provide ART for all children, but also as soon as possible for the youngest children, who are at a higher risk of death compared to older children. women do not receive antenatal care. The conditions of access to prenatal and infant HIV diagnosis and antiretroviral treatment upstream of the MONOD project were discussed elsewhere [10]. These results demonstrated the difficulties in the implementation of PMTCT interventions in West Africa. The linkage between PMTCT and postnatal care services needs to be strengthened in order to identify HIV infection and initiate EART at the earliest opportunity [38]. Authors’ contributions VL, MTK, NM, VA, RS and PL were co-investigators of the MONOD project and contributed to data interpretation. DD and VL contributed to the design, data analysis and interpretation; DD and VL drafted the first report of the manuscript, which all authors subsequently reviewed, edited and approved. MAF, MC, DA, MTK, DY, CAB and DD were involved in the conduct of the field study. Acknowledgements The authors warmly thank the children and their families involved in this project, the staff, the national AIDS programme in Coˆte d’Ivoire and the national AIDS committee in Burkina Faso for their support and Dr Nina Deschamps for the language corrections. De´sire´ Dahourou is a PhD student fellow funded by the ANRS (ANRS12206-B89). Authors’ affiliations 1 1MONOD Project, ANRS 12206, Centre de Recherche Internationale pour la Sante´, Ouagadougou, Burkina Faso; 2Inserm, U1219, Institut de Sante´ Publique, Epide´miologie et De´veloppement, University of Bordeaux, Bordeaux, France; 3Centre Muraz, Bobo-Dioulasso, Burkina Faso; 4PACCI Programme, Site ANRS, Projet Monod, Abidjan, Coˆte d’Ivoire; 5Pediatric Department, CHU of Cocody, Abidjan, Coˆte d’Ivoire; 6University of Ouagadougou, Ouagadougou, Burkina Faso; 7Pediatric Department, Centre Hospitalier Universitaire (CHU) de Yopougon, Abidjan, Coˆte d’Ivoire; 8Department of Infection and Immunity, Luxembourg Institute of Health, Luxembourg; 9Pediatric Department, CHU Charles de Gaulle, Ouagadougou, Burkina Faso; 10Pediatric Department, Hoˆpital Universitaire des Enfants de la Reine Fabiola, Universite´ Libre de Bruxelles, Brussels, Belgium; 11Inserm, U1027, Universite´ Toulouse, Toulouse, France Our study has limitations. First, our data represent only urban settings, probably reflecting a better access to early infant diagnosis and antiretroviral treatment compared to rural settings, where the delay of care could be even longer than that reported in our study. A few variables presented a high number of missing data (data on vital status of the father, his HIV status and antiretroviral treatment, which were recorded at the pre-inclusion visit). These missing data were mainly related to children whose parents did not accept the inclusion in the MONOD project or those who died before inclusion. Although that selection bias might have affected the precision of our estimate, we feel that it might have underestimated the OR estimates. Finally, the need to get either both parents’ consent (or the mother’s if she was the only legal guardian, or the legal caregiver if parents were not alive) before inclusion of the children and EART initiation in this research context could have also underestimated the true EART acceptability rate. Those children who did not have parental consent were included in the ART programme but not in the research cohort. This may not depict the real life situation, but this allows us to highlight the place of the father in the family’s interaction with the field reality and the mothers’ difficulties of coping with a long-term daily ART strategy for themselves and their children without the father knowing it. Despite these limitations, our findings are original. This is the first study to document the pre-ART cascade and family acceptability of EART in children less than two years in the West African context. Discussion It is urgent to raise awareness of both the community and healthcare workers in order to increase the number of mothers and children receiving PMTCT and achieve the goal of ‘‘zero new paediatric infections’’ [39]. Few cases of tuberculosis were identified in our settings (5/161; 3%), reflecting a difference of contextual morbidity in West Africa compared to South Africa [40]. Finally, our results highlight the delayed pre-ART cascade with a low coverage of EID mentioned in West Africa compared to South Africa [41]. The ANRS 12206 MONOD Collaboration Study Group (as of 1 February 2015) Participating sites The ANRS 12206 MONOD Collaboration Study Group (as of 1 February 2015) Participating sites Burkina Faso, Ouagadougou: Centre de Recherche International pour la Sante´: Malik Coulibaly, De´sire´ Lucien Dahourou, Nicolas Meda (co-investigator), Colette Oue´draogo, Mamadou Sawadogo, Wilfried Some´, De´sire´ Sondo, Elisabeth Thio. CHU Charles De Gaulle: Mamadou Barry, William Hiembo, Fla Koue´ta, Adama Ouattara, Moussa Oue´draogo, Rasmata Oue´draogo, Sylvie Oue´draogo, Bernadette Congo, Rose Barry, Diarra Ye´. CHU Yalgado Oue´draogo: Malika Congo, Edouard Mine´ne´, Marie Coulibaly, Pierre Innocent Guissou Ange`le Kalmogho, Ludovic Kam, Emile Oue´draogo, Lassana Sangare´, Caroline Yonaba. Programme Sectoriel Sante´ de Lutte contre le SIDA et les IST: Ramatou W. Sawadogo. Programme d’Appui au Monde Associatif et Communautaire: Odette Ky-Zerbo. Competing interests The authors have no conflicts of interest to disclose. Disclaimer 20. Dube Q, Dow A, Chirambo C, Lebov J, Tenthani L, Moore M, et al. Implementing early infant diagnosis of HIV infection at the primary care level: experiences and challenges in Malawi. Bull World Health Organ. 2012;90(9): 699704. The content is solely the responsibility of the authors and does not necessarily represent the official views of the French INSERM-ANRS, EDCTP or University of Bordeaux. 21. Berhan Y. Age and CD4 count of vertically HIV-infected children at the time of diagnosis: what are independent predictors for being symptomatic and CD4 counts drop? J Trop Pediatr. 2011;57(1):1423. 21. Berhan Y. Age and CD4 count of vertically HIV-infected children at the time of diagnosis: what are independent predictors for being symptomatic and CD4 counts drop? J Trop Pediatr. 2011;57(1):1423. Funding Centre pour la Recherche en Sante´ (CRP-Sante´), Luxembourg, European and Developing Countries Clinical Trials Partnership (EDCTP, reference: IP.2007.33011.002), French INSERM-ANRS, Institut de Sante´ Publique, d’E´pi- de´miologie et de De´veloppement, University of Bordeaux, France. Clinical Trial Number: ClinicalTrial.gov registry number NCT01127204. 18. WHO. 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Administrative Team: Elodie Vernoux (Bordeaux, France), Aminata Pare´- Karambiri (Ouagadougou, Burkina Faso), Zouma Tinto (Ouagadougou, Burkina Faso), Adoulaye CISSE (Abidjan, Coˆte d’Ivoire), Madikona Dosso (Abidjan, Coˆte d’Ivoire). 13. Desmonde S, Dicko F, Koueta F, Eboua T, Balestre E, Amani-Bosse C, et al. Association between age at antiretroviral therapy initiation and 24-month immune response in West-African HIV-infected children. AIDS Lond Engl. 2014;28(11):164555. MONOD ANRS 12206 Scientific Steering Committee: Roger Salamon (chair, Bordeaux, France),Vale´riane Leroy (coordinating investigator, Bordeaux, France), MONOD ANRS 12206 Scientific Steering Committee: Roger Salamon (chair, Bordeaux, France),Vale´riane Leroy (coordinating investigator, Bordeaux, France), MONOD ANRS 12206 Scientific Steering Committee: Roger Salamon (chair, Bordeaux, France),Vale´riane Leroy (coordinating investigator, Bordeaux, France), Nicolas Meda (co-investigator, Ouagadougou, Burkina Faso), Marguerite Timite- Konan (co-investigator, Abidjan, Coˆte d’Ivoire), Vic Arendt (co-investigator, Luxembourg), Ste´phane Blanche (co-investigator, Paris, France), Philippe Lepage (co-investigator, Brussels, Belgium), Philippe Van de Perre (co-investigator, Montpellier, France), Franc¸ois Dabis (Bordeaux, France), Jean-Claude Schmit (CRP-Sante´, Luxembourg). ) y ( g g ) Nicolas Meda (co-investigator, Ouagadougou, Burkina Faso), Marguerite Timite- Konan (co-investigator, Abidjan, Coˆte d’Ivoire), Vic Arendt (co-investigator, Luxembourg), Ste´phane Blanche (co-investigator, Paris, France), Philippe Lepage (co-investigator, Brussels, Belgium), Philippe Van de Perre (co-investigator, Montpellier, France), Franc¸ois Dabis (Bordeaux, France), Jean-Claude Schmit (CRP-Sante´, Luxembourg). 14. Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 FSU Abobo-Avocatier: Gbame´ne´ Kouassi. Pharmacie de la Sante´ Publique: Carine Kodo. Implementers: Toure´ Siaka, Pety Toure´ (ACONDA), Fassinou Ekouevi (EGPAF), Ida Viho (ICAP), Anthony Richard Tanoh, Olivier Ble´ (Fondation ARIEL GLASER). Community representatives: Yaya Coulibaly (RIP), Philome`ne Takouo (ONG Bayema). Programme ESTHER: Jean Marie Massumbuko. CIRBA: Kouadio Kouakou. Programme National de Sante´ Infantile: Dorothe´e Koumi. Programme Elargi de Vaccination: Berte´ Kone´. Methodology and Data Management Center: Inserm U897, Institut de Sante´ Publique, d’E´pide´miologie et de De´veloppement, University of Bordeaux, France: Sophie Dattez, Sophie Karcher, Je´roˆme Le Carrou, Vale´riane Leroy (coordinating investigator), Karen Malateste, Pierre Touret. Methodological Support: Caroline Bouyssou, Genevie`ve Cheˆne, Vale´rie Conte, Sophie Des- monde, Delphine Gabillard, Vale´rie Journot, Roger Salamon. MEREVA, Bordeaux. Website: http://mereva.isped.u-bordeaux2.fr/monod/ Accueil.aspx FSU Abobo-Avocatier: Gbame´ne´ Kouassi. Pharmacie de la Sante´ Publique: Carine Kodo. Implementers: Toure´ Siaka, Pety Toure´ (ACONDA), Fassinou Ekouevi (EGPAF), Ida Viho (ICAP), Anthony Richard Tanoh, Olivier Ble´ (Fondation ARIEL GLASER). Community representatives: Yaya Coulibaly (RIP), Philome`ne Takouo (ONG Bayema). Programme ESTHER: Jean Marie Massumbuko. CIRBA: Kouadio Kouakou. Programme National de Sante´ Infantile: Dorothe´e Koumi. Programme Elargi de Vaccination: Berte´ Kone´. Methodology and Data Management Center: Inserm U897, Institut de Sante´ Publique, d’E´pide´miologie et de De´veloppement, University of Bordeaux, France: Sophie Dattez, Sophie Karcher, Je´roˆme Le Carrou, Vale´riane Leroy (coordinating investigator), Karen Malateste, Pierre Touret. Methodological Support: Caroline Bouyssou, Genevie`ve Cheˆne, Vale´rie Conte, Sophie Des- monde, Delphine Gabillard, Vale´rie Journot, Roger Salamon. MEREVA, Bordeaux. Website: http://mereva.isped.u-bordeaux2.fr/monod/ Accueil aspx FSU Abobo-Avocatier: Gbame´ne´ Kouassi. Pharmacie de la Sante´ Publique: Carine Kodo. Implementers: Toure´ Siaka, Pety Toure´ (ACONDA), Fassinou Ekouevi (EGPAF), Ida Viho (ICAP), Anthony Richard Tanoh, Olivier Ble´ (Fondation ARIEL GLASER). Community representatives: Yaya Coulibaly (RIP), Philome`ne Takouo (ONG Bayema). Programme ESTHER: Jean Marie Massumbuko. CIRBA: Kouadio Kouakou. Programme National de Sante´ Infantile: Dorothe´e Koumi. Programme Elargi de Vaccination: Berte´ Kone´. Methodology and Data Management Center: Inserm U897, Institut de Sante´ Publique, d’E´pide´miologie et de De´veloppement, University of Bordeaux, France: Sophie Dattez, Sophie Karcher, Je´roˆme Le Carrou, Vale´riane Leroy (coordinating investigator), Karen Malateste, Pierre Touret. Methodological Support: Caroline Bouyssou, Genevie`ve Cheˆne, Vale´rie Conte, Sophie Des- monde, Delphine Gabillard, Vale´rie Journot, Roger Salamon. MEREVA, Bordeaux. Website: http://mereva.isped.u-bordeaux2.fr/monod/ A il 5. Newell M-L, Coovadia H, Cortina-Borja M, Rollins N, Gaillard P, Dabis F, et al. Conclusions Competing early mortality of HIV-infected infants and fear of stigma within the parental couple play a key role in the missed opportunities of EART for children in West Africa. A public health effort must be made in order to take care of HIV-exposed infants and to identify HIV-infected infants earlier by strengthening linkages between PMTCT and paediatric HIV facilities, increasing community awareness on the need to treat children early, providing a global package of care for HIV-positive mothers (access to ART, child nutrition, improvement of socio-economic conditions), involving the father at the earliest opportunity according to maternal willingness, facilitating communication within Coˆte d’Ivoire, Abidjan: Programme PACCI: Xavier Anglaret, Clarisse Amani- Bosse´, Divine Avit, Christine Danel, Serge Eholie´, Didier Ekoue´vi, Eulalie Kanga, Suzanne Kouadio, Se´verin Lennaud, Maxime Aime´ Oga, The´re`se N’Dri-Yoman. CHU Cocody: Madeleine Amorissani-Folquet, Evelyne Dainguy, Beugre Kouassi, Jean-Claude Kouassi, Gladys Oka. CHU Yopougon: Kader Keita, Jean Yves Lambin, Franc¸ois Eboua Tanoh, Marguerite Timite´-Konan (co-investigator). Site Abobo-Avocatier: Ve´ronique Mea-Assande. Site CePReF-enfants: Addi Edmond Aka, Hortense Aka-Dago, Sylvie N’Gbeche, Euge`ne Messou. Lab CeDReS: Arlette Emieme, Fatoumata Kone´, Herve´ Menan, Thomas Toni, Vincent Yapo. Programme National de Prise en Charge: Kouame´ Abo, Irma Ahoba, David Aka. 9 9 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 Dahourou DL et al. Journal of the International AIDS Society 2016, 19:20601 http://www.jiasociety.org/index.php/jias/article/view/20601 \| http://dx.doi.org/10.7448/IAS.19.1.20601 35. Yeap AD, Hamilton R, Charalambous S, Dwadwa T, Churchyard GJ, Geissler PW, et al. Factors influencing uptake of HIV care and treatment among children in South Africa a qualitative study of caregivers and clinic staff. AIDS Care. 2010;22(9):11017. antiretroviral therapy at primary health care facilities in zambia. JAMA. 2007;298(16):188899. 35. Yeap AD, Hamilton R, Charalambous S, Dwadwa T, Churchyard GJ, Geissler PW, et al. Factors influencing uptake of HIV care and treatment among children in South Africa a qualitative study of caregivers and clinic staff. AIDS Care. 2010;22(9):11017. antiretroviral therapy at primary health care facilities in zambia. JAMA. 2007;298(16):188899. 26. Anaky M-F, Duvignac J, Wemin L, Kouakoussui A, Karcher S, Toure´ S, et al. Scaling up antiretroviral therapy for HIV-infected children in Coˆte d’Ivoire: determinants of survival and loss to programme. Bull World Health Organ. 2010;88(7):4909. 26. Anaky M-F, Duvignac J, Wemin L, Kouakoussui A, Karcher S, Toure´ S, et al. Scaling up antiretroviral therapy for HIV-infected children in Coˆte d’Ivoire: determinants of survival and loss to programme. Bull World Health Organ. 2010;88(7):4909. 36. 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https://openalex.org/W2799316437	https://europepmc.org/articles/pmc5971305?pdf=render	English	null	NADPH Oxidase Signaling Pathway Mediates Mesenchymal Stem Cell-Induced Inhibition of Hepatic Stellate Cell Activation	Stem cells international	2,018	cc-by	10,645	Haowen Qiao,1 Yu Zhou ,2 Xingping Qin,1 Jing Cheng,1 Yun He ,3 and Yugang Jiang 2 1Department of Physiology, School of Basic Medicine, Wuhan University School of Medicine, 185 Donghu Street, Wuhan 430071 China Q , , gp g Q , J g g, , g g J g 1Department of Physiology, School of Basic Medicine, Wuhan University School of Medicine, 185 Donghu Street, Wuhan 430071, China 2Department of Neurosurgery, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410000, China 3Department of Infectious Diseases, The Third People’s Hospital of Shenzhen, No. 29 Bulan Road, Shenzhen 518000, China 2Department of Neurosurgery, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410000, China 3Department of Infectious Diseases, The Third People’s Hospital of Shenzhen, No. 29 Bulan Road, Shenzhen 518000, China Correspondence should be addressed to Yun He; heyun9185@aliyun.com and Yugang Jiang; jiangyugang@ali Received 13 December 2017; Accepted 28 March 2018; Published 10 May 2018 Academic Editor: Silvia Brunelli Academic Editor: Silvia Brunelli Copyright © 2018 Haowen Qiao 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. Copyright © 2018 Haowen Qiao et al. This is an open access article distributed under the Creative Commons A which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is Background. Bone marrow-derived mesenchymal stem cells (BMSCs) have blossomed into an eﬀective approach with great potential for the treatment of liver ﬁbrosis. The aim of this study was to investigate the underlying antiﬁbrosis mechanisms by which the BMSC inhibit activated hepatic stellate cells (HSCs) in vivo and in vitro. Methods. To study the eﬀect of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) on activated HSCs, we used HSCs and the coculture systems to evaluate the inhibition of activated HSCs from the aspects of the apoptosis of activated HSCs. In addition, activation of NADPH oxidase pathway and the changes in liver histopathology were tested by using the carbon tetrachloride- (CCl4-) induced liver ﬁbrosis in mice. Results. Introduction of hBM-MSCs signiﬁcantly inhibited the proliferation of activated HSCs by inducing the apoptosis process of activated HSCs. The eﬀect of hBM-MSCs reduced the signaling pathway of NADPH oxidase in activated HSCs. Besides, the signaling pathway of NADPH oxidase mediated hBM-MSC upregulation of the expression of the peroxisome proliferator-activated receptor γ and downregulation of the expression of α1(I) collagen and alpha-smooth muscle actin (α-SMA) in activated HSCs. Haowen Qiao,1 Yu Zhou ,2 Xingping Qin,1 Jing Cheng,1 Yun He ,3 and Yugang Jiang 2 1Department of Physiology, School of Basic Medicine, Wuhan University School of Medicine, 185 Donghu Street, Wuhan 430071 China Moreover, the hBM-MSC-induced decrease in the signaling pathway of NADPH oxidase was accompanied by the decrease of the activated HSC number and liver ﬁbrosis in a mouse model of CCl4-induced liver ﬁbrosis. Conclusion. The hBM-MSCs act as a promising drug source against liver ﬁbrosis development with respect to hepatopathy as a therapeutic target. Hindawi Stem Cells International Volume 2018, Article ID 1239143, 13 pages https://doi.org/10.1155/2018/1239143 Hindawi Stem Cells International Volume 2018, Article ID 1239143, 13 pages https://doi.org/10.1155/2018/1239143 2. Materials and Methods 2.1. Reagents and Antibodies. Diphenyleneiodonium chloride (DPI, an inhibitor for NADPH oxidase signaling pathway), Cell Counting Kit-8, TRI-Reagent, and Hoechst 33342 were from Sigma (St. Louis, MO, USA), and Annexin V-FITC/PI Apoptosis Detection Kit were purchased from Beyotime (Jiangsu, China). Carbon tetrachloride (CCI4) (concentration 100%) and olive oil were obtained from Algomhoria Com- pany. Enhanced BCA Protein Assay Kit was from Beyotime Biotechnology (Haimen, China). The primary antibodies including Bax, Bcl-2, cleaved caspase-3, α-smooth muscle actin (α-SMA), and α1(I) collagen were purchased from Abcam (Cambridge, MA, USA), and PPARγ, p47phox (serine 359), p-p47phox, 4-hydroxynonenal (4-HNE), and β-actin were purchased from Santa Cruz (Santa Cruz, CA, USA). 2.5. Cell Proliferation Assay and Apoptosis Analysis. Cell pro- liferation was determined using a sensitive colorimetric assay, the Cell Counting Kit-8 (CCK-8; Sigma-Aldrich, St. Louis, MO, USA). The coculture cells were collected after culturing for 24, 48, or 76 hours. The CCK-8 reagent was added to the lower chamber containing activated HSCs in the HSC group and the coculture group and incubated for 2 hours according to the manufacturer’s protocol. For the apo- ptosis analysis, after culturing for 76 hours, the coculture cells were stained with Annexin V-FITC/PI Apoptosis Detection Kit by BD FACS Calibur (BD Biosciences, San Jose, CA, USA) and the test methods were implemented according to the manufacturer’s instructions. 2.2. Preparation of Human Bone Marrow-Derived Mesenchymal Stem Cells. Human bone marrow-derived mes- enchymal stem cells were from healthy persons between 40 and 45 years of old who voluntarily donated bone marrow stem cells. Brieﬂy, mesenchymal stem cells were collected into appropriately supplemented DMEM culture medium (Dulbecco’s Modiﬁed Eagle’s Medium, Gibco, San Diego, CA, USA), at 37°C/5% CO2 fully humidiﬁed atmosphere. The expressions of hBM-MSC surface markers cluster of dif- ferentiation (CD14, CD34, CD45, CD90, CD73, and CD105) were detected by BD FACS Calibur™(BD Biosciences, San Jose, CA, USA), and cells were characterized as having high expression of these positive markers (CD90, CD73, and CD105) and low expression of the negative markers (CD14, CD34, and CD45) [11]. 2.6. Assay of NADPH Oxidase Activity. NADPH oxidase (NOX) activity was measured using lucigenin as described elsewhere [13]. Brieﬂy, the coculture cells or the activated HSCs were harvested after culturing for 24, 48, or 76 hours and centrifuged at 400 ×g for 10 min at 4°C, and the cell pellet was kept on ice after resuspended by 35 μl/per well of the ice-cold RPMI-1640 medium. 1. Introduction and diﬀerentiation capacity [6] possess an attractive option in the preclinical and clinical studies [7]. Moreover, multipo- tent MSCs can diﬀerentiate into numerous tissue lineages including hepatocytes [8]. Encouragingly, some clinical trials have proved that BMSCs can eﬀectively alleviate end-stage liver disease and improve symptoms and liver function [9]. Additionally, human bone marrow-derived mesenchy- mal stem cells (hBM-MSCs) have the advantages of autol- ogous sources and abundance of cells [10], suggesting being considered a promising tool for cell-based therapies of liver cirrhosis treatment. However, the underlying mecha- nisms are not yet well understood. Therefore, further explo- ration is required to better understand how MSCs regulate HSCs activation in the occurrence and development of liver cirrhosis. Liver ﬁbrosis, the result of chronic liver injury caused by various factors, is characterized by overexpression and accumulation of extracellular matrix (ECM) proteins and is associated with the activation of hepatic stellate cells (HSCs) [1, 2]. Since activated HSCs are the major collagen- producing cells in liver injury and the activation of HSCs is considered to be a crucial step in the development of liver ﬁbrosis [3–5], thus, it is essential and urgent to seek the potential target to reduce the secretion of ECM or collagen synthesis and control liver ﬁbrosis by inhibiting the activa- tion of HSCs. Bone marrow-derived mesenchymal stem cells (BM- MSCs) which are known to have plasticity, high proliferation, 2 Stem Cells International chamber (24 mm diameter, 0.4 mm pore size; Corning) was used. The activated HSCs were mixed and cocultured with hBM-MSCs in supplemented DMEM containing 10% fetal bovine serum (FBS) at a 1 : 1 ratio for activated HSCs and hBM-MSCs. The activated HSC cells were inoculated in the lower part, and the hBMSCs were inoculated in the upper part. The cell numbers were evaluated using microscopy, and viability was determined by the trypan blue exclusion assay. As a comparison to the coculture system, the activated HSC group was seeded in the lower chamber and treated with or without 1 μM DPI [12] or DMEM containing 10% FBS. Samples were collected after culturing for 24, 48, or 72 hours. In this study, hBM-MSCs were evaluated for their eﬀectiveness in liver cirrhosis treatment by transplantation. We used mouse model of CCl4-induced liver cirrhosis to assess pathological eﬀectiveness. 1. Introduction We intended to investigate whether hBM-MSCs accommodate HSC activation and as well as possible repair mechanisms for liver ﬁbrosis, focusing on its role in the signaling pathway mediating the eﬀect of hBM-MSCs on HSC activation. 2. Materials and Methods Then the NOX activity was measured in cell homogenates in a reaction mixture that con- tained NADPH (1μM) or lucigenin (20 μM), 5 μl of cell sus- pension, and a ﬁnal 200 μl volume of prewarmed (37°C) RPMI-1640 medium. The chemiluminescence was continu- ously recorded for 12min, and the speciﬁc enzyme activity was calculated as counts per million cells. 2.7. Animals and Treatments. Male mice (10 to 12 weeks old, 20 to 28 g) were obtained from the animal center of the Central South University (Changsha, China). In brief, CCl4 was usually used for induction of mouse liver ﬁbrosis [14]. Male mice were randomly separated into three groups (ten mice/each group): group 1 (olive oil +vehicle), group 2 (CCl4 +vehicle), and group 3 (CCl4 +hBM-MSCs). At ﬁrst, the group 2 and group 3 were treated with CCl4 (diluted 1 : 5 in olive oil, 5 μl/g) intraperitoneally twice weekly for 11 weeks. For the normal control group (group 1), the mice were injected with the same volume of olive oil. 2.3. HSC Isolation and Culture. HSCs were isolated from adult Sprague-Dawley (SD) rats (8 weeks of age) or mice and primary rat HSCs were isolated using a two-step collage- nase perfusion. Brieﬂy, the rat liver was minced and incu- bated in 0.02% pronase with deoxyribonuclease (Sigma) after the rat liver perfused in situ with collagenase (Sigma) and streptavidin (Sigma). Then, the mixture was centrifuged to remove parenchymal cells and subsequently HSCs were recovered by density gradient centrifugation of nonparench- ymal cells. HSCs were activated for 10–14 days by culturing in a 25 cm2 ﬂask with DMEM (Gibco) containing 10% fetal bovine serum (Gibco) at 37°C/5% CO2 fully humidiﬁed atmosphere. The activated HSCs between passages 3 and 8 were used for experiments. 2.3. HSC Isolation and Culture. HSCs were isolated from adult Sprague-Dawley (SD) rats (8 weeks of age) or mice and primary rat HSCs were isolated using a two-step collage- nase perfusion. Brieﬂy, the rat liver was minced and incu- bated in 0.02% pronase with deoxyribonuclease (Sigma) after the rat liver perfused in situ with collagenase (Sigma) and streptavidin (Sigma). Then, the mixture was centrifuged to remove parenchymal cells and subsequently HSCs were recovered by density gradient centrifugation of nonparench- ymal cells. HSCs were activated for 10–14 days by culturing in a 25 cm2 ﬂask with DMEM (Gibco) containing 10% fetal bovine serum (Gibco) at 37°C/5% CO2 fully humidiﬁed atmosphere. 2.4. Coculture System. To evaluate the eﬀects of the hBM- MSCs on activated HSCs, indirect coculture in Transwell 2. Materials and Methods p47phox, p-p47phox, NOX2, 4-HNE and PPARγ, and subse- quently by horseradish peroxidase-conjugated secondary antibody. β-Actin was used as an internal control. 2.11. RNA Isolation and Real-Time PCR. Total RNA was extracted from cells by using TRI-Reagent according to the manufacturer’s instructions. Equal amounts of RNA were reverse transcribed to cDNA with a cDNA reverse transcrip- tion kit (Takara Biotechnology Co, Ltd.). Real-time PCR was performed with iTaqTM Universal SYBR Green (Bio-Rad Laboratories Inc., Hercules, CA, USA). The target gene was ampliﬁed according to the following conditions: initial dena- turation at 94°C for 10 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 15sec, and extension at 70°C for 15 sec; and a ﬁnal extension at 70°C for 5 min. Sirius red staining of collagen was used to stain collagen on tissue sections for histological analysis of liver ﬁbrosis. In short, paraformaldehyde-ﬁxed liver tissue sections were incubated with picric acid-fast green (Amresco, Solon, USA), and then stained with picric acid Sirius red (Amresco, Solon, USA) for 1 hour. Images were captured by using a light microscope. The cycle threshold (Ct) values were normalized against the endogenous cyclophilin control and analyzed by using the ΔΔCt method [17]. The value was quantiﬁed by normalization to the cyclophilin mRNA level. Primers were listed in Table 1. 2.9. Measurement of Malondialdehyde, Glutathione Peroxidase, Superoxide Dismutase, Catalase, and Hydroxyproline in Liver Tissues. The malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) in liver tissues were analyzed according to the protocol provided by the commercial kits (Nanjing Jiancheng Bioengineering Institute, China). The results were shown as mmol/mg pro- tein (MDA and GSH), U/mg protein (SOD and CAT), or mg/mg in wet liver (hydroxyproline). 2.12. Statistical Analysis. The results are obtained as the mean ±standard deviation of at least three assays and the dif- ferences between means were evaluated by using an unpaired two-sided Student’s t-test. P < 0 05 was considered as signif- icant diﬀerence. 2.10. Western Blot Analysis. The HSCs were harvested and centrifuged (10000g) at 4°C for 20 min to prepare the whole cell lysate. Next, by using an Enhanced BCA Protein Assay Kit according to the manufacturer’s protocol. The super- natants were used for quantiﬁcation of the total protein concentration. The denatured samples were analyzed by 10% SDS-PAGE gels and transferred onto nitrocellulose membrane. 2. Materials and Methods The blots were incubated with antibodies against Bax, Bcl-2, cleaved caspase-3, α-SMA, α1(I) collagen, 2. Materials and Methods The activated HSCs between passages 3 and 8 were used for experiments. After 11 weeks of CCl4 or olive oil administration, group 1 and group 2 received 0.1 mL PBS/mouse via the tail vein; group 3 was infused with 8× 106 hBM-MSCs in PBS/mouse via the liver portal vein. CCl4 administration was continued after hBM-MSC transplantation in accordance with earlier standards in case of spontaneous ﬁbrosis recovery [15]. After 3 weeks of hBM-MSC transplantation, the liver was ﬁxed in 2.4. Coculture System. To evaluate the eﬀects of the hBM- MSCs on activated HSCs, indirect coculture in Transwell Stem Cells International 3 Table 1: Sequences of DNA primers for real-time PCR in this study. Gene Primer sequences Rat p47phox Forward 5′-CAGCCATGG GGGACACCTTCATT-3′ Reverse 5′-GCCTCAATGGG GAACATCTCCTTCA-3′ Rat α-SMA Forward 5′-ACAACGTGC CTATCTATGAGGGCT-3′ Reverse ′-AGCGACATA GCACAGCTTCTCCTT-3′ Rat α1(I) collagen Forward 5′-TGGTCCCAA AGGTTCTCCTGGT-3′ Reverse 5′-TTAGGTCCA GGGAATCCCATCACA-3′ Rat cyclophilin Forward 5′-TGGATGGCA AGCATGTGGTCTTTG-3′ Reverse 5′-CTTCTTGCT GGTCTTGCCATTCCT-3′ 4% buﬀered paraformaldehyde for immunostaining analysis or HSCs were isolated from mice for Western blot analysis. All animal experiments were performed in accordance with guidelines from the Central South University Institutional Animal Care and Use Committee. Table 1: Sequences of DNA primers for real-time PCR in this study. 2.8. Immunoﬂuorescence Staining and Sirius Red Staining. For examination of the expressions of p47phox (a subunit of NADPH oxidase) and 4-hydroxynonenal (4-HNE) in HSCs in the mouse liver, a double ﬂuorescent staining was performed as described elsewhere [12]. After being blocked with normal serum, tissue sections were incubated with primary rabbit anti-p47phox (1: 100, Cat. SC-14015, Santa Cruz, CA, USA), rabbit anti-4-HNE (1: 50, Cat. Ab46546, Abcam, Cambridge, MA, USA), and primary mouse anti-synaptophysin (SYP, 1 : 10, Cat. SC-365488, Santa Cruz, CA, USA), a marker for quiescent and acti- vated HSCs [16]. The secondary antibodies, including DyLight 594-conjugated secondary antibody (1 : 500, Cat. GtxRb-003-D5948NHSX, ImmunoReagents Inc., Raleigh, USA) and DyLight 488-conjugated secondary antibody (1 : 500, Cat. GtxMu-003-D488NHSX, ImmunoReagents Inc., Raleigh, USA), were used according to the manufac- turer’s instructions. The nuclei were counterstained with Hoechst 33342. For single ﬂuorescence staining of α- smooth muscle actin (α-SMA) on the liver sections, the paraformaldehyde-ﬁxed liver sections were blocked with normal serum and then incubated with primary antibody against α-SMA (1 : 100) and the DyLight 594-conjugated secondary antibody, followed by counterstaining with the nuclear dye haematoxylin. Images were captured with the ﬂuorescence microscope. 3. Results 3.1. Preparation of Human Bone Marrow-Derived Mesenchymal Stem Cells. The hBM-MSCs in culture are deﬁned by the expression of cell surface markers as shown in Figure 1, we found that the hBM-MSCs exhibited strong expression of classical markers including, CD73, CD90 and CD105 expression and lack the expression of CD14, CD34, Stem Cells International 97.0% 99.7% CD14 FITC 0 101 102 103 104 105 CD34 FITC 0 101 102 103 104 105 CD45 FITC 0 101 102 103 104 105 CD90 FITC 0 101 102 103 104 105 CD105 FITC 0 101 102 103 104 105 CD73 FITC 0 101 102 103 104 105 0.1% 1.8% 0.3% 99.6% 0 50 100 150 200 Counts 0 50 100 150 200 Counts 0 60 120 240 300 Counts 0 60 120 240 300 Counts 0 60 120 240 300 Counts 0 50 100 150 200 Counts Figure 1: Preparation of human bone marrow-derived mesenchymal stem cells. The expressions of hBM-MSC surface markers’ cluster of diﬀerentiation (CD14, CD34, CD45, CD90, CD73, and CD105) were detected by BD FACS Calibur. CD14 FITC 0 101 102 103 104 105 0.1% 0 50 100 150 200 Counts CD34 FITC 0 101 102 103 104 105 0.3% 0 50 100 150 200 Counts Counts CD14 FITC CD45 FITC 0 101 102 103 104 105 1.8% 0 50 100 150 200 Counts 0 101 102 103 104 105 99.6% 0 60 120 240 300 Counts Counts 99.7% CD90 FITC 0 CD73 FITC 0 101 102 103 104 105 0 60 120 240 300 Counts 97.0% CD45 FITC CD105 FITC 0 101 102 103 104 105 0 60 120 240 300 Counts Counts Counts Figure 1: Preparation of human bone marrow-derived mesenchymal stem cells. The expressions of hBM-MSC surface markers’ cluster of diﬀerentiation (CD14, CD34, CD45, CD90, CD73, and CD105) were detected by BD FACS Calibur. and CD45.The data presented here support that these hBM- MSCs in culture are consistent with the characteristics of stem cells [11]. whether the antiproliferative activity of hBM-MSCs is related to apoptosis. The ﬂow cytometry was used after double stain- ing with Annexin V-FITC/PI to determine the cell apoptosis. 3. Results The cells of coculture group and the activated HSC group were collected after culturing for 48 hours, and then Figures 3(a) and 3(b) suggested that the apoptotic cell num- ber of activated HSCs in the coculture group was signiﬁcantly increased at 48 hours when compared with that in the acti- vated HSC group at 48 hours. Furthermore, we also investi- gated the expression of Bcl-2, Bax, and cleaved caspase-3 [18] to investigate the underlying mechanism of the apopto- sis process of hBM-MSCs on activated HSCs. The proteins Bcl-2, Bax, and cleaved caspase-3 in single cultures and cocultures at 48 hours were tested by Western blot analysis (Figure 3(c)). The results showed in the coculture system protein that the signal of antiapoptotic protein Bcl-2 was weakened. In contrast, the proapoptotic proteins Bax and cleaved caspase-3 protein Bax were gradually increased. 3.2. The Inhibitory Eﬀects of hBM-MSCs on Activated HSC Proliferation. The Transwell coculture was used to determine the eﬀects of hBM-MSC transplantation on the HSC prolif- eration; the amount of growing cells was visualized under an inverted microscope (Figure 2(a)). Next, to assess the eﬀects of hBM-MSCs on the activated HSCs, as shown in Figure 2(b), CCK-8 assays were performed to measure the activated HSC proliferation, and there were no signiﬁcant diﬀerences between single and cocultured activated HSCs at 24 hours; however, hBM-MSCs revealed the signiﬁcant eﬀect of suppressing the proliferation of activated HSCs at 48 hours and 72 hours when compared with those in the activated HSC group. 3.3. hBM-MSCs Induce Apoptosis Process in Activated HSCs. Since we have conﬁrmed that the inhibitory eﬀects of hBM- MSCs on activated HSC proliferation, next, we explored 3.4. NADPH Oxidase Pathway Mediates hBM-MSC Inhibition of HSC Activation. We previously determined that 5 Stem Cells International ⁎⁎ ⁎ 0 50 100 150 200 250 Relative cell growth rate 24 h 48 h 72 h 0 h HSCs HSCs + hBM-MSCs (a) Activated HSCs Coculture cells 24 h 48 h 72 h (b) Figure 2: The inhibitory eﬀects of hBM-MSCs on activated HSC proliferation. (a) The inhibitory eﬀects of hBM-MSCs on activated HSC proliferation examined using CCK-8 assays in single cultures and cocultures at 24, 48, and 72 hours. Data are expressed as the mean ± standard deviation of three independent experiments. ∗P < 0 05 versus the control group and ∗∗P < 0 001 versus the control group. 3. Results (b) Representative images of activated HSCs in single cultures and cocultures at 24, 48, and 72 hours. Scale bar = 200 μm. ⁎⁎ ⁎ 0 50 100 150 200 250 Relative cell growth rate 24 h 48 h 72 h 0 h HSCs HSCs + hBM-MSCs (a) Activated HSCs Coculture cells 24 h 48 h 72 h (b) activity of NADPH oxidase and the regulation of HSC activ- ity and liver ﬁbrosis [21]. Thereby, p47phox was used to examine the respective signaling pathway. As shown in Figure 4(b), the coculture cells revealed a signiﬁcant decrease in p47phox mRNA level as compared with the control. As expected in Figures 4(c) and 4(d), hBM-MSCs caused the decrease in the levels of NADPH oxidase transmembrane subunit NOX2 and its ligand p47phox in a time-dependent manner on HSC activation. Considering that p47phox phosphorylation regulates activation of NADPH oxidase [22], we examined the phosphorylation of p47phox. This approach revealed that the serine 359 phosphorylation of the p47phox subunits was signiﬁcantly decreased with the treatment of hBM-MSCs compared with the control group. This ﬁnding encouraged us to speculate that NADPH oxi- dase was required for the inhibition eﬀect of hBM-MSCs on HSC activation. Relative cell growth rate (a) 48 h (b) Activated HSCs Coculture cells 24 h 72 h 3.5. The Pathways of NADPH Oxidase Is Involved in hBM- MSC-Induced Reduction in Liver Fibrosis. The signaling pathways of NADPH oxidase were suggested to mediate hBM-MSC reduction of HSCs; thereby, to evaluate the potential contribution of NADPH oxidase in liver ﬁbrosis, the expression of Peroxisome proliferator-activated receptor γ (PPARγ) (a hallmark of adipocytes), α-SMA, and α1(I) col- lagen (two markers of activated HSCs) was tested. After the activated HSCs or the cocultured were pretreated with 1 μM DPI (an inhibitor for NADPH oxidase signaling path- way), the mRNA and protein levels of PPARγ, α-SMA, and α1(I) collagen were detected. The results in Figure 5 showed that after the activated HSC incubation with DPI or with hBM-MSCs, the mRNA and protein expression of α-SMA and α1(I) collagen decreased, whereas the expression of PPARγ, considerably increased. What is more, the inhibitor for NADPH oxidase signaling pathway strengthened the hBM-MSCs, the promotion eﬀect on PPARγ and inhibition of α1(1) procollagen and α-SMA expressions. These consis- tent ﬁndings in two diﬀerent cell treatment supported the critical role of NADPH oxidase and hBM-MSCs in regulating HSC activation. 3. Results (b) Figure 2: The inhibitory eﬀects of hBM-MSCs on activated HSC proliferation. (a) The inhibitory eﬀects of hBM-MSCs on activated HSC proliferation examined using CCK-8 assays in single cultures and cocultures at 24, 48, and 72 hours. Data are expressed as the mean ± standard deviation of three independent experiments. ∗P < 0 05 versus the control group and ∗∗P < 0 001 versus the control group. (b) Representative images of activated HSCs in single cultures and cocultures at 24, 48, and 72 hours. Scale bar = 200 μm. 3.6. hBM-MSCs Lead to the Decrease in Liver Fibrosis in the Mouse Model of CCl4-Induced Liver Injury. Based on the above results, to further investigate whether hBM-MSCs inﬂuenced liver ﬁbrosis in vivo, we examined the expressions of α-SMA and collagen in vivo. CCl4 can induce liver ﬁbrosis in normal mice, and this is a well-established model of liver injury [14]. Brieﬂy, mice were randomly separated into three groups: group 1 (olive oil+ vehicle), group 2 (CCl4 + vehicle), and group 3 (CCl4 +hBM-MSCs). Almost all the mice develop liver ﬁbrosis throughout the 11-week period of CCl4 treatment, three groups of mice were coadministered, with or without hBM-MSCs. Next, the α-SMA-positive cells in the livers were detected by immunoﬂuorescence to evalu- ate the HSC activation and the collagen stained by Sirius red was examined to appraise liver ﬁbrosis. Figure 6(a) shows that α-SMA-positive cells or the signs of liver ﬁbrosis were barely detectable in group 1 (olive oil +vehicle) whereas HSC activation (the number of α-SMA-positive cells) and hBM-MSCs resulted in the loss of activated HSC prolifera- tion and induced the apoptosis of activated HSCs. However, the detailed mechanism involved in this phenomenon remains unclear. Accumulating evidence indicates ROS are critical intermediates in liver physiology and pathology [19]. Moreover, the NADPH oxidase (NOX) family, which acts as a predominant mediator of redox homeostasis, may contribute to ROS production during liver ﬁbrosis [20]. Thus, we attempted to investigate the roles of NADPH oxi- dase involved in hBM-MSC regulation of activated HSCs. The coculture cells or the activated HSCs were cultured for 24, 48, and 72 h; Figure 4(a) showed that the eﬀects of hBM-MSCs reduced the NOX activity in a time-dependent manner, suggesting that the pathways of NADPH oxidase might contribute the eﬀect of hBM-MSCs on HSC activation. 3. Results ∗∗P < 0 01 versus the control group. (c) The protein Bax, cleaved caspase-3, and Bcl-2 protein n single cultures and cocultures at 72 hours were, respectively, analyzed by Western blot analysis. ∗P < 0 05 versus the control group and ∗P < 0 001 versus the control group (a) Bax HSCs HSCs + hBM-MSCs 훽-Actin Bcl-2 Cleaved caspase-3 (a) (c) Figure 3: hBM-MSCs induce apoptosis process in activated HSCs. (a, b) The apoptosis of the cells was measured using ﬂow cytometry in single cultures and cocultures at 72 hours. ∗∗P < 0 01 versus the control group. (c) The protein Bax, cleaved caspase-3, and Bcl-2 protein in single cultures and cocultures at 72 hours were, respectively, analyzed by Western blot analysis. ∗P < 0 05 versus the control group and ∗∗P < 0 001 versus the control group. signaling pathway and reduced liver ﬁbrosis in HSCs in mouse model of CCl4-induced liver injury; the role of this pathway in mouse model of CCl4-induced liver ﬁbrosis was further investigated. To assess whether hBM-MSCs inﬂu- enced NADPH oxidase in the same model in vivo, we exam- ined the productions of 4-HNE (a lipid peroxidation product as a general marker of ROS) and p47phox in HSCs. The mice were given the administration of vehicle or hBM-MSCs throughout the 11-week period of CCl4 treatment, the posi- tive HSCs for 4-HNE and p47phox in livers were examined by double ﬂuorescence staining. SYP can be used as a marker for quiescent and activated HSCs [23]. Figure 7(a) showed representative photomicrographs of immunoﬂuorescent analysis for 4-HNE and p47phox (red colour) and SYP (green colour) in the liver sections from each group. Double ﬂuorescent staining the relative number of 4-HNE and p47phox-positive HSCs demonstrated that, compared with group 1 (olive oil +vehicle), CCl4 increased 4-HNE and p47phox-positive HSCs in group 2 (CCl4 + vehicle), which was partially counteracted by hBM-MSC transplantation in liver ﬁbrosis sharply increased in group 2 (CCl4 + vehicle). However, when mice in group 3 received hBM-MSCs, the promoting eﬀect of CCl4 was suppressed and the α-SMA- positive cells and liver ﬁbrosis were partially suppressed. Western blot analysis further proved the protective eﬀect of hBM-MSCs on liver ﬁbrosis. We isolated the HSCs from groups 1–3 and examined the protein levels of α-SMA and α1(I) collagen. 3. Results The functional active component of the NADPH oxidase complex, p47phox, is considered to play a central role in the 6 6 Stem Cells International UR 3.03% UR 0.73% PI 101 102 103 104 100 Annexin V-FITC 100 101 102 103 104 101 102 103 104 100 100 101 102 103 104 (a) ⁎⁎ HSCs HSCs + hBM-MSCs 0 1 2 3 4 5 UR cell proportion (%) (b) HSCs HSCs + hBM-MSCs ⁎⁎ ⁎ ⁎ 0.0 0.5 1.0 1.5 2.0 The relative expression of protein Bcl-2 Cleaved caspase-3 Bax Bax HSCs HSCs + hBM-MSCs 훽-Actin Bcl-2 Cleaved caspase-3 (c) Figure 3: hBM-MSCs induce apoptosis process in activated HSCs. (a, b) The apoptosis of the cells was measured using ﬂow cytometry in single cultures and cocultures at 72 hours. ∗∗P < 0 01 versus the control group. (c) The protein Bax, cleaved caspase-3, and Bcl-2 protein in single cultures and cocultures at 72 hours were, respectively, analyzed by Western blot analysis. ∗P < 0 05 versus the control group and ∗∗P < 0 001 versus the control group. UR 3.03% UR 0.73% PI 101 102 103 104 100 Annexin V-FITC 100 101 102 103 104 101 102 103 104 100 100 101 102 103 104 HSCs HSCs + hBM-MSCs 0 1 2 3 4 UR cell proportion (%) (a) hBM-MSCs (b) HSCs HSCs + hBM-MSCs ⁎⁎ ⁎ ⁎ 0.0 0.5 1.0 1.5 2.0 The relative expression of protein Bcl-2 Cleaved caspase-3 Bax Bax HSCs HSCs + hBM-MSCs 훽-Actin Bcl-2 Cleaved caspase-3 (c) Figure 3: hBM-MSCs induce apoptosis process in activated HSCs. (a, b) The apoptosis of the cells was measured using ﬂow cytometry in ingle cultures and cocultures at 72 hours. ∗∗P < 0 01 versus the control group. (c) The protein Bax, cleaved caspase-3, and Bcl-2 protein n single cultures and cocultures at 72 hours were, respectively, analyzed by Western blot analysis. ∗P < 0 05 versus the control group and ∗P < 0 001 versus the control group. (a) hBM MSCs (b) HSCs HSCs + hBM-MSCs ⁎⁎ ⁎ ⁎ 0.0 0.5 1.0 1.5 2.0 The relative expression of protein Bcl-2 Cleaved caspase-3 Bax Bax HSCs HSCs + hBM-MSCs 훽-Actin Bcl-2 Cleaved caspase-3 (c) igure 3: hBM-MSCs induce apoptosis process in activated HSCs. (a, b) The apoptosis of the cells was measured using ﬂow cytometry in ngle cultures and cocultures at 72 hours. 3. Results The results (Figure 6(b)) revealed that, com- pared with group 1, CCl4 increased the expressions of α- SMA and α1(I) collagen (group 2), and these eﬀects were attenuated by the hBM-MSC transplantation (group 3). Accordingly, the extent of liver ﬁbrosis changed in concor- dance with the alteration of HSC activation. liver ﬁbrosis sharply increased in group 2 (CCl4 + vehicle). However, when mice in group 3 received hBM-MSCs, the promoting eﬀect of CCl4 was suppressed and the α-SMA- positive cells and liver ﬁbrosis were partially suppressed. Western blot analysis further proved the protective eﬀect of hBM-MSCs on liver ﬁbrosis. We isolated the HSCs from groups 1–3 and examined the protein levels of α-SMA and α1(I) collagen. The results (Figure 6(b)) revealed that, com- pared with group 1, CCl4 increased the expressions of α- SMA and α1(I) collagen (group 2), and these eﬀects were attenuated by the hBM-MSC transplantation (group 3). Accordingly, the extent of liver ﬁbrosis changed in concor- dance with the alteration of HSC activation. Collectively, these in vivo observations provide further support to the role of hBM-MSCs in HSC activation and liver ﬁbrogenesis. 3.7. hBM-MSCs Decrease the Levels of 4-HNE and p47phox in HSCs in Mouse Model of CCl4-Induced Liver Fibrosis. We have shown that hBM-MSCs can restrain NADPH oxidase 7 Stem Cells International ⁎ ⁎ 0.0 0.5 1.0 1.5 NOX activity as % of control 48 h 72 h 24 h HSCs HSCs + hBM-MSCs (a) ⁎ 0.0 0.5 1.0 1.5 p47 phox mRNA fold change 48 h 72 h 24 h HSCs HSCs + hBM-MSCs (b) ⁎⁎ ⁎ 0.0 0.5 1.0 1.5 The relative expression of NOX2 48 h 72 h 24 h HSCs HSCs + hBM-MSCs NOX2 훽-Actin 48 h 24 h 72 h HSCs HSCs + hBM-MSCs HSCs HSCs + hBM-MSCs HSCs HSCs + hBM-MSCs (c) ⁎⁎ ⁎ ⁎⁎ ⁎ 0.0 0.5 1.0 1.5 The relative expression of p-p47 phox 0.0 0.5 1.0 1.5 The relative expression of p47 phox 48 h 72 h 24 h 48 h 72 h 24 h HSCs HSCs + hBM-MSCs HSCs HSCs + hBM-MSCs HSCs HSCs + hBM-MSCs p47 phox p-p47 phox 훽-Actin 48 h 72 h 24 h HSCs HSCs + hBM-MSCs HSCs HSCs + hBM-MSCs (d) gure 4: NADPH oxidase pathway mediates hBM-MSC inhibition of HSC activation. The cocultures and activated HSCs arvested after being cultured for 24, 48, and 72 hours. 3. Results 8 Stem Cells International Stem Cells International 8 HSCs HSCs + hBM MSCs HSCs + DPI HSCs + hBM MSCs ⁎ ⁎ ⁎ 0.0 0.5 1.0 1.5 훼-SMA mRNA fold change HSCs HSCs + hBM MSCs HSCs + DPI HSCs + hBM MSCs ⁎ ⁎⁎ ⁎ 0.0 0.5 1.0 1.5 훼1(I) collagen mRNA fold change HSCs HSCs + hBM MSCs HSCs + DPI HSCs + hBM MSCs ⁎⁎ ⁎ ⁎ 0.0 0.5 1.0 1.5 2.0 2.5 PPAR훾 mRNA fold change PPAR훾 mRNA fold change + hBM-MSCs + DPI + hBM-MSCs + DPI (a) + hBM-MSCs + DPI + hBM-MSCs + DPI (b) + hBM-MSCs + DPI + hBM-MSCs + DPI (c) ⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎ 0.0 0.5 1.0 1.5 The relative expression of protein HSCs HSCs + hBM-MSCs HSCs + DPI HSCs + hBM-MSCs + DPI 훼-SMA PPAR훾 훼1(I) collagen 훼-SMA 훼1(I) collagen PPAR훾 훽-Actin (d) Figure 5: The pathways of NADPH oxidase are involved in hBM-MSC-induced reduction in liver ﬁbrosis. The cocultures and activated HSCs were pretreated with or without 1 μM DPI for 1 hour, and then cells were harvested after cultured for 72 hours. (a–c) The mRNA levels of PPARγ, α-SMA, and α1(I) collagen were, respectively, detected by real-time PCR (n = 3). ∗P < 0 05 versus the control group. ∗∗P < 0 001 versus the control group. ∗P < 0 05 versus activated HSCs plus DPI. (d) The proteins of PPARγ, α-SMA, and α1(I) collagen were evaluated by Western blot. ∗P < 0 05 versus the control group. ∗∗P < 0 001 versus the control group. + DPI (a) + DPI (b) + DPI (c) ⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎ 0.0 0.5 1.0 1.5 The relative expression of protein HSCs HSCs + hBM-MSCs HSCs + DPI HSCs + hBM-MSCs + DPI 훼-SMA훾 훼1(I) collagen 훼-SMA 훼1(I) collagen PPAR훾 훽-Actin (d) Figure 5: The pathways of NADPH oxidase are involved in hBM-MSC-induced reduction in liver ﬁbrosis. The cocultures and activated HSCs were pretreated with or without 1 μM DPI for 1 hour, and then cells were harvested after cultured for 72 hours. (a–c) The mRNA levels of PPARγ, α-SMA, and α1(I) collagen were, respectively, detected by real-time PCR (n = 3). ∗P < 0 05 versus the control group. ∗∗P < 0 001 versus the control group. ∗P < 0 05 versus activated HSCs plus DPI. 3. Results (d) The proteins of PPARγ, α-SMA, and α1(I) collagen were evaluated by Western blot. ∗P < 0 05 versus the control group. ∗∗P < 0 001 versus the control group. 3. Results (a) NADPH oxidase activity was measured. (b) p47phox mRNA level re cyclophilin was analyzed by real-time PCR (n = 3). ∗P < 0 05 versus the control group. (c, d) NOX2, p47phox, and p-p47phox valuated by Western blot. Experiments were performed in triplicate. ∗P < 0 05 versus the control group and ∗∗P < 0 001 versu ontrol group. tem Cells International (d) Figure 4: NADPH oxidase pathway mediates hBM-MSC inhibition of HSC activation. The cocultures and activated HSCs were harvested after being cultured for 24, 48, and 72 hours. (a) NADPH oxidase activity was measured. (b) p47phox mRNA level relative to cyclophilin was analyzed by real-time PCR (n = 3). ∗P < 0 05 versus the control group. (c, d) NOX2, p47phox, and p-p47phox were evaluated by Western blot. Experiments were performed in triplicate. ∗P < 0 05 versus the control group and ∗∗P < 0 001 versus the control group. 3.8. Eﬀects of hBM-MSCs on MDA, GSH, SOD, and CAT Level in Liver Tissues. Reports have indicated that liver injury can improve oxidative stress through regulating glutathione (GSH) metabolism and attenuating oxidative damage to lipids and proteins, resulting in an antioxidative response [24]. In the present study, in Figure 8, CCl4 injection mark- edly increased malondialdehyde (MDA) level and decreased GSH, superoxide dismutase (SOD), and catalase (CAT) levels, whereas hBM-MSCs signiﬁcantly protected the liver against this eﬀect. group 3 (CCl4 + hBM-MSCs), which suggested the inhibitory role of hBM-MSCs in inductions of 4-HNE and p47phox in HSCs in the model. group 3 (CCl4 + hBM-MSCs), which suggested the inhibitory role of hBM-MSCs in inductions of 4-HNE and p47phox in HSCs in the model. To conﬁrm the above results, HSCs were isolated from each group and directly used for detecting 4-HNE and p47phox protein levels by Western blot analysis (Figure 7(b)). As expected, CCl4 clearly increased the 4- HNE and p47phox protein level. These results further support the role of the NADPH oxidase pathway in the inhibitory eﬀects of hBM-MSCs on HSCs in vivo. To conﬁrm the above results, HSCs were isolated from each group and directly used for detecting 4-HNE and p47phox protein levels by Western blot analysis (Figure 7(b)). As expected, CCl4 clearly increased the 4- HNE and p47phox protein level. These results further support the role of the NADPH oxidase pathway in the inhibitory eﬀects of hBM-MSCs on HSCs in vivo. 4. 3. Results Discussion Hepatic ﬁbrosis is known to be a common clinical symptom. Th iti l l f HSC ti ti i l d l t h excellent clinical candidates in the treatment of hepatic ﬁbrosis, this study has been concerned on establishing proofs for seeking the molecular mechanisms underlying hBM MSC i hibiti f HSC ti ti d th li i HSCs HSCs + hBM-MSCs HSCs + DPI HSCs + hBM-MSCs + DPI ⁎ ⁎ ⁎ 0.0 0.5 1.0 1.5 훼-SMA mRNA fold change (a) HSCs HSCs + hBM-MSCs HSCs + DPI HSCs + hBM-MSCs + DPI ⁎ ⁎⁎ ⁎ 0.0 0.5 1.0 1.5 훼1(I) collagen mRNA fold change (b) HSCs HSCs + hBM-MSCs HSCs + DPI HSCs + hBM-MSCs + DPI ⁎⁎ ⁎ ⁎ 0.0 0.5 1.0 1.5 2.0 2.5 PPAR훾 mRNA fold change (c) ⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎⁎ ⁎ 0.0 0.5 1.0 1.5 The relative expression of protein HSCs HSCs + hBM-MSCs HSCs + DPI HSCs + hBM-MSCs + DPI 훼-SMA PPAR훾 훼1(I) collagen 훼-SMA 훼1(I) collagen PPAR훾 훽-Actin (d) Figure 5: The pathways of NADPH oxidase are involved in hBM-MSC-induced reduction in liver ﬁbrosis. The cocultures and activated HSCs were pretreated with or without 1 μM DPI for 1 hour, and then cells were harvested after cultured for 72 hours. (a–c) The mRNA levels of PPARγ, α-SMA, and α1(I) collagen were, respectively, detected by real-time PCR (n = 3). ∗P < 0 05 versus the control group. ∗∗P < 0 001 versus the control group. ∗P < 0 05 versus activated HSCs plus DPI. (d) The proteins of PPARγ, α-SMA, and α1(I) collagen were evaluated by Western blot. ∗P < 0 05 versus the control group. ∗∗P < 0 001 versus the control group. 4. Discussion excellent clinical candidates in the treatment of hepatic ﬁbrosis, this study has been concerned on establishing proofs for seeking the molecular mechanisms underlying hBM-MSC inhibition of HSCs activation and thus relieving the pathological liver ﬁbrosis in vitro and vivo. These data focus on talking over three aspects: (1) the hBM-MSCs inhibit HSC proliferation and induce apoptosis process in activated HSCs; (2) the positive eﬀect of the hBM-MSCs- downregulated NADPH oxidase pathway inﬂuences the levels of PPARγ, α-SMA, and α1(I) collagen in vitro; (3) and the hBM-MSCs play an important role in protecting CCI4-induced liver ﬁbrosis via inhibition of the NADPH oxidase pathway. Hepatic ﬁbrosis is known to be a common clinical symptom. The critical role of HSC activation in early development has been previously found in liver ﬁbrosis [25] In recent years, the mesenchymal stem cells (MSCs) due to their practical advantages in regenerative medicine have received more and more attention. The bone marrow-derived- (BM-) MSCs are readily obtained and have favorable characteristics including ease of handling in vitro, self-renewal, multipotent diﬀerentiation, and low immunogenicity [26]. Given that the inhibition of the activated HSCs is the central event of the reversal of hepatic ﬁbrosis and BM-MSCs are the 9 Stem Cells International 훼-SMA Nuclei CCl4 + hBM-MSCs Olive oil + vehicle CCl4 + vehicle Merge Collagen CCl4 + hBM-MSCs Olive oil + vehicle CCl4 + vehicle ⁎⁎ # 0 20 40 60 80 100 The numbers of 훼-SMA positive HSCs (a) CCl4 + hBM-MSCs Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs Olive oil + vehicle CCl4 + vehicle * ⁎⁎ ⁎⁎ # # 0.0 0.5 1.0 1.5 2.0 The relative expression of protein 훼-SMA 훼-1(I) collagen 훼-SMA Collagen 훽-Actin (b) Figure 6: hBM-MSCs lead to the decrease in liver ﬁbrosis in the mouse model of CCI4-induced liver injury. Mice were divided into three groups and were, respectively, given administration of olive oil plus vehicle, CCI4 (5 μl/g body weight, two times a week) plus vehicle or CCI4 (5 μl/g body weight, two times a week) plus hBM-MSCs (8 × 106/mouse). (a) Single ﬂuorescence staining of α-SMA on the liver sections was detected (red ﬂuorescence); the nuclei (blue ﬂuorescence) were counterstained with Hoechst 33342. The images were captured with the ﬂuorescence microscope. Scale bar = 100 μm. And stain collagen was examined by Sirius red staining of collagen. The representative images were captured with a light microscope. 4. Discussion Scale bar = 50 μm. The number of α-SMA-positive HSCs in six randomly chosen ﬁelds was counted at 100-fold magniﬁcation, and the average values were shown. ∗∗P < 0 001 versus the control group. #P < 0 05 versus the mice of group treatment with CCI4 plus vehicle. (b) The protein levels of α-SMA and α1(I) collagen were examined by Western blot analysis after the HSCs were isolated from each group. ∗∗P < 0 001 versus the control group. #P < 0 05 versus the mice of group treatment with CCI4 plus vehicle. 9 Stem Cells International 훼-SMA Nuclei CCl4 + hBM-MSCs Olive oil + vehicle CCl4 + vehicle Merge Collagen CCl4 Olive oil CCl4 ⁎⁎ # 0 20 40 60 80 100 The numbers of 훼-SMA positive HSCs + hBM-MSCs + vehicle + vehicle (a) CCl4 + hBM-MSCs Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs Olive oil + vehicle CCl4 + vehicle * ⁎⁎ ⁎⁎ # # 0.0 0.5 1.0 1.5 2.0 The relative expression of protein 훼-SMA 훼-1(I) collagen 훼-SMA Collagen 훽-Actin (b) Figure 6: hBM-MSCs lead to the decrease in liver ﬁbrosis in the mouse model of CCI4-induced liver injury. Mice were divided into three groups and were, respectively, given administration of olive oil plus vehicle, CCI4 (5 μl/g body weight, two times a week) plus vehicle or CCI4 (5 μl/g body weight, two times a week) plus hBM-MSCs (8 × 106/mouse). (a) Single ﬂuorescence staining of α-SMA on the liver sections was detected (red ﬂuorescence); the nuclei (blue ﬂuorescence) were counterstained with Hoechst 33342. The images were captured with the ﬂuorescence microscope. Scale bar = 100 μm. And stain collagen was examined by Sirius red staining of collagen. The representative images were captured with a light microscope. Scale bar = 50 μm. The number of α-SMA-positive HSCs in six randomly chosen ﬁelds was counted at 100-fold magniﬁcation, and the average values were shown. ∗∗P < 0 001 versus the control group. #P < 0 05 versus the mice of group treatment with CCI4 plus vehicle. 4. Discussion (b) The protein levels of α-SMA and α1(I) collagen were examined by Western + hBM-MSCs + vehicle + vehicle (a) CCl4 + hBM-MSCs Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs Olive oil + vehicle CCl4 + vehicle * ⁎⁎ ⁎⁎ # # 0.0 0.5 1.0 1.5 2.0 The relative expression of protein 훼-SMA 훼-1(I) collagen 훼-SMA Collagen 훽-Actin (b) (b) Figure 6: hBM-MSCs lead to the decrease in liver ﬁbrosis in the mouse model of CCI4-induced liver injury. Mice were divided into three groups and were, respectively, given administration of olive oil plus vehicle, CCI4 (5 μl/g body weight, two times a week) plus vehicle or CCI4 (5 μl/g body weight, two times a week) plus hBM-MSCs (8 × 106/mouse). (a) Single ﬂuorescence staining of α-SMA on the liver sections was detected (red ﬂuorescence); the nuclei (blue ﬂuorescence) were counterstained with Hoechst 33342. The images were captured with the ﬂuorescence microscope. Scale bar = 100 μm. And stain collagen was examined by Sirius red staining of collagen. The representative images were captured with a light microscope. Scale bar = 50 μm. The number of α-SMA-positive HSCs in six randomly chosen ﬁelds was counted at 100-fold magniﬁcation, and the average values were shown. ∗∗P < 0 001 versus the control group. #P < 0 05 versus the mice of group treatment with CCI4 plus vehicle. (b) The protein levels of α-SMA and α1(I) collagen were examined by Western blot analysis after the HSCs were isolated from each group. ∗∗P < 0 001 versus the control group. #P < 0 05 versus the mice of group treatment with CCI4 plus vehicle. indicated that hBM-MSCs inhibited the proliferation of acti- vated HSCs by inducing the apoptosis process of activated HSCs. In this regard, the BM-MSCs exerted a promoting eﬀect in apoptosis process of activated HSCs by two-cell apoptotic proteins, antiapoptotic protein Bcl-2, and proa- poptotic protein Bax [18]. The Bcl-2/Bax signaling is the key to mitochondrial-mediated pathway and an important The BM-MSCs could reduce the proliferative capability of activated HSCs and have the potential to act as a prolifer- ative function through changing cell-cycle distribution [27]. The beneﬁcial eﬀects of MSCs on liver ﬁbrosis may be medi- ated by autocrine and paracrine mechanisms. It is reported that BMSCs can enhance the apoptosis of activated HSCs through secreting paracrine factors [28]. 4. Discussion In fact, our report 10 10 Stem Cells International ⁎⁎ # Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs 0.0 0.5 1.0 1.5 The relative number of HSCs Total HSCs p47-phox-positive HSCs Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs ⁎⁎ # 0.0 0.5 1.0 1.5 The relative number of HSCs Total HSCs 4-HNE-positive HSCs 4-HNE SYP Nuclei Merge Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs SYP p47-phox Nuclei Merge Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs (a) ⁎⁎ ⁎⁎ ## ## Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs 0.0 0.5 1.0 1.5 2.0 The relative expression of protein p47 phox 4-HNE p47 phox 4-HNE 훽-Actin Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs (b) Figure 7: hBM-MSCs decrease the levels of 4-HNE and p47phox in HSCs in a mouse model of CCl4-induced liver ﬁbrosis. As descr Figure 6, male mice were randomly separated into three groups; the mice of group 1 served as normal control group, and the other two of mice, respectively, received hBM-MSCs (8 × 106/mouse) or vehicle throughout the 11-week period of CCI4 (5 μl/g body weight, two a week). (a) Double-staining was performed on liver section: synaptophysin (SYP, a marker for quiescent and activated HSCs) ﬂuorescence); 4-HNE and p47phox (markers for positive HSCs) (red ﬂuorescence); nuclei (blue ﬂuorescence). The images captured with the ﬂuorescence microscope. Scale bar = 50 μm. The total HSCs (SYP-positive HSCs) and the relative number of 4 and p47phox-positive HSCs in six randomly chosen ﬁelds were counted at 100-fold magniﬁcation, and the average value shown. ∗∗P < 0 001 versus the control group. #P < 0 05 versus the mice of group treatment with CCI4 plus vehicle. (b) The p levels of 4-HNE and p47phox were examined by Western blot analysis after the HSCs were isolated from each group. ∗∗P < versus the control group. ##P < 0 001 versus the mice of group treatment with CCI4 plus vehicle. 4. Discussion The levels of MDA, GSH, SOD, and CAT in liver tissues in the three groups were detected with chromatometry. ∗P < 0 05 versus the control group. ∗∗P < 0 001 versus the control group. #P < 0 05 versus the mice of group treatment with CCI4 plus vehicle. 11 Stem Cells International Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs ⁎ # 0 1 2 3 4 5 MDA protein (nmol/mg) Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs ⁎ # 0 5 10 15 GSH protein (nmol/mg) MDA protein (nmol/mg) vehicle vehicle hBM MSCs (a) Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs ⁎⁎ # 0 50 100 150 SOD protein (U/mg) (c) (b) Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs # ⁎ 0 10 20 30 40 CAT protein (U/mg) (d) (a) (b) Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs ⁎⁎ # 0 50 100 150 SOD protein (U/mg) (c) Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs # ⁎ 0 10 20 30 40 CAT protein (U/mg) (d) Figure 8: Eﬀects of hBM-MSCs on MDA, GSH, SOD, and CAT level in liver tissues. The levels of MDA, GSH, SOD, and CAT in liver t in the three groups were detected with chromatometry. ∗P < 0 05 versus the control group. ∗∗P < 0 001 versus the control group. #P versus the mice of group treatment with CCI4 plus vehicle. (a) (b) (c) (d) Figure 8: Eﬀects of hBM-MSCs on MDA, GSH, SOD, and CAT level in liver tissues. The levels of MDA, GSH, SOD, and CAT in liver tissues in the three groups were detected with chromatometry. ∗P < 0 05 versus the control group. ∗∗P < 0 001 versus the control group. #P < 0 05 versus the mice of group treatment with CCI4 plus vehicle. may a better understanding of the inhibition eﬀect of hBM- MSCs on NADPH oxidase signaling pathway. regulator of the intrinsic apoptosis [29]. Importantly, the results were consistent with those of other antiﬁbrotic drugs [30], which have been shown to promote the apoptosis of activated HSCs by downregulating Bcl-2 and upregulating Bax and cleaved caspase-3. The accumulation of extracellular matrix ECM which is mainly produced by activated HSCs is one of the important characteristics for the reaction of ﬁbrosis [3]. 4. Discussion ⁎⁎ # Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs 0.0 0.5 1.0 1.5 The relative number of HSCs Total HSCs p47-phox-positive HSCs Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs ⁎⁎ # 0.0 0.5 1.0 1.5 The relative number of HSCs Total HSCs 4-HNE-positive HSCs 4-HNE SYP Nuclei Merge Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs SYP p47-phox Nuclei Merge Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs (a) The relative number of HSCs (a) (b) Figure 7: hBM-MSCs decrease the levels of 4-HNE and p47phox in HSCs in a mouse model of CCl4-induced liver ﬁbrosis. As described in Figure 6, male mice were randomly separated into three groups; the mice of group 1 served as normal control group, and the other two groups of mice, respectively, received hBM-MSCs (8 × 106/mouse) or vehicle throughout the 11-week period of CCI4 (5 μl/g body weight, two times a week). (a) Double-staining was performed on liver section: synaptophysin (SYP, a marker for quiescent and activated HSCs) (green ﬂuorescence); 4-HNE and p47phox (markers for positive HSCs) (red ﬂuorescence); nuclei (blue ﬂuorescence). The images were captured with the ﬂuorescence microscope. Scale bar = 50 μm. The total HSCs (SYP-positive HSCs) and the relative number of 4-HNE and p47phox-positive HSCs in six randomly chosen ﬁelds were counted at 100-fold magniﬁcation, and the average values were shown. ∗∗P < 0 001 versus the control group. #P < 0 05 versus the mice of group treatment with CCI4 plus vehicle. (b) The protein levels of 4-HNE and p47phox were examined by Western blot analysis after the HSCs were isolated from each group. ∗∗P < 0 001 versus the control group. ##P < 0 001 versus the mice of group treatment with CCI4 plus vehicle. 11 Stem Cells International 11 Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs ⁎ # 0 1 2 3 4 5 MDA protein (nmol/mg) (a) Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs ⁎ # 0 5 10 15 GSH protein (nmol/mg) (b) Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs ⁎⁎ # 0 50 100 150 SOD protein (U/mg) (c) Olive oil + vehicle CCl4 + vehicle CCl4 + hBM-MSCs # ⁎ 0 10 20 30 40 CAT protein (U/mg) (d) Figure 8: Eﬀects of hBM-MSCs on MDA, GSH, SOD, and CAT level in liver tissues. 4. Discussion Peroxisome proliferator-activated receptor γ (PPARγ) exerts a key role in the inhibition of HSC activation [34]. Our researches showed that using either DPI (an inhibitor for NADPH oxi- dase pathway) or the hBM-MSCs would result in a dramatic drop of αSMA (a well-established marker for HSC activation) and α(I) collagen (the major component of ECM) in acti- vated HSCs in vitro and a rocket increasing of the PPARγ expression. The inhibitor for NADPH oxidase pathway, DPI, signiﬁcantly strengthened the hBM-MSCs, the promo- tion eﬀect on PPARγ, and inhibition of α1(1) collagen and α-SMA expressions. This result suggested that hBM-MSCs might regulate activated HSCs expression, at least in part, by inhibiting the NADPH oxidase pathway. In addition, multiple signaling pathways are involved in the eﬀect of the MSC-induced HSC growth inhibition [31]. We noticed that the generation of reactive oxygen species (ROS) is interrelated and interacts on hepatic ﬁbrosis pro- cesses, and NADPH oxidases (NOX) work as a key source of ROS [32]. What is more, NADPH oxidase signaling path- way is closely related in experimental models of liver ﬁbrosis and in patients with chronic HCV-derived infection [33]. There was a possibility that a potentially eﬀective mecha- nisms between hBM-MSCs and HSCs might exist. In in vitro experiments, hBM-MSCs would signiﬁcantly reduce the NOX activity. p47phox, a functional active component of the NADPH oxidase complex, is considered to play a crucial role in the activity of NADPH oxidase [20]. In our study, a slight reduction in p47phox expression had been found. Interestingly, the phosphorylation of the p47phox subunits was signiﬁcantly decreased by the eﬀect of the hBM-MSCs. Thus, the results that the hBM-MSCs may restrain the NADPH oxidase activation by phosphorylation of p47phox Multiple signaling pathways or channels have been con- ﬁrmed to participate in the regulatory mechanism of hBM- MSCs on HSC activation [35, 36], and our study may provide a new clue and possibility for explaining the protective eﬀect of hBM-MSCs on liver ﬁbrosis. The antiﬁbrotic eﬀects of the hBM-MSCs were also investigated by carbon tetrachloride- 12 Stem Cells International (CCl4-) induced liver ﬁbrotic mouse model via the liver portal vein; the hBM-MSCs recover liver function markers at 21 days after transplantation. [6] M. A. Puglisi, V. Tesori, W. Lattanzi et al., “Therapeutic impli- cations of mesenchymal stem cells in liver injury,” Journal of Biomedicine and Biotechnology, vol. 2011, article 860578, 8 pages, 2011. 4. Discussion Our results revealed that NADPH oxidase was required for hBM-MSC-induced decrease in HSC activation in vitro and in vivo. 4-Hydroxynonenal (HNE) mediates oxidative stress-linked pathological processes [27], and a large number of positive HSCs for 4-HNE and p47phox was tested in mouse cirrhosis liver [12]. As we have demonstrated, CCl4 increased 4-HNE and p47phox-positive HSCs whereas the expression of 4-HNE and p47phox-positive HSCs was reduced with the treatment of hBM-MSCs. In combination with the above in vivo experiments, hBM-MSCs deduce the NADPH oxidase pathway, accompanied by the inhibitions of HSC activation and liver ﬁbrosis in CCl4-induced liver injury of mice. The stimulation of GSH synthesis and the inhibition of GSH depletion are major contributors to the antioxidative mechanism in liver injury. In our study, CCl4 injection markedly decreased GSH levels, whereas hBM- MSCs signiﬁcantly reversed this eﬀect. [7] V. Volarevic, J. Nurkovic, N. Arsenijevic, and M. Stojkovic, “Concise review: therapeutic potential of mesenchymal stem cells for the treatment of acute liver failure and cirrhosis,” Stem Cells, vol. 32, no. 11, pp. 2818–2823, 2014. [8] C. A. Gregory, D. J. Prockop, and J. L. Spees, “Non-hematopoi- etic bone marrow stem cells: molecular control of expansion and diﬀerentiation,” Experimental Cell Research, vol. 306, no. 2, pp. 330–335, 2005. [9] S. Gaia, A. Smedile, P. Omedè et al., “Feasibility and safety of G-CSF administration to induce bone marrow-derived cells mobilization in patients with end stage liver disease,” Journal of Hepatology, vol. 45, no. 1, pp. 13–19, 2006. [10] Z. J. Yan, Y. Q. Hu, H. T. Zhang et al., “Comparison of the neural diﬀerentiation potential of human mesenchymal stem cells from amniotic ﬂuid and adult bone marrow,” Cellular and Molecular Neurobiology, vol. 33, no. 4, pp. 465–475, 2013. [11] T. H. Shin, S. Lee, K. R. Choi et al., “Quality and freshness of human bone marrow-derived mesenchymal stem cells decrease over time after trypsinization and storage in phosphate-buﬀered saline,” Scientiﬁc Reports, vol. 7, no. 1, p. 1106, 2017. In summary, we have shown that hBM-MSCs are able to suppress the HSC activation and hBM-MSCs were shown to induce the apoptosis of activated HSCs. Furthermore, in vitro and in vivo, we showed that the antiﬁbrotic eﬀect of hBMSCs was mediated at least through the signaling pathways of NADPH oxidase. The authors declare that they have no conﬂicts of interest. The authors declare that they have no conﬂicts of interest. [15] F. Varga, G. Méhes, and Z. Molnár, “Reversibility of hepatic ﬁbrosis induced by carbon tetrachloride in the rat,” Acta Phy- siologica Academiae Scientiarum Hungaricae, vol. 29, no. 1, pp. 69–74, 1966. 4. Discussion These data suggested that hBMSCs play an important role in protecting CCI4-induced HSC activa- tion via inhibition of the CCI4 NADPH oxidase pathway and may represent a novel therapeutic way for the disease. These ﬁndings here not only greatly clariﬁed the mechanism of hepatic ﬁbrosis and provided a novel treatment option for the liver ﬁbrosis but also made a thorough inquiry into the preclinical mechanisms of MSC’s regenerative medicine. [12] H. Qiao, Q. Cao, Y. Fu et al., “Sex-determining region Y-box 9 acts downstream of NADPH oxidase to inﬂuence the eﬀect of leptin on PPARγ1 expression in hepatic stellate cells,” Biochi- mica et Biophysica Acta (BBA) - Molecular Basis of Disease, vol. 1862, no. 11, pp. 2186–2196, 2016. [13] C. C. Lin, W. N. Lin, R. L. Cho, C. Y. Wang, L. D. Hsiao, and C. M. Yang, “TNF-α-induced cPLA2 expression via NADPH oxidase/reactive oxygen species-dependent NF-κB cascade on human pulmonary alveolar epithelial cells,” Frontiers in Phar- macology, vol. 7, p. 447, 2016. [14] N. H. Truong, N. H. Nguyen, T. V. le et al., “Comparison of the treatment eﬃciency of bone marrow-derived mesenchymal stem cell transplantation via tail and portal veins in CCl4- induced mouse liver ﬁbrosis,” Stem Cells International, vol. 2016, Article ID 5720413, 13 pages, 2016. Authors’ Contributions Haowen Qiao and Yu Zhou contributed equally to this work. Haowen Qiao and Yu Zhou contributed equally to this work. [16] D. Cassiman, J. van Pelt, R. de Vos et al., “Synaptophysin: a novel marker for human and rat hepatic stellate cells,” The American Journal of Pathology, vol. 155, no. 6, pp. 1831– 1839, 1999. Conflicts of Interest The authors declare that they have no conﬂicts of interest. References [1] S. L. Friedman, “Hepatic stellate cells: protean, multifunc- tional, and enigmatic cells of the liver,” Physiological Reviews, vol. 88, no. 1, pp. 125–172, 2008. [1] S. L. Friedman, “Hepatic stellate cells: protean, multifunc- tional, and enigmatic cells of the liver,” Physiological Reviews, vol. 88, no. 1, pp. 125–172, 2008. [17] X. Zhai, H. Qiao, W. Guan et al., “Curcumin regulates perox- isome proliferator-activated receptor-γ coactivator-1α expres- sion by AMPK pathway in hepatic stellate cells in vitro,” European Journal of Pharmacology, vol. 746, pp. 56–62, 2015. [2] A. Geerts, “History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells,” Seminars in Liver Disease, vol. 21, no. 3, pp. 311–336, 2001. [2] A. Geerts, “History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells,” Seminars in Liver Disease, vol. 21, no. 3, pp. 311–336, 2001. [18] J. C. Martinou and R. J. Youle, “Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics,” Develop- mental Cell, vol. 21, no. 1, pp. 92–101, 2011. [3] V. Hernandez-Gea and S. L. Friedman, “Pathogenesis of liver ﬁbrosis,” Annual Review of Pathology: Mechanisms of Disease, vol. 6, no. 1, pp. 425–456, 2011. [19] E. Crosas-Molist and I. Fabregat, “Role of NADPH oxidases in the redox biology of liver ﬁbrosis,” Redox Biology, vol. 6, pp. 106–111, 2015. [4] M. Sarem, R. Znaidak, M. Macías, and R. Rey, “Hepatic stellate cells: it’s role in normal and pathological conditions,” Gastro- enterología y Hepatología, vol. 29, no. 2, pp. 93–101, 2006. [20] R. Bataller, R. F. Schwabe, Y. H. Choi et al., “NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic ﬁbrosis,” The Journal of Clinical Investiga- tion, vol. 112, no. 9, pp. 1383–1394, 2003. [5] D. Schuppan and Y. O. Kim, “Evolving therapies for liver ﬁbrosis,” The Journal of Clinical Investigation, vol. 123, no. 5, pp. 1887–1901, 2013. 13 Stem Cells International 13 [21] S. De Minicis and D. A. Brenner, “NOX in liver ﬁbrosis,” Archives of Biochemistry and Biophysics, vol. 462, no. 2, pp. 266–272, 2007. [22] H. Kono, I. Rusyn, M. Yin et al., “NADPH oxidase–derived free radicals are key oxidants in alcohol-induced liver disease,” The Journal of Clinical Investigation, vol. 106, no. 7, pp. 867– 872, 2000. [23] X. Zhai, K. Yan, J. References Fan et al., “The β-catenin pathway contrib- utes to the eﬀects of leptin on SREBP-1c expression in rat hepatic stellate cells and liver ﬁbrosis,” British Journal of Phar- macology, vol. 169, no. 1, pp. 197–212, 2013. [24] H. Shi, A. Shi, L. Dong et al., “Chlorogenic acid protects against liver ﬁbrosis in vivo and in vitro through inhibition of oxidative stress,” Clinical Nutrition, vol. 35, no. 6, pp. 1366–1373, 2016. [25] S. L. Friedman, “Molecular regulation of hepatic ﬁbrosis, an integrated cellular response to tissue injury,” The Journal of Biological Chemistry, vol. 275, no. 4, pp. 2247–2250, 2000. [26] S. Terai, T. Ishikawa, K. Omori et al., “Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy,” Stem Cells, vol. 24, no. 10, pp. 2292– 2298, 2006. [27] S. Qin, H. Jiang, S. Su et al., “Inhibition of hepatic stellate cell proliferation by bone marrow mesenchymal stem cells via regulation of the cell cycle in rat,” Experimental and Therapeu- tic Medicine, vol. 4, no. 3, pp. 375–380, 2012. [28] B. Parekkadan, D. van Poll, Z. Megeed et al., “Immunomodu- lation of activated hepatic stellate cells by mesenchymal stem cells,” Biochemical and Biophysical Research Communications, vol. 363, no. 2, pp. 247–252, 2007. [29] A. Shamas-Din, S. Bindner, X. Chi, B. Leber, D. W. Andrews, and C. Fradin, “Distinct lipid eﬀects on tBid and Bim activa- tion of membrane permeabilization by pro-apoptotic Bax,” The Biochemical Journal, vol. 467, no. 3, pp. 495–505, 2015. [30] Y. Wang, J. Gao, D. Zhang, J. Zhang, J. Ma, and H. Jiang, “New insights into the antiﬁbrotic eﬀects of sorafenib on hepatic stellate cells and liver ﬁbrosis,” Journal of Hepatology, vol. 53, no. 1, pp. 132–144, 2010. [31] S. Chen, L. Xu, N. Lin, W. Pan, K. Hu, and R. Xu, “Activation of Notch1 signaling by marrow-derived mesenchymal stem cells through cell–cell contact inhibits proliferation of hepatic stellate cells,” Life Sciences, vol. 89, no. 25-26, pp. 975–981, 2011. [32] S. De Minicis, E. Seki, Y.-H. Paik et al., “Role and cellular source of nicotinamide adenine dinucleotide phosphate oxidase in hepatic ﬁbrosis,” Hepatology, vol. 52, no. 4, pp. 1420–1430, 2010. [33] P. Sancho, J. Mainez, E. Crosas-Molist et al., “NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver ﬁbrosis development,” PLoS One, vol. 7, no. 9, article e45285, 2012. [34] H. References Tsukamoto, “Adipogenic phenotype of hepatic stellate cells,” Alcoholism: Clinical & Experimental Research, vol. 29, no. S2, pp. 132S-133S, 2005. [35] P. P. Wang, D. Y. Xie, X. J. Liang et al., “HGF and direct mesenchymal stem cells contact synergize to inhibit hepatic stellate cells activation through TLR4/NF-kB pathway,” PLoS One, vol. 7, no. 8, article e43408, 2012. [36] X. E. Sun, X. Q. Zhang, and M. M. Liu, “Eﬀect of bone marrow mesenchymal stem cells on the TGF-β1/Smad signaling path- way of hepatic stellate,” Genetics and Molecular Research, vol. 14, no. 3, pp. 8744–8754, 2015.
https://openalex.org/W4388981927	https://storage.googleapis.com/jnl-lse-j-lselr-files/journals/1/articles/485/submission/proof/485-1-2770-1-10-20230313.pdf	English	null	Sovereign Therefore Limited: The Unconstitutionality of Ouster Clauses for Errors of Law Under the British Constitution	LSE law review	2,023	cc-by	9,432	2023 2023 LSE LAW REVIEW 429 * BCL Candidate (Oxon) ’23, LLB (UCL) ’22. I thank Professor Rick Rawlings (UCL) for his precious feedback on earlier versions of this piece. The views expressed in this article are mine only. Address for correspondence: vincent.lafortune@wadham.ox.ac.uk. Sovereign Therefore Limited: The Unconstitutionality of Ouster Clauses for Errors of Law under the British Constitution Vincent Lafortune* ABSTRACT This paper makes a radical claim: it argues that ouster clauses that seek to prevent judicial review for errors of law are incompatible with, and thus inadmissible under, the British Constitution. In other words, the claim is that judicial review for illegality is a constitutional fundamental that not even Parliament is competent to abolish. Two arguments are deployed to support that contention. Firstly, it is argued that a full grasp of the core constitutional principle of parliamentary sovereignty logically entails Parliament’s inability to oust the judicial review jurisdiction of the courts with respect to errors of law, as parliamentary sovereignty and ouster clauses are mutually exclusive concepts. Secondly, it is argued that this limitation to Parliament’s legislative remit is the manifestation of a higher order law that necessarily constrains the exercise of legislative power in a democracy. Vol. VIII Sovereign Therefore Limited 430 In exceptional circumstances involving an attempt to abolish judicial review or the ordinary role of the courts, the Appellate Committee of the House of Lords or a new Supreme Court may have to consider whether this is a constitutional fundamental which even a sovereign Parliament acting at the behest of a complaisant House of Commons cannot abolish.1 4 John A.G. Griffith, ‘The Political Constitution’ (1979) 42 MLR 1, 19. 3 Reference to ‘review’ in this paper is intended to include all the components involved in the process of judicially reviewing an administrative act for errors of law: from the scrutinising of the act’s fit under the empowering primary legislation to the nullification ab initio of the administrative act for want of fit (whether by way of quashing order or declaration). 2 Ouster clauses are defined as ‘[s]tatutory provisions which seek to limit [or indeed outright oust] the ordinary jurisdiction of the court’: Anisminic Ltd v Foreign Compensation Commission [1969] 2 AC 147 170. y 1 R (Jackson) v Attorney General [2005] UKHL 56; [2006] 1 AC 262 [302]-[303] by Lord Steyn. 3 Reference to ‘review’ in this paper is intended to include all the components involved in the process of judicially reviewing an administrative act for errors of law: from the 2 Ouster clauses are defined as ‘[s]tatutory provisions which seek to limit [or indeed outright oust] the ordinary jurisdiction of the court’: Anisminic Ltd v Foreign Compensation Commission [1969] 2 AC 147 170. 4 John A.G. Griffith, ‘The Political Constitution’ (1979) 42 MLR 1, 19. 5 ‘Jurisdiction’ is used here in its enlarged, post-Anisminic sense: see O’Reilly v Mackman [1983] 2 AC 237, 278. Moreover, the reader may legitimately query the decision to restrict this analysis to statutorily derived public powers. Why not address public powers derived from other sources, such as prerogative powers? To echo what is said further down in the main text, this omission should not be treated as an admission. The decision to so restrict the analysis is due to multiple factors. First, statute-derived powers undeniably constitute the bulk of administrative powers in use today making their treatment of the utmost relevance. Second, the core contention is radical and controversial enough as it is that it would be unhelpful in the circumstances not to adopt as narrow a scope as possible. Third, and relatedly, the nature of prerogative executive powers is in itself a controversial topic and an analysis of ouster clauses pertaining to those powers would necessarily make assumptions that could attract criticism and thus further detract from the core case. We shall return to prerogative powers in the future. A. Aim of this Article This article contends that ouster clauses2 (‘ousters’) that purport to oust the jurisdiction of the courts to review executive decisions for errors of law (or, interchangeably, ‘illegality’ or ‘classic ultra vires’) are incompatible with, and therefore inadmissible under, the Constitution of the United Kingdom.3 To be sure, the contention is not merely that ousters should not be enacted by Parliament because they are inimical to a healthy constitutional balance. This would be to implicitly accede to a view of the constitutional order that this paper rejects, namely that ‘[e]verything that happens [in the United Kingdom] is constitutional’4 and that where the constitutional equilibrium is to be struck is ultimately a matter for the political arena. The contention of this piece is more radical, as statutory provisions that unequivocally purport to bar the courts from submitting decisions taken by public bodies to judicial review for illegality ought to be denied legal effect by the courts, notwithstanding unambiguous intentions LSE LAW REVIEW 2023 431 to the contrary by Parliament, because such statutory provisions are unconstitutional. to the contrary by Parliament, because such statutory provisions are unconstitutional. This argument will be advanced in two parts. In the first part, I will argue that the core constitutional principle of parliamentary sovereignty itself logically necessitates Parliament’s inability to oust judicial review for errors of law. Thus, the very thing that is often invoked as the ultimate justification for the constitutionality of ouster clauses — the notion of parliamentary sovereignty — will be shown to act as a logical bar to the abolition by Parliament of judicial review for illegality. In the second part, I will argue that this ‘legislative disability’, which will have been shown in the first part to be derivable from the notion of sovereignty, is in the final analysis the consequence of a higher order law that necessarily constrains the exercise of legislative power in a democracy. B. The Type of Ouster Clauses to Be Discussed This article deals exclusively with ouster clauses that are designed to preclude the review of executive decisions or acts falling outside the jurisdiction (in the classic ultra vires sense) afforded to the decision-maker by the empowering legislation.5 In other words, this paper tackles ouster clauses that purport to bar the courts from scrutinising and, if need be, impeaching a public body’s decision for illegality. Of course, ouster clauses may also be designed to limit the applicability of the various common law principles of good administration, such as natural justice, reasonableness, proper purpose, etc. However, this paper does not address the constitutional position of these other Vol. VIII Sovereign Therefore Limited 432 ‘precepts of legality enforced by judicial review against public agencies’.6 This omission should not, however, be treated as an admission that these are undeserving of the same fundamental status. It will undoubtedly be important to return to these matters in a future contribution. For now, it is simply considered that tackling ouster clauses that purport to shield administrative decisions from judicial review for errors of law is an undertaking to which one entire contribution can properly be devoted. In principle, review for errors of law is a straightforward affair. Consider the underpinning theory to begin with, where Parliament, the legislative sovereign, grants powers to a body or a person. That body or person possesses that power only by virtue of grant of that power by the legislature; absent such a conferral, the body or person is no more legally empowered, nor no less legally burdened, than a private, lay individual. The picture that emerges is that of a primordial legal ‘black hole’, incrementally populated with ‘jurisdictional spotlights’ as Parliament empowers bodies or persons with various public administrative capacities. This imagery vividly brings to life the ‘fundamental principle, inherent throughout the legal system, that powers can be validly exercised only within their true limits’.7 If a power is not exercised according to its limits, the exercise of the power is but a purported one as, in truth, it is an act carried out by the body or person that falls within the legal nothingness, that is, the black hole, and however close it might have gotten to the jurisdictional spotlight, it does not benefit from its vitalising effect. 10 R (UNISON) v Lord Chancellor [2017] UKSC 51, [2020] AC 869. 9 HM Treasury v Ahmed (No 2) [2010] UKSC 5, [2010] 2 AC 534 [1]. 8 ibid. 7 William Wade and Christopher Forsyth, Administrative Law (11th edn, OUP 2014) Chapter 2, 11. 6 T.R.S. Allan, The Sovereignty of Law: Freedom, Constitution and Common Law (OUP, 2013) 217. 14 ibid [54]. 13 R (Privacy International) v Investigatory Powers Tribunal [2019] UKSC 22; [2020] AC 491. 12 Anisminic (n 2) 170. 11 ibid [87]. B. The Type of Ouster Clauses to Be Discussed The law, in other words, does not ‘see’ the act, as the ‘authority has in law done nothing’.8 Review for errors of law is one important way of giving effect to the above theoretical underpinnings. The court looks at the empowering statute and compares its terms with the action purportedly taken on that basis by a public body. If it turns out that the public body has acted inconsistently with the terms of the applicable law, that body has acted ‘without authority’ and its actions are in effect writ in water, having no effect at law.9 UNISON10 is instructive in that 10 R (UNISON) v Lord Chancellor [2017] UKSC 51, [2020] AC 869. 9 HM Treasury v Ahmed (No 2) [2010] UKSC 5, [2010] 2 AC 534 [1]. LSE LAW REVIEW 2023 433 respect. In that case, the question was whether fees of access to tribunals imposed by the Lord Chancellor pursuant to Section 42(1) of the Tribunals, Courts and Enforcement Act 2007 were ultra vires (i.e., were mistaken as to the applicable law) because they had the effect of creating a ‘real risk that persons will effectively be prevented from having access to justice’,11 an effect which the 2007 Act was interpreted as not empowering fees set under it to produce. Lord Reed found that the court fees had such an effect and that, therefore, the Order responsible for those fees was to be quashed and treated as unlawful ab initio. The kind of ouster clause that this article contends is repugnant to the Constitution; it is the kind of ouster clause that seeks to prevent the courts from engaging in the process just described. 19 ibid [43]. 18 R (Cart) v Upper Tribunal [2011] UKSC 28, [2012] 1 AC 663. 17 This is for two main reasons. First, it is clear from the second ground of appeal in Privacy International (n 13) that there is an appetite to run a ‘strike down’ argument. Second, it is my hope that a flurry of academic commentary prompted by the enactment of s 2 JRCA 2022 will convince practitioners to bring such a case before the court. 16 s 2(7) JRCA 2022. 15 This also applies to s 3 of the Dissolution and Calling of Parliament Act 2022 which predates s 2 JRCA 2022, but the ouster in Section 2 JRCA 2022 is more likely to make waves and thus is the one discussed here. C. The Trigger for this Article Until very recently, this paper’s relevance to public and administrative lawyers might have been legitimately doubted. That it was concerned with a question of academic interest only would have been fair criticism — although, to my mind at least, not a fatal criticism, for the quest to uncover the true nature and implications of our Constitution is surely an intrinsically worthy enterprise. The reason why the utility of this paper may have heretofore been doubted is well known to public lawyers, as ever since the case of Anisminic the courts have drawn the distinction between a decision and a purported decision.12 This distinction, which was reaffirmed as recently as in 2019 in the Privacy International case,13 has effectively rendered nugatory statutory provisions seeking to preclude the courts from judicially reviewing ‘decisions’ taken by public bodies. That was so because a ‘decision’ afflicted by an error of law was in fact not a ‘decision’ but a ‘nullity’, or a ‘purported decision’, which was, as such, not captured by the ouster clause.14 In providing for the screening of ‘decisions’ from judicial review, Parliament was deemed not to have intended to preclude the review and quashing of ‘purported decisions’. Hence, there was no need to run a constitutional ‘strikedown’ argument in order to neutralise an ouster clause. Vol. VIII Vol. VIII 434 Sovereign Therefore Limited Today, the relevance of the question at the heart of this paper — namely, whether Parliament has the competence to prevent review and quashing of executive decisions taken under an empowering statute but afflicted by one or more errors of law — cannot be in doubt. Section 2 of the Judicial Review and Courts Act 2022 (‘JRCA 2022’) is an ouster clause worded in such a way that immunises it from the linguistic distinction just discussed.15 Indeed, in providing that ‘“decision” includes any purported decision’,16 Parliament closes the interpretative gap through which the courts have, until now, bypassed ouster clauses. It follows that if, and most likely when,17 s 2 JRCA 2022 is litigated, the arguments will necessarily focus on what is perhaps the most profound, contentious, and divisive of constitutional questions. Indeed, although in normal times it is certainly preferable to avoid asking whether Parliament’s legislative remit knows any limits, here the Supreme Court is bound to be asked to directly address and answer the question of Parliament’s ability to enact ouster clauses. C. The Trigger for this Article The object of this article is therefore to suggest an answer to this critically important question. D. Caveat An important caveat must be borne in mind throughout this paper. As will become clear further down, one assumption upon which this contribution in part rests is that tribunals are not constituent entities of the judicial system. Tribunals, instead, are ‘judicialised’ executive bodies, but nothing more than that. Thus, in subscribing to the position as it was laid down by Lady Hale in Cart18 — where Her Ladyship clearly held the view that tribunals were not part of the ‘legal system’ (it is submitted that ‘judicial system’ is slightly more helpful terminology)19 — this article conforms to the current authoritative legal characterisation of tribunals. My view is that Lady Hale is right to say that tribunals are not judicial entities properly so called. Of course, one may take issue LSE LAW REVIEW 2023 435 with that assumption and with Lady Hale’s characterisation of tribunals,20 but it is not the aim of this paper to participate in that debate — that is a matter for another enterprise. Should the reader reject the aforementioned characterisation of tribunals, then for them the arguments in this paper will only apply to ouster clauses designed to preclude the judicial review of decisions taken by executive bodies other than tribunals. To be clear, if one takes the view that tribunals are proper executive bodies, then the following discussion applies to ouster clauses shielding tribunal decisions as much as it does to ouster clauses shielding decisions from other, more traditional, executive bodies. But, if one takes the view that tribunals are constitutionally distinct from executive bodies and should be considered as constituents of the legal (or judicial) system, then to that person the arguments herein apply to ouster clauses attached to decisions taken by all executive bodies, but not to tribunals. Finally, this paper should not lose its prima facie appeal from the standpoint of the reader who disagrees with the view that tribunals are constitutional equivalents of other executive bodies. Indeed, although for this reader the ouster clause in s 2 JRCA 2022 is immune from the arguments that will be made in this contribution because it protects a decision made by a tribunal, there are two reasons why she might still be interested in reading on. D. Caveat Firstly, as just mentioned, the law currently stands in favour of the view that tribunals are not judicial bodies, which means that legal arguments can confidently be run on that basis. Secondly, the government openly intends to use ‘[t]he explicit Cart ouster clause … as an example to guide the development of effective legislation in the future’.21 It is likely that such future legislation will not only seek to shield decisions made by tribunals from judicial review but also decisions taken by more ‘traditional’ executive decision-makers. The arguments hereunder can be considered as a pre-emptive strike on such constitutional aberrations. 21 Judicial Review Reform Consultation, ‘The Government Response’ (2021) CP 477 [5 21 20 Timothy Endicott, Administrative Law (5th edn, OUP 2021) 490. 21 Judicial Review Reform Consultation, ‘The Government Response’ (2021) CP 477 [55]. Timothy Endicott, Administrative Law (5th edn, OUP 2021) 490. 26 Conor Gearty, ‘In the Shallow End’ (2022) 42 London Review of Books. 25 ibid [169]. 24 Privacy International (n 13) [207]-[211]. 23 Lady Hale and Lords Kerr, Carnwath and Lloyd-Jones. 22 Privacy International (n 13) [207]. E. Angle of Attack It will have become apparent to the reader acquainted with the case law on ouster clauses that this article aims to show why the second question Vol. VIII Vol. VIII Sovereign Therefore Limited 436 which arose but ultimately was not answered in the Privacy International case — namely ‘whether … Parliament may by statute “oust” the supervisory jurisdiction of the High Court to quash the decision of an inferior court or tribunal of limited statutory jurisdiction’22 — should be answered in the negative. In the Privacy International litigation, it ultimately proved unnecessary for the majority23 to answer that question. Applying the Anisminic interpretive approach to the construction of Section 67(8) of the Regulation of Investigatory Powers Act 2000, the majority found that Parliament had not in fact intended to oust the courts’ judicial review jurisdiction for errors of law made by the Investigatory Powers Tribunal (‘IPT’). The second question did not therefore arise for consideration. Nevertheless, some justices in the majority provided substantive obiter dicta on the second question, and the dissentients, having found that Parliament had in fact intended to exclude the jurisdiction of the courts to review decisions of the IPT for errors of law, necessarily had to grapple with it. Accordingly, the approach I have chosen for this paper is to frame my arguments around Lord Sumption’s discussion of and answer to that question in his dissenting judgment.24 This approach, I believe, maximises the impact of this contribution on the law. Indeed, while Lord Sumption is no longer in the Supreme Court, it is noteworthy and, in fact, of paramount importance that Lord Reed unqualifiedly agreed with Lord Sumption’s judgment in Privacy International.25 Since Lord Reed is the acting President of the Supreme Court, His Lordship is, incontestably, an influential figure on the bench.26 As things currently stand, Lord Reed’s Supreme Court would most likely follow Lord Sumption’s reasoning. Thus, instead of arguing against ouster clauses in isolation and leaving it to others to pluck and apply the substantive reasoning hereunder to the case law, I have decided to apply it myself to Lord Sumption’s judgment to show where His Lordship (respectfully) erred in concluding that Parliament did have the power to oust judicial review in that case. There are other reasons why direct engagement with Lord Sumption’s judgment is valuable, and these will become clear later. E. Angle of Attack 2023 LSE LAW REVIEW 437 Finally, as will be seen below, it is noteworthy that nothing in Lord Sumption’s answer to the second question turns on the distinction between tribunals and other executive bodies.27 Therefore, while the question in Privacy International is narrowly framed and refers only to ‘inferior courts’ and ‘tribunals of limited statutory jurisdiction’ — understandably, as the decision challenged in that case was that of the IPT — Lord Sumption’s answer can safely be read as answering the broader question of whether Parliament may, by statute, oust the supervisory jurisdiction of the High Court to quash the decision of any decision-making body empowered by statute. This only further emphasises the pertinence of framing the substantive arguments of this paper around Lord Sumption’s (and thus Lord Reed’s) reasoning. Indeed, an attack on His Lordship’s disposal of arguments against ouster clauses in a case involving tribunal decisions is, at one and the same time, a pre-emptive attack on his disposal of arguments against ouster clauses in a case involving more traditional executive decision-makers. 30 ibid. 29 ibid [208]. 28 Privacy International (n 13) [209]. 27 In contradistinction to Lord Wilson’s answer, see Privacy International (n 13) [238]-[253]. F. Signposting In outline, Lord Sumption thought there were two possible arguments available to someone trying to persuade a court to deny legal effect to ouster clauses. The first, the conceptual argument, argued that, by denying effect to ousters, the courts would be, far from violating parliamentary sovereignty, affirming it.28 Second, the radical argument argued that a higher law, to be ‘ascertained and applied by the court[s]’,29 required the courts to outrightly preclude Parliament from ousting judicial review.30 Ultimately, for Lord Sumption, none of these arguments proved successful, as the question of whether Parliament was competent to oust judicial review was, in the final analysis, to be answered in the affirmative. 1. Conceptual Argument/Argument for Sovereignty 1. Conceptual Argument/Argument for Sovereignty In Section II, I argue that Lord Sumption’s conceptual argument — which is really an argument from sovereignty (and which will henceforth bear that name), ‘conceptual’ merely reflecting the conclusion arrived at by Lord Sumption in the Vol. VIII Vol. VIII Sovereign Therefore Limited 438 light of his understanding of the nature and implications of parliamentary sovereignty — is rooted in an incomplete understanding of the notion of parliamentary sovereignty. Therefore, and however unintentionally, Lord Sumption erects, and then attacks, a strawman. In truth, a fully-fledged grasp of the notion of parliamentary sovereignty leads to the conclusion that ouster clauses are logically incompatible with that notion, as parliamentary sovereignty and ouster clauses for illegality are mutually exclusive concepts. Consequently, ouster clauses cannot survive in the statute book so long as parliamentary sovereignty remains the core constitutional principle of the British legal system.31 This conclusion will be arrived at via a close reading, and an intertwined discussion of, Laws LJ’s judgment in Cart32 and Lord Sumption’s judgment in Privacy International.33 Contrary to Lord Sumption’s claim in Privacy International that he was merely reiterating Laws LJ’s argument in Cart,34 it will be shown that Laws LJ’s version of the argument from sovereignty is not only fundamentally different from Lord Sumption’s, but is also one which displays a true grasp of the nature, and implications, of parliamentary sovereignty. The decision to utilise and, in a sense, revitalise Laws LJ’s judgment in Cart has important practical ramifications. With the obfuscatory veil draped by Lord Sumption over Laws LJ’s argument in Cart lifted, the version of the argument from sovereignty advocated for in this article is revealed to have endorsement at the Court of Appeal level, making it a more realistic contender for the Supreme Court’s seal of approval. This is another reason why direct engagement with Lord Sumption’s judgment is valuable. 33 Privacy International (n 13). 32 R (Cart) v Upper Tribunal [2011] QB 120. 31 A constitutional fact which was recently unqualifiedly reaffirmed in R (Miller) v Secretary of State for Exiting the European Union [2017] UKSC 5, [2018] AC 61 [43]. 34 ibid [208] in conjunction with [210]. 33 Privacy International (n 13). 2. Higher law argument In Section III, I argue that the fundamental problem with Lord Sumption’s summary dismissal of the radical or higher law argument is that His Lordship equates the absence of a written constitution with the absence of a higher law patrolling the boundaries of the vast territory afforded to primary LSE LAW REVIEW 2023 439 legislation.35 I will endeavour to show that this is a non sequitur: a higher law anterior to, and immune from, primary legislation will be shown to exist and to require the striking-down of ouster clauses notwithstanding the lack of a constitutional text. A. Introduction As mentioned in sub-section F above, Lord Sumption in his dissenting judgment in Privacy International claimed that he was reiterating Laws LJ’s argument from sovereignty in Cart.36 The initial aim of this section is to rebut that claim by demonstrating that the two arguments are fundamentally different. Indeed, close analysis of the relevant passages in the two judgments will reveal that Lord Sumption was not in fact reiterating Laws LJ’s position, as the conclusions reached by Their Lordships with respect to the possibility of ousting judicial review are actually diametrically opposed. The upshot is that there are two versions of the argument from sovereignty available in the case law for the Supreme Court to choose between. Once this initial task is completed, the section will go on to demonstrate why Laws LJ’s conception of parliamentary sovereignty — and therefore the Lord Justice’s version of the argument from sovereignty — is the right one. In proceeding as such, not only is Lord Sumption’s argument from sovereignty substantively countered, but a better alternative is revealed to have judicial endorsement. 37 ibid [210]. 36 ibid [208]. 35 ibid [209]. B. The Judgments 1. Lord Sumption: Privacy International According to Lord Sumption, refusing to give effect to an ouster clause would, ‘up to a point’, go towards vindicating ‘the sovereignty of Parliament, and not limiting it’.37 This is because it is difficult to imagine Parliament at once intending ‘to create a tribunal with [both] limited jurisdiction Vol. VIII Sovereign Therefore Limited 440 and unlimited power to determine such limit at its own will and pleasure’.38 However, the contradiction in statutory terms produced by an ouster clause, far from being conclusive, was but one factor among others to be considered when interpreting an ouster clause.39 The argument was, in the final analysis, ‘a variant of the claimant’s primary case [i.e., first ground of appeal] about Parliamentary intention’40 and in that sense ‘[a] sufficiently clear and all-embracing ouster clause might demonstrate that Parliament had indeed intended to [abolish the court’s judicial review jurisdiction]’.41 While it would certainly be unorthodox and ‘a strange thing for Parliament to intend’, it was nevertheless ‘conceptually possible’ for Parliament to provide as much.42 47 ibid [38]. 46 ibid [36]. 45 ibid [35]. 44 ibid [34]. 43 Cart (n 32) [33]. 42 ibid. 41 ibid. 40 ibid [210]. 39 Privacy International (n 13) [208]. 38 Lord Sumption in Privacy International (n 13) [208] citing Farwell LJ in R v Shoreditch Assessment Committee, Ex p Morgan [1910] 2 KB 859, 880 (emphasis added). 44 ibid [34] 43 Cart (n 32) [33]. 42 ibid. 41 ibid. 40 ibid [210]. 39 Privacy International (n 13) [208]. 38 Lord Sumption in Privacy International (n 13) [208] citing Farwell LJ in R v Shoreditch Assessment Committee, Ex p Morgan [1910] 2 KB 859, 880 (emphasis added). 39 Privacy International (n 13) [208] 38 Lord Sumption in Privacy International (n 13) [208] citing Farwell LJ in R v Shoreditch Assessment Committee, Ex p Morgan [1910] 2 KB 859, 880 (emphasis added). 2. Lord Justice Laws: R (Cart) v Upper Tribunal Germane to our purposes is the passage between [32] and [39] of the Lord Justice’s judgment. Laws LJ begins with the avowal of a discomfort, as His Lordship is not at ease to circumvent an ouster simply by invoking the weighty presumption, repeatedly established since Anisminic, against an intention of Parliament to oust the review and quash nullities. Instead, Laws LJ felt the need to explain the underlying rationale that gives rise to the presumption, lest it become nothing more than ‘an article of faith’.43 According to Laws LJ, the reason for the presumption is that judicial review is fundamental to the rule of law.44 The requirements of the rule of law are many,45 but the one that is contravened by, and which triggers the presumption against, ouster clauses is that ‘statute law has to be mediated by an authoritative judicial source’46 lest the meaning of the law ‘be degraded to nothing more than a matter of opinion’.47 Thus, ‘the very effectiveness of statute law [i.e., vindication of Parliament’s 47 ibid [38]. 46 ibid [36]. 45 ibid [35]. 44 ibid [34]. 43 Cart (n 32) [33]. 42 ibid. 41 ibid. 40 ibid [210]. 39 Privacy International (n 13) [208]. 38 Lord Sumption in Privacy International (n 13) [208] citing Farwell LJ in R v Shoreditch Assessment Committee, Ex p Morgan [1910] 2 KB 859, 880 (emphasis added). LSE LAW REVIEW 2023 441 legislative sovereignty] … requires’ that an independent and impartial body, namely, a court, be the ultimate interpreter of statutes.48 legislative sovereignty] … requires’ that an independent and impartial body, namely, a court, be the ultimate interpreter of statutes.48 Having laid down the foundations atop which the robust presumption against ouster clauses rests, Laws LJ shifts gears: ‘the need for … an authoritative judicial source cannot be dispensed with by Parliament’.49 These are unequivocal terms from the Lord Justice as they amount to saying that ‘judicial review cannot be abolished by Parliament’. At this point, a potential charge of internal inconsistency must be countered on behalf of Laws LJ to circumvent ambiguity and cherry-picking of his judgment. The charge is as follows: it is difficult to see how Laws LJ can simultaneously speak of a presumption against the ousting of judicial review and of a categorical impossibility for Parliament to dispense with judicial review. 50 E.g., Anisminic (n 2), O’Reilly (n 5). 49 Ibid (emphasis added). 48 ibid. 2. Lord Justice Laws: R (Cart) v Upper Tribunal The nature of the presumption is that it can be displaced and yet we are told Parliament cannot even do so, as the two propositions appear mutually exclusive. I suggest, however, that these propositions can readily be reconciled. For Laws LJ, the presumption operates merely as a first line of defence against the ousting of judicial review; it is perfectly legitimate to presume both (i) that it is the legislature’s intention to conduct its business in a constitutionally sound manner by keeping with the rule of law and (ii) that it is Parliament’s intention for the limits it sets to be respected. In other words, the presumption is the manifestation of (i) a routine application of the principle of legality in conjunction with (ii) the same rule of interpretation as adopted by Lord Sumption. This presumption has clear operational potency as evidenced by Anisminic and its progeny,50 and it is also the least contentious path to the nullification of an ouster clause. But it is not, according to Laws LJ, the ultimate justification for denying legal effect to ouster clauses, as if push came to shove and the presumption is displaced, Parliament would have to be declared outright incompetent to abolish judicial review. In other words, a presumption and a hard-edged denial of validity are not mutually exclusive, but rather two mechanisms of different intensities invoked to reach the same result, namely the nullification of ouster clauses and with it the preservation of judicial review. Whilst in most cases the presumption will be sufficient to neutralise an ouster Vol. VIII 442 Sovereign Therefore Limited clause, in others it will be defeated by unequivocal language — such as an ouster clause modelled on s 2 JRCA 2022 — and recourse to hard-edged limitation of Parliament’s legislative competency will be necessary, on Laws LJ’s account. 55 ibid. 54 Cart (n 32) [38] (emphasis added). 53 Privacy International (n 13) [210]. 52 Cart (n 32) [38] (emphasis added). 51 ibid [208]. C. The Fork in the Road and Challenge Posed Despite Lord Sumption’s claim that in laying down his conceptual argument he was merely reiterating ‘the view suggested by Laws LJ in the Divisional Court in [Cart]’,51 the two judgments plainly arrive at drastically different conclusions. As just seen, for Laws LJ ‘the need for … an authoritative judicial source cannot be dispensed with by Parliament’.52 In stark contrast, for Lord Sumption, Parliament may, in the ultimate analysis, dispense with judicial review for errors of law by enacting a clear enough ‘all-embracing ouster’.53 A choice must therefore be made between Lord Sumption’s and Laws LJ’s version of the argument from sovereignty. To inform that choice, it is necessary to appreciate that the fork in the road between the two views is the result of a fundamental disagreement as to the nature, and therefore the implications of, parliamentary sovereignty. First, for Lord Sumption, ‘parliamentary sovereignty’ is shorthand for ‘what Parliament intends to be law is law’. This is clear from the way His Lordship reduces the conundrum posed by ouster clauses to an interpretative one. I will call this the ‘thin’ version of parliamentary sovereignty. Laws LJ, on the other hand, advances a substantive understanding of the notion of parliamentary sovereignty. This is clearest when two of His Lordship’s claims are considered together, namely that an ‘authoritative judicial source cannot be dispensed with by Parliament’54 and that to limit Parliament’s legislative power is in fact to ‘affirm’ its sovereignty.55 Thus, for Laws LJ, the notion of parliamentary sovereignty cannot be reduced to, and thus (what comes to the same) is at least not fully captured by, the notion of giving effect to Parliament’s expressed intentions without question. I will call this the ‘thick’ version of parliamentary sovereignty. 2023 LSE LAW REVIEW 443 At first glance, Laws LJ’s substantive version of sovereignty seems like judicial supremacism cloaked under the pretence of affirming parliamentary sovereignty. It is true that, without more, His Lordship’s assertions could appear rather arbitrary in light of the Diceyan conception of parliamentary sovereignty which was, once again, recently reaffirmed by the apex court.56 And yet, there is one crucial clue rather casually revealed by Laws LJ which, it is argued, plants the signpost to the fundamental and true rationale underpinning the idea of a substantive conception of parliamentary sovereignty that entails Parliament’s inability to enact ouster clauses. 61 ibid 187. 60 William Wade, ‘The Basis of Legal Sovereignty’ (1955) Cambridge Law Journal 172. 59 ibid [210]. 58 Privacy International (n 13) [208] 57 Cart (n 32) [38]. 56 R (Miller) v SS for Exiting the EU [2017] UKSC 5; [2017] 2 WLR 583 [43]. C. The Fork in the Road and Challenge Posed Critically, His Lordship draws a direct comparison between the ‘old rule that Parliament cannot bind itself’ and his rule that judicial review cannot be ousted by Parliament.57 The implicit proposition that inheres in this direct comparison drawn by Laws LJ is the rule that Parliament cannot bind itself and shares the same rationale as the rule that Parliament cannot dispense with judicial review. Now, the fact that Parliament is disabled from binding its future self is a truism that no one, let alone Lord Sumption, would seek to deny — and indeed His Lordship acknowledges it.58 Thus, the challenge appears straightforward: if the analogy drawn by Laws LJ is revealed to be sound, by parity of reasoning, the proponents of the ‘thin’ version of sovereignty have no choice but to admit the validity of Laws LJ’s ‘thick’ version. They must also concede that however ‘clear and all-embracing’59 the ouster clause, it cannot succeed in ousting judicial review for errors of law. D. Challenge Answered Our inquiry into the rationale behind the rule that Parliament cannot bind itself starts with Sir William Wade’s seminal article, The Basis of Legal Sovereignty.60 The crucial passage for our present purposes is the following, in which Wade quotes Salmond on Jurisprudence:61 But whence comes the rule that Acts of Parliament have the force of law? This is legally ultimate; its source is historical only, not legal … It is the law because it is the Vol. VIII 444 Sovereign Therefore Limited law, and for no other reason that it is possible for the law itself to take notice of. No statute can confer this power upon Parliament, for this would be to assume and act on the very power that is to be conferred.62 This is a rule of logic. Wade, developing on Salmond’s observations, rightly points out that, ‘if no statute can establish the rule that the courts obey Acts of Parliament[, then] no statute can alter or abolish that rule’.63 Therefore, one readily notices that the fundamental reason why Parliament cannot bind its future self is that to do so would undermine parliamentary supremacy, for some later Acts would need to be struck down or ignored if they were incompatible with earlier, ‘enshrined’ ones. A further corollary deducible from Salmond’s rule of logic must be that it cannot be possible for Parliament to divest itself of, transfer, or share its legal sovereignty — this too would be to proceed on the assumption that Parliament’s legal sovereignty is a product of its own will, a proposition which Salmond has just shown is absurd as a matter of logic. Thus, in Wade’s words, the ‘apparent paradox that [those rules are] unalterable by [an all-powerful] Parliament turns out to be a truism’ that logically flows from the ultimately political, and not legal, fact of parliamentary sovereignty.64 The final piece of the puzzle is to demonstrate why it would be an infringement of the same rule of logic — again, that legislative sovereignty is not a product of Parliament’s will — for Parliament to shield decisions afflicted by errors of law from judicial review and therefore prevent them from being nullified, whether by a quashing order or a declaration. Again, if this can somehow be demonstrated, the challenge is answered. It is helpful to picture the following scenario. 65 These are essentially the facts of Anisminic (n 2). 64 ibid 188. 63 Wade (n 60) 187. 62 ibid; Sir John Salmond, Salmond on jurisprudence (10th edn, Sweet & Maxwell 1947) 155 (emphasis added). 65 These are essentially the facts of Anisminic (n 2). 69 ibid 212. 68 ibid. 67 Aileen McHarg, ‘Giving Substance to Sovereignty: Parliamentary Sovereignty and Parliamentary Effectiveness’ in Brice Dickson and Conor McCormick (eds), The Judicial Mind: A Festschrift for Lord Kerr of Tonaghmore (Bloomsbury 2021) 211. 66 Cart (n 32) [37]. D. Challenge Answered An executive body acts outside jurisdiction because it has, for example, considered a matter which it was not entitled to consider on its way to reaching a determination.65 Now, realise that for as long as its decision stands, it has in essence both crafted its own jurisdiction — or, what comes to the same, legislated it — and acted pursuant to said sui generis legislation. In the usual course of things, of course, the public body’s decision would be quashed or declared unlawful ab initio by a court for want of a legal basis. Yet, ex hypothesi, LSE LAW REVIEW 2023 445 this otherwise impeachable act is now rendered unimpeachable by operation of an ouster clause enacted by Parliament which precludes the court from reviewing a flawed decision. What has just occurred? Effectively, Parliament has vested the notional public body with de facto legislative competence; the executive has been allowed to ‘write [its] own laws’.66 And in granting another body legislative competence, Parliament has done the very thing which the rule of logic expounded above has shown it is logically disabled from doing — because, once again, it is not the source of its own legislative supremacy — namely, transferring or sharing its legislative sovereignty with another body. The challenge has therefore been answered. Ouster clauses are plainly incompatible with an orthodox Diceyan conception of parliamentary sovereignty. What is often invoked as the ultimate justification for Parliament’s power to enact ouster clauses if it so wishes, namely, the core constitutional principle of parliamentary sovereignty, has been shown to disable Parliament from doing that very thing. E. Briefly Addressing Criticism Some commentators have expressed scepticism at Laws LJ’s analogy between the old and trite rule that Parliament cannot bind itself, and His Lordship’s strict rule that Parliament cannot oust judicial review. In particular, Aileen McHarg objects that Laws LJ’s analogy is ‘far from compelling’ and a tad deceiving.67 That is so because, in her view, while the former rule is derived from the fact that every iteration that Parliament enjoys ‘unlimited legislative competence’, the latter rule is one which stems from the sacrosanctity of the ‘rule of law’.68 Therefore, to McHarg, while the former rule is a product of the core constitutional notion of parliamentary sovereignty, to suggest as much of the latter rule by drawing an analogy with the former is to ‘turn the doctrine [of parliamentary sovereignty] on its head’.69 Vol. VIII Vol. VIII 446 Sovereign Therefore Limited In this section, I have attempted to show that the analogy is actually sound and that both rules share the same source. In other words, McHarg’s ‘distinction of sources’ argument has been shown to be unfounded. Indeed, both the rule that says Parliament cannot bind its future self and the one that says Parliament cannot dispense with judicial review for errors of law are corollaries of, and thus are derived from, the notion of parliamentary sovereignty. Ouster clauses are not denied effect because ‘they are deemed to be incompatible with the rule of law’.70 They are denied effect because they run counter to parliamentary sovereignty when that notion is properly understood. As shown, once it is realised that the rule that courts continuously obey Acts of Parliament (i.e., parliamentary sovereignty) is of extra-legal nature (i.e., that it is not a rule that emanates from Parliament itself), it becomes clear that Parliament cannot meddle with that rule, which it would be doing if it purported to alter its own legislative competence (which gives us the rule against entrenchment of legislation), or to transfer or share its legislative authority (which gives us the rule against ouster clauses for errors of law). In other words, Parliament’s legislative authority must remain both limitless and exclusive. And since an ouster clause for errors of law is tantamount to Parliament transferring its legislative authority to another body, denying it legal effect is not to violate but to preserve Parliament’s exclusive legislative authority. 70 McHarg (n 67) 211. 74 Allan (n 6) 219 (emphasis added). 73 R (Daly) v Secretary of State for the Home Department [2001] UKHL 26; [2001] 2 AC 532, 548. 72 ibid [209] (emphasis added). 71 Privacy International (n 13) [208]. F. Conclusion There are two key takeaways from this section. Firstly, the veil cast over Laws LJ’s argument by Lord Sumption has been lifted. It is simply not the case that for Laws LJ too the dilemma is merely ‘conceptual’ and interpretative. There are two starkly contrasting versions of the argument from sovereignty to be found in the case law. This is important, as the Supreme Court has a choice between two alternatives that have curial support. Secondly, this article has shown that the correct choice is Laws LJ’s strict version of the argument since it flows from a ‘thick’, and true, conception of sovereignty. Indeed, a careful examination of the notion of parliamentary sovereignty reveals inbuilt disabilities derived from the fact that Parliament is not the source of its own legislative supremacy. One of the said disabilities is that Parliament cannot bind its future self. Another is that it cannot transfer, or share, its legislative competence. And since ouster clauses for errors of law result in Parliament LSE LAW REVIEW 2023 447 effectively granting executive legislative competence, such ouster clauses must be denied legal effect so long as parliamentary sovereignty continues to reign supreme. In the name of parliamentary sovereignty, ouster clauses must not be given legal effect in this legal system. 74 Allan (n 6) 219 (emphasis added). A. Introduction According to Lord Sumption, the radical argument contends that courts may, in exceptional circumstances, strike down statute in the name of a ‘higher law’.71 His Lordship rejects that argument on the basis that: ‘in the absence of a written constitution capable of serving as a higher source of law’, primary legislation is supreme.72 The purpose of this section is to show that this is a non sequitur: the lack of a textual constitution does not entail a completely limitless legislative remit for Parliament. Written constitutions recognise, rather than create, principles and rights fundamental to, and ever-present in, democratic societies.73 Accordingly, these principles and fundamental rights are also present in democratic societies with unwritten constitutions, such as the Constitution of the United Kingdom. The challenge this section sets out to meet can be formulated as follows. If it can be shown, as it will be, that in attempting to preclude the quashing of executive decisions or acts that are afflicted by an error of law, Parliament is seeking to use its legislative powers in a way that runs counter to the justification for its very own legislative empowerment, then it must follow that it is disabled from doing so. Importantly, the contention here is not merely that ‘we should not attribute to Parliament intentions that flout the very conditions on which the legitimacy of its enactments depends’.74 Rather, it is that notwithstanding such intentions, eminently clear as they may be, they cannot prevail. Vol. VIII 448 Sovereign Therefore Limited The argument made in this section buttresses the argument from sovereignty made in the previous section. It serves to show that the notion of parliamentary sovereignty and its logical ramifications — one of which being that Parliament may not oust the judicial review jurisdiction of the courts to quash decisions afflicted by errors of law — far from being products of sheer happenstance, are manifestations of the necessary limitations on legislative power that operate in democratic political systems. 78 Ritchie Robertson, The Enlightenment (Penguin 2020), 833. 77 Laws (n 75) 31: ‘Without democracy the government is by definition autocratic’. 76 Allan (n 6) 218. 75 John Laws, The Constitutional Balance (Bloomsbury 2015) 31. B. Democracy and its Underpinning Ideals The starting point, and one fundamental assumption for the purpose of this section, is that the United Kingdom is a democracy. This is not a contentious assumption. It is supported by the observable fact that the lawmaker is Parliament, an entity constituted of representatives elected from time to time via universal suffrage. In Britain, therefore, the wielding of political power is contingent on the continuing assent of the people, as ‘[t]he electorate can throw out the government’.75 That is the cornerstone feature of the democratic form of government. It follows that it is the ideals that underpin democracy which need unearthing to assess whether, in light of ‘the constraints inherent in the best conception of democracy’,76 ouster clauses are admissible under the British Constitution. It is helpful, in seeking to identify democracy’s underpinning ideals, to take as a starting point its antithesis, autocracy.77 To understand the motivation behind the shift from autocracy to democracy — and therefore to grasp the ideals that underpin, and that are sought to be given effect to by, democratic forms of government — we must understand, at least in outline, the lessons often attributed to the Age of Enlightenment. Distilled to its essence, one of the main takeaways from the philosophical revolution of the 17th and 18th centuries was that no person is invested with inherent moral supremacy and that any political regime that is premised on such a notion is absurd and, ultimately, wicked. Diderot, for example, made the point trenchantly when he proclaimed that ‘the arbitrary government of a just and enlightened prince is always bad’.78 To be sure, ‘arbitrary government’ is here used to mean the wielding of political LSE LAW REVIEW 2023 449 authority justified purely on the footing of a title derived from lineage or any other factual circumstances. The vice in all such claims to authority was that normative statements (‘you ought to do this’) could not, without being absurd, rest on mere statements of fact (‘because I am X, Y, Z.’). 83 Hugo Grotius, The Rights of War and Peace (Liberty Fund, 2005), 79. 82 ibid 7. 81 ibid. 80 Laws (n 75) 31. 79 David Hume, Treatise of Human Nature (Clarendon Press, 1896), 469: ‘You cannot derive an ought from an is’. 80 79 David Hume, Treatise of Human Nature (Clarendon Press, 1896), 469: ‘You cannot derive an ought from an is’. 82 ibid 7. B. Democracy and its Underpinning Ideals Indeed, as Hume famously remarked, it was ‘altogether inconceivable’ (and here ‘inconceivable’ should be understood in the sense of ‘impossible’) that normative statements could be deduced from factual premises, and this was precisely the rule of logic that the autocratic form of government so blatantly flouted.79 Instead, a normative statement may only validly be rooted in an appeal to reasons (i.e., by an appeal to the goodness of the consequences which it is thought will be produced by the act). But as soon as one person claims that she has good reasons to demand that others act as she wills, the natural response, from those who disagree, is to argue that the expected consequences of the act are not in fact beneficial; or that they are but that the act suggested is not the proper way to achieve them; or that those actions should not take priority over others, etc. The ability to reason is universal and ‘difference and disputation’ about what ought to be done is inevitable.80 Indeed, ‘it is an inherent feature of unruly humankind that what benefits the people will always be contentious’.81 Once this is grasped, the path to democracy starts to take shape. There is, however, one last condition of the human kind (the universality of reason and the plurality of normative outlooks being the two just identified) that remains to be acknowledged. Man is a gregarious species. No doubt there are solitary individuals, ‘but society … is the paradigm’.82 Indeed, ‘amongst the Things peculiar to Man is his Desire of Society’.83 This is an observable fact; it is hardwired in our condition as much as the ability to reason is. The upshot is that a structure to regulate collective life needs to be erected lest cacophony ensues because, as we have seen above, individuals will inevitably advance conflicting normative propositions — we need a structure to solve our unsocial sociability without falling foul of the autocratic fallacy. In other words, we need a mechanism to Vol. VIII Sovereign Therefore Limited 450 invest political power in a ruler (whose political decisions for the community are authoritative and ultimate) which does not make a mockery of the realisations made above, namely (i) that only reasons can validly ground normative propositions and (ii) that the faculty of reason is a universal feature of the human condition. The answer, of course, is the democratic machinery. 85 ibid 79. 84 John Laws, ‘Law and Democracy’ [1995] Public Law 72, 85. B. Democracy and its Underpinning Ideals Democracy provides a political battleground, the outcome of the tussle occurring in which is dictated by a candidate, and her normative outlook having garnered the greatest share of support from reasoning-capable human peers. That way, and for the time being, the exercise of the ruler’s political authority is cured of arbitrariness in the sense that the exercise of her political authority, which manifests itself through the enactment of laws, is taken to have the tacit assent of the polity. It is only through the polling booth that ‘the measures passed by government’ may acquire the ‘crucial moral authority’ which is so blatantly lacking in autocracies.84 2. Classic ultra vires or illegality The necessity of the ultra vires or illegality ground of review, and thus the inadmissibility of ouster clauses that prevent the quashing of executive decisions afflicted by the vice of illegality, is very straightforwardly distilled from the foregoing discussion. As this article has demonstrated earlier, a ‘decision … that flouts the limits of [the] empowering legislation’ is tantamount to the executive crafting its own jurisdiction or, what comes to the same, legislating.90 But this is pure arbitrariness: the executive does not benefit from the same democratic cure as the legislature; it cannot craft sui generis purposes but must give effect to Parliament’s own. If Parliament were allowed to preclude the quashing of decisions plagued by errors of law, it would be actively sabotaging the democratic rationale upon which its own legislative power rests, something which, as we have seen, the ground rule necessarily prohibits. 1. Generally 1. Generally Having established in outline the ideals that motivate the establishment of, and therefore underpin, the democratic form of government, we are now equipped to ascertain the ground rule in accordance with which the exercise of democratically acquired political power must be conducted. That ground rule, in turn, shapes to the most minute detail the ‘conditions under which [those who wield political power] are permitted to do so’.85 The foregoing entails the following. Since it is only because it accedes to political power via a democratic procedure that the lawmaker’s (Parliament’s) promulgations are cured from the scourge of arbitrariness — such cure being, again, the chief aim of the democratic machinery — it must follow that the lawmaker is disabled from empowering those taking decisions pursuant to, and acting in accordance with, its promulgations to act in an arbitrary fashion. That is the ground rule. Had it been otherwise, the legislature would be allowing an exercise of public powers which does not advance the purpose (which is what ‘to act arbitrarily’ LSE LAW REVIEW 2023 451 means in this context) of the only legitimate source of public or executive powers in a democracy, namely the legislature’s own promulgations. In other words, the ruler’s law would ‘stifle its own justification’.86 Thus, ‘any delegation of power to a public authority must be applied to the furtherance of legitimate public purposes’ which Parliament has authored.87 It follows that Parliament does not draft its laws on a blank canvas. Instead, indelible terms and conditions invariably populate every single Act of Parliament. These provide that Parliament cannot violate the democratic justification that lends its words the power of law, which it would be doing if it permitted arbitrary acts under the law it promulgates to withstand judicial scrutiny. Law in a democracy, therefore, has a baseline substance that is ‘impervious to any purported legislative abrogation’;88 law is the sovereign.89 90 Allan (n 6) 220. 89 As the title of Allan’s latest book, The Sovereignty of Law (n 6), rightly suggests. 88 ibid 217. 87 Allan (n 6) 224. 86 Laws (n 75) 32. 90 Allan (n 6) 220. 89 As the title of Allan’s latest book, The Sovereignty of Law (n 6), rightly suggests. 92 Privacy International (n 13) [144]. 91 Allan (n 6) 217. 91 Allan (n 6) 217. IV. CONCLUSION The chief aim of this article was to mount a frontal attack on ouster clauses. Particularly, this article has focused on ouster clauses designed to prevent the review and quashing of executive decisions purportedly taken Vol. VIII Sovereign Therefore Limited 452 pursuant to an empowering statute, but ultimately afflicted by one or more errors of law. With the argument from sovereignty, this article has shown that a fully-fledged understanding of the notion of parliamentary sovereignty, the core constitutional principle of the United Kingdom’s legal system, leads to the rejection of ouster clauses. One cannot at once espouse the principle of parliamentary sovereignty and accept the validity of ouster clauses. With the higher law argument, I have sought to show that the foundations upon which the exercise of political power rests in a democracy entail Parliament’s inability to allow decision-makers acting pursuant to its laws to act arbitrarily. In conclusion, if this analysis has found favour with the reader, they are invited to ponder whether the above arguments can in some way be extended to capture all the grounds of judicial review currently recognised by the courts. In other words, can an argument be made, using what has been said above and especially in Section III, to say that Parliament should not be able to oust judicial review for grounds other than illegality? There is scholarship that argues for the fundamentality of all grounds of judicial review — and ‘fundamentality’ here should be understood to mean imperviousness to unilateral riddance by Parliament of these grounds of review.91 There is also recent high-level obiter dicta that points this way.92 No particular claims to that effect are made here, but there is no reason why we should not now direct our efforts to the inquiry of whether such a claim can be made in light of the constitutional arrangement of the United Kingdom where Parliament may make the law it wishes, so long of course as it does not purport to give the executive legislative competence.
https://openalex.org/W1984907440	https://orbi.uliege.be/bitstream/2268/175154/1/Bouche%cc%81_et_al_2015.pdf	English	null	Heat can erase epigenetic marks of vernalization in<i>Arabidopsis</i>	Plant signaling & behavior/Plant signalling & behavior	2,015	cc-by	3,740	Plant Signaling & Behavior Plant Signaling & Behavior To cite this article: Frédéric Bouché, Nathalie Detry & Claire Périlleux (2015) Heat can erase epigenetic marks of vernalization in Arabidopsis, Plant Signaling & Behavior, 10:3, e990799, DOI: 10.4161/15592324.2014.990799 To cite this article: Frédéric Bouché, Nathalie Detry & Claire Périlleux (2015) Heat can erase epigenetic marks of vernalization in Arabidopsis, Plant Signaling & Behavior, 10:3, e990799, DOI: 10.4161/15592324.2014.990799 To link to this article: http://dx.doi.org/10.4161/15592324.2014.990799 To cite this article: Frédéric Bouché, Nathalie Detry & Claire Périlleux (2015) Heat can erase epigenetic marks of vernalization in Arabidopsis, Plant Signaling & Behavior, 10:3, e990799, DOI: 10.4161/15592324.2014.990799 li k hi i l h //d d i /10 4161/15592324 2014 990799 y y g On: 23 April 2015, At: 01:28 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK y y g On: 23 April 2015, At: 01:28 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Click for updates Plant Signaling & Behavior Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kpsb20 Heat can erase epigenetic marks of vernalization in Arabidopsis Frédéric Bouchéa, Nathalie Detrya & Claire Périlleuxa a University of Liège; Laboratory of Plant Physiology; PhytoSYSTEMS; Liège, Belgium Accepted author version posted online: 03 Feb 2015. To cite this article: Frédéric Bouché, Nathalie Detry & Claire Périlleux (2015) Heat can erase epigenetic marks of vernalization in Arabidopsis, Plant Signaling & Behavior, 10:3, e990799, DOI: 10.4161/15592324.2014.990799 To link to this article: http://dx.doi.org/10.4161/15592324.2014.990799 Keywords: Arabidopsis, devernalization, epigenetic modiﬁcations, FLOWERING LOCUS C, Flowering, H3K27me3, H3K4me3, vernalization Keywords: Arabidopsis, devernalization, epigenetic modiﬁcations, FLOWERING LOCUS C, Flowering, H3K27me3, H3K4me3, vernalization © Frederic Bouche, Nathalie Detry, and Claire Perilleux Correspondence to: Claire Perilleux; Email: cperilleux@ulg.ac.be Correspondence to: Claire Perilleux; Email: cperilleux@ulg.ac.be Submitted: 08/10/2014 Revised: 06/11/2014 Vernalization-requiring plants are unable to ﬂower or ﬂower very late unless they are exposed to prolonged cold tem- peratures. The ecological signiﬁcance of this trait is to block ﬂowering prior to winter and permit the ﬂoral transition under the favorable conditions of follow- ing spring, when ambient temperature and daylength increase.1 In Arabidopsis thaliana, vernalization requirement is largely due to the high expression level of the FLOWERING LOCUS C (FLC) gene encoding a MADS-domain protein.2,3 FLC acts as a transcriptional inhibitor of the ﬂowering integrator genes FLOWER- ING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1).4-7 Vernalization leads to stable p In Arabidopsis as well as some other species such as Henbane, vernalization is both quantitative – short cold spells have no effect on ﬂowering – and stable, since cold exposure and the subsequent ﬂoral transition may be separated by several months.8 There are, however, species in which vernalization is not stable.9,28 In species with a stable vernalized state, the memory of cold is only maintained after long cold exposure, and this behavior reﬂects the quantitative nature of the response. At the molecular level, it has been shown that the accumulation of H3K27me3 during vernalization reﬂects the length of the cold exposure and cell- autonomously regulates the transition Accepted: 06/11/2014 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Versions of published Taylor & Francis and Routledge Open articles and Taylor & Francis and Routledge Open Select articles posted to institutional or subject repositories or any other third-party website are without warranty from Taylor & Francis of any kind, either expressed or implied, including, but not limited to, warranties of merchantability, fitness for a particular purpose, or non-infringement. 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Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions It is essential that you check the license status of any given Open and Open Select article to confirm conditions of access and use. ADDENDUM Plant Signaling & Behavior 10:3, e990799; March 2015; Published with license by Taylor & Francis Heat can erase epigenetic marks of vernalization in Arabidopsis Frederic Bouche, Nathalie Detry, and Claire Perilleux* University of Liege; Laboratory of Plant Physiology; PhytoSYSTEMS; Liege, Belgium Frederic Bouche, Nathalie Detry, and Claire Perilleux* University of Liege; Laboratory of Plant Physiology; PhytoSYSTEMS; Liege, Belgium silencing of FLC and so relieves FT and SOC1, which can then be activated by a range of environmental or endogenous signals stimulating ﬂowering (reviewed in refs. 8–12). V ernalization establishes a memory of winter that must be maintained for weeks or months in order to promote ﬂowering the following spring. The sta- bility of the vernalized state varies among plant species and depends on the dura- tion of cold exposure. In Arabidopsis thaliana, winter leads to epigenetic silencing of the ﬂoral repressor gene FLOWERING LOCUS C (FLC) and the duration of cold is measured through the dynamics of chromatin modiﬁcations during and after cold. The growing con- ditions encountered post-vernalization are thus critical for the maintenance of the vernalized state. We reported that high temperature leads to devernalization and, consistently, to FLC reactivation in Arabidopsis seedlings. Here we show that the repressive epigenetic mark H3K27me3 decreases at the FLC locus when vernalized seedlings are grown at 30C, unless they were ﬁrst exposed to a stabilizing period at 20C. Ambient tem- perature thus controls the epigenetic memory of winter. V The initial level of FLC expression depends on the balance between activation and repression activities of FRIGIDA (FRI) and the autonomous ﬂowering pathway, respectively, which regulate antisense FLC transcripts (called COOLAIR) and histone modiﬁcations (reviewed in ref. 13). Allelic variation at FRI and FLC loci largely explain the differences in vernalization requirement between winter and summer accessions of Arabidopsis.14-18 Prior to ver- nalization, histone modiﬁcations associated with actively transcribed genes, such as H3K4me3 and H3K36me2/me3, are enriched at FLC whereas repressive marks such as H3K27me3 are present but low abundant.19-24 During vernalization, grad- ual accumulation of the repressive mark H3K27me3 occurs at the nucleation region, just downstream of the transcrip- tion initiation site, and thereafter spreads over the whole region of FLC upon return to warm temperature.19,25-27 Downloaded by [University of Liege] at 01:28 23 April 2015 Downloaded by [University of Liege] at 01:2 http://dx.doi.org/10.4161/15592324.2014.990799 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. Addendum to: Perilleux C, Pieltain A, Jacquemin G, Bouche F, Detry N, D’Aloia M, Thiry L, Aljochim P, Delansnay M, Mathieu AS, Lutts S, Tocquin P. A root chicory MADS-box sequence and the Ara- bidopsis ﬂowering repressor FLC share common features that suggest conserved function in ver- nalization and de-vernalization responses. Plant Journal 2013; 75:390–402. Plant Signaling & Behavior e990799-1 www.tandfonline.com Figure 1. Experimental set-up. Col-FRI seeds were either stratiﬁed for 3 days at 4C or vernalized for 6 weeks at 2–4C. After the cold period, seedlings were transferred to 20C (10:14 h light:dark cycles, 100 mE.m¡2.s¡1) or were ﬁrst exposed to a devernalization treatment of 1 week at 30C before transfer to 20C conditions. The effects of these different post-vernalization conditions on FLC transcript level and ﬂowering are summarized on the right according to Perilleux et al.28 return to warm temperature, fast deposi- tion of repressive histone marks across the whole gene allows and is required for maintenance of the silenced state,30 which is limited to mitotically active tissues.31 At the physiological level though, the stability of vernalization does not only depend on its duration, but also on the growing conditions prevailing post-vernal- ization.32 ‘Warm temperatures’ - as used in experimental work on Arabidopsis - are around 20C and stabilize the vernalized state. By contrast, ‘hot temperatures’ can devernalize the plants, as reported in early work on Arabidopsis.33,34 We showed pre- viously that one week at 30C given directly after a saturating vernalization period completely suppressed the Figure 1. Experimental set-up. Col-FRI seeds were either stratiﬁed for 3 days at 4C or vernalized for 6 weeks at 2–4C. After the cold period, seedlings were transferred to 20C (10:14 h light:dark cycles, 100 mE.m¡2.s¡1) or were ﬁrst exposed to a devernalization treatment of 1 week at 30C before transfer to 20C conditions. http://dx.doi.org/10.4161/15592324.2014.990799 H3K4me3 proﬁle across FLC, for non vernalized seedlings (NV) and after 6 weeks of vernalization (V) followed by 3 weeks of warm (20C), or 1 week of heat (30C) given before (30–20C) or after (20–30C) 2 weeks of warm. (A) H3K4me3 proﬁle expressed as % of chro- matin input retrieved for the NV/20C sample. (B) H3K4me3 quantiﬁcations (§ SE of 3 technical replicates) relative to constitutive marks at ACTIN7 and UBIQUITIN10 genes. Two independent biological replicates are shown. Primers used are listed in Table S2. Downloaded by [University of Liege] at 0 vernalized state and reactivated FLC in Arabidopsis.28 By contrast, the same treat- ment given after 2 ‘stabilizing’ weeks of 20C had no effect. As a complementary experiment, we analyzed the effect of one week at 30C on the chromatin mark pro- ﬁles at the FLC locus. vernalization when it occurs straight after cold. By contrast, when the heat treatment was given after 2 weeks at 20C, the repres- sive H3K27me3 marks of vernalization remained on the chromatin and the vernal- ized state was maintained (Fig. 2). Angel et al.25 evaluated to 3 days the time taken after cold to achieve maximum H3K27me3 deposition at FLC locus, and hence the growing conditions occurring then are critically important for mainte- nance of the vernalized state. The erasing effect of heat (Fig. 4) highlights a risk for winter plants if we think of the extreme and erratic weather events that are becom- ing more common as the climate changes. abundant in vernalized than in non-vernal- ized seedlings (Fig. 2), and distributed across the FLC gene as previously reported.25,26 By contrast, vernalization had a very weak effect on the abundance of the H3K4me3 activation marks (Fig. 3). Most interestingly, the 30C treatment given straight after vernalization completely prevented the accumulation of H3K27me3 marks at all the tested regions of the FLC gene, including the nucleation site (regions I to III in Fig. 2). A slight increase in H3K4me3 activation marks was also detected (Fig. 3). These results are fully consistent with the fact that this 30C treatment reactivates FLC and inhibits ﬂowering28 (Fig. 1). We can thus conclude that heat removes the epigenetic marks of Downloaded by [University of L ChIP analyses were performed on high-expressing FLC seedlings (Col- FRI)35 harvested 3 weeks after the end of a 6-week vernalization period or a 3-day stratiﬁcation as a control (Fig. 1). http://dx.doi.org/10.4161/15592324.2014.990799 The effects of these different post-vernalization conditions on FLC transcript level and ﬂowering are summarized on the right according to Perilleux et al.28 and thus the overall FLC level is gradually turned down29 and reﬂects the average state of a cell population.25 Then, upon from an ‘active’ to a ‘silenced’ state of FLC.25 After longer cold treatment, an increasing number of cells are switched off gure 2. H3K27me3 ChIP experiments. H3K27me3 proﬁle across FLC, for non vernalized seedlings (NV) and after 6 weeks of vernalization (V) followed by weeks of warm (20C), or 1 week of heat (30C) given before (30–20C) or after (20–30C) 2 weeks of warm. (A) H3K27me3 proﬁle expressed as % of romatin input retrieved for the V/20C sample. (B) H3K27me3 quantiﬁcations (§ SE of 3 technical replicates) relative to constitutive marks at AGAMOUS d SHOOT MERISTEMLESS genes. Two independent biological replicates are shown; similar results were obtained in a third experiment (Supplemental g. S1). Primers used are listed in Table S1. Figure 2. H3K27me3 ChIP experiments. H3K27me3 proﬁle across FLC, for non vernalized seedlings (NV) and after 6 weeks of vernalization (V) followed by 3 weeks of warm (20C), or 1 week of heat (30C) given before (30–20C) or after (20–30C) 2 weeks of warm. (A) H3K27me3 proﬁle expressed as % of chromatin input retrieved for the V/20C sample. (B) H3K27me3 quantiﬁcations (§ SE of 3 technical replicates) relative to constitutive marks at AGAMOUS and SHOOT MERISTEMLESS genes. Two independent biological replicates are shown; similar results were obtained in a third experiment (Supplemental Fig. S1). Primers used are listed in Table S1. Plant Signaling & Behavior Volume 10 Issue 3 e990799-2 Figure 3. H3K4me3 ChIP experiments. H3K4me3 proﬁle across FLC, for non vernalized seedlings (NV) and after 6 weeks of vernalization (V) followed by 3 weeks of warm (20C), or 1 week of heat (30C) given before (30–20C) or after (20–30C) 2 weeks of warm. (A) H3K4me3 proﬁle expressed as % of chro- matin input retrieved for the NV/20C sample. (B) H3K4me3 quantiﬁcations (§ SE of 3 technical replicates) relative to constitutive marks at ACTIN7 and UBIQUITIN10 genes. Two independent biological replicates are shown. Primers used are listed in Table S2. Downloaded by [University of Liege] at 01:28 23 April 2015 Figure 3. H3K4me3 ChIP experiments. Disclosure of Potential Conﬂicts of Interest Disclosure of Potential Conﬂicts of Interest No potential conﬂicts of interest were disclosed. No potential conﬂicts of interest were disclosed. Figure 4. Model of the devernalization process. Vernalization represses the expression of FLC through chromatin modiﬁcations, mainly an increase in H3K27me3. Heat occurring just after vernal- ization restores FLC activity and this is at least partly due to the removal of H3K27me3 repressive marks. http://dx.doi.org/10.4161/15592324.2014.990799 Proto- cols for chromatin extraction and use of anti-H3K27me3 and -H3K4me3 anti- bodies followed manufacturer’s instruc- tions (www.diagenode.com). We observed that the repressive H3K27me3 marks were much more References Downloaded by [University of Liege] at 01:28 23 April 2015 1. Chouard P. Vernalization and its relations to dormancy. Annu Rev Plant Physiol 1960; 11:191–238; http://dx.doi. org/10.1146/annurev.pp.11.060160.001203 27. Sung S, Amasino RM. Vernalization in Arabidopsis thaliana is mediated by the PHD ﬁnger protein VIN3. Nature 2004; 427:159-64; PMID:14712276; http:// dx.doi.org/10.1038/nature02195 15. Johanson U, West J, Lister C, Michaels S, Amasino R, Dean C. 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Vernalization-induced trime- thylation of histone H3 lysine 27 at FLC is not main- tained in mitotically quiescent cells. Curr Biol 2007; 17:1978-83; PMID:17980595; http://dx.doi.org/ 10.1016/j.cub.2007.10.026 19. Bastow R, Mylne JS, Lister C, Lippman Z, Martienssen RA, Dean C. Vernalization requires epigenetic silencing of FLC by histone methylation. Nature 2004; 427:164- 7; PMID:14712277; http://dx.doi.org/10.1038/ nature02269 6. Michaels SD, Himelblau E, Kim SY, Schomburg FM, Amasino RM. Integration of ﬂowering signals in win- ter-annual Arabidopsis. Plant Physiol 2005; 137:149- 56; PMID:15618421; http://dx.doi.org/10.1104/ pp.104.052811 j 32. Bernier G, Kinet JM, Sachs RM. The Physiology of Flowering Vol. I : Initiation of Flowers. CRC Press, 1981. 20. Cao Y, Dai Y, Cui S, Ma L. Histone H2B monoubiqui- tination in the chromatin of FLOWERING LOCUS C regulates ﬂowering time in Arabidopsis. Plant Cell 2008; 20:2586-602; PMID:18849490; http://dx.doi. org/10.1105/tpc.108.062760 33. Chintraruck B, Ketellapper HJ. Interaction of vernaliza- tion, photoperiod and high temperature in ﬂowering of Arabidopsis thaliana (L.) HEYNH. Plant Cell Physiol 1969; 10:271-6. 7. Searle I, He Y, Turck F, Vincent C, Fornara F, Krober S, Amasino RA, Coupland G. The transcription factor FLC confers a ﬂowering response to vernalization by repressing meristem competence and systemic signaling in Arabidopsis. Genes Dev 2006; 20:898-912; PMID:16600915; http://dx.doi.org/10.1101/gad. 373506 34. Napp-Zinn K. Untersuchungen €uber das Vernalisa- tionsverhalten einer winterannuellen Rasse von Arabi- dopsis thaliana. Planta 1957; 50:177-210; http://dx.doi. org/10.1007/BF01930342 21. He Y, Doyle MR, Amasino RM. PAF1-complex-medi- ated histone methylation of FLOWERING LOCUS C chromatin is required for the vernalization-responsive, winter-annual habit in Arabidopsis. Genes Dev 2004; 18:2774-84; PMID:15520273; http://dx.doi.org/ 10.1101/gad.1244504 8. Amasino R. Seasonal and developmental timing of ﬂowering. Plant J 2010; 61:1001-13; PMID:20409274; http://dx.doi.org/10.1111/j.1365- 313X.2010.04148.x 35. Lee I, Amasino RM. Effect of vernalization, photope- riod, and light quality on the ﬂowering phenotype of Arabidopsis plants containing the FRIGIDA gene. Plant Physiol 1995; 108:157-62; PMID:12228459 22. Oh S, Park S, van Nocker S. Genic and global functions for Paf1C in chromatin modiﬁcation and gene e990799-4 Plant Signaling & Behavior Volume 10 Issue 3
W4292630697.txt	https://zenodo.org/records/5884471/files/Seiten%20aus%20SZH_10_2015.pdf	de		Holzweg oder Königsroute? Ein Blick auf die Umsetzung der Behindertenrechtskonvention, ein Jahr nach ihrer Ratifizierung	Zenodo (CERN European Organization for Nuclear Research)	2,015	cc-by	2,504	BEHINDERTENRECHTSKONVENTION – TEILHABE UND BETEILIGUNG 7 Corinne Wohlgensinger Holzweg oder Königsroute? Ein Blick auf die Umsetzung der Behindertenrechtskonvention, ein Jahr nach ihrer Ratiﬁzierung Zusammenfassung Die Schweiz hat die Behindertenrechtskonvention (BRK) ratiﬁziert und damit Ja gesagt zu einer Gewährleistung von gleichen Rechten, auch für Menschen mit einer Behinderung. Nun gilt es für die Heilpädagogik, der Richtung, welche die BRK vorgibt, zu folgen, dabei aber die Augen vor den eigenen Schwächen und jenen der Konvention nicht zu verschliessen. Résumé La Convention relative aux droits des personnes handicapées (CDPH) a été ratiﬁée par la Suisse, qui a ainsi manifesté son attachement à la notion d’égalité des droits, pour les personnes en situation de handicap également. Il s’agit à présent pour la pédagogie spécialisée de suivre la direction déﬁnie par la CDPH, sans pour autant fermer les yeux sur ses propres points faibles et ceux de la Convention. Die Behindertenrechtskonvention hat einiges zu bieten: Sie reisst Barrieren nieder, befreit aus Abhängigkeiten, schafft Wahlmöglichkeiten und öffnet damit Türen zu einem Leben, wie es andere auch führen. Sie macht Menschen mit Behinderungen und ihre Bedürfnisse sichtbar. Zudem ist sie deutlicher Ausdruck dafür, dass die Betroffenen nicht mehr als Objekte der Fürsorge, sondern als Subjekte mit Rechten wahrgenommen und verstanden werden, oder vielmehr: verstanden werden sollen. Mit der Ratiﬁzierung der BRK im April vergangenen Jahres erklärte die Schweiz den Beitritt zu diesem völkerrechtlichen Vertrag als Ergänzung und Konkretisierung des bestehenden schweizerischen Behindertengleichstellungsrechts. Die damit verfolgten Ziele waren nichts weniger, als ein Bewusstsein für das Thema «gleiche Rechte für Menschen mit Behinderungen» zu schaffen und das bestehende Diskriminierungsverbot und die rechtliche Gleichstellung zu stärken. Im Zentrum der Diskussion Schweizerische Zeitschrift für Heilpädagogik, Jg. 21, 10 / 2015 stand die Stärkung des selbstbestimmten Lebens und der gesellschaftlichen Teilhabe von Menschen mit einer Behinderung. Die BRK schien dafür das Mittel der Wahl zu sein (vgl. Wohlgensinger, 2014, S. 58ff.). Bloss ein modisches Accessoire? Die BRK wurde im deutschsprachigen Raum als Meilenstein der Behindertengleichstellungspolitik gefeiert. Die hohen Erwartungen schlugen sich in diversen Begriffs-Superlativen nieder. Von einem «epochalen Dokument monumentaler Grösse» war die Rede (Wocken, 2010, S. 130), vom «Klangzauber», der die Richtung weisen soll (Wacker, 2011, S. 239) oder «Katalysator», welcher es ermöglicht, stärker auf individuelle Problemlagen zu reagieren (Kahle, 2012, S. 149). Von Seiten der Heilpädagogik bestand die Hoffnung, die BRK möge ihre Wirkung entfalten und Chancen eröffnen, Diskussionen beﬂügeln und Druck ausüben, um nicht nur das veränderte Verständnis von Behinderung durchzusetzen, sondern auch Forderungen 8 BEHINDERTENRECHTSKONVENTION – TEILHABE UND BETEILIGUNG nach Unterstützung zu bestärken (vgl. Wohlgensinger, 2014, S. 83). Tatsächlich ist es aufgrund der thematischen Breite der Konvention einfach, sich als heilpädagogische Fachperson mit seinen Anliegen wiederzuerkennen: Sei es, wenn es ganz allgemein um das Verständnis von Behinderung geht, um Forderungen wie jene nach schulischer Inklusion oder selbstbestimmtem Leben oder auch die Legitimation von Forschungsvorhaben, deren Inhalte in den einzelnen Artikeln der Konvention verortet werden können. Gerade jene Artikel, welche schulische Inklusion oder selbstbestimmte Lebensführung fordern, sind nicht frei von Widersprüchen. Die blosse und manchmal auch voreilige Verknüpfung des eigenen Anliegens mit dem Konventionstext bringt allerdings die Gefahr mit sich, dass der Anschein entsteht, man wolle einem Trend nacheifern, sich die BRK als modisches Accessoire umhängen und dabei den eigentlichen Inhalt beim Alten lassen. Dies, ohne weiter der Frage nachzugehen, was denn eine menschenrechtliche Orientierung für das eigene Thema bedeuten könnte. Eine ähnliche Gefahr für die Umsetzung der Konvention droht auch von staatlicher Seite: Die Feststellung, dass die bestehenden Ziele der Behindertenpolitik und die gesetzlichen Regelungen der BRK entsprechen, sagt nichts darüber aus, ob die getroffenen Massnahmen und Leistungen auch genügen. Dies merkt Nicklas-Faust (2011) in ihrem Kommentar zum ersten Staatenbericht Deutschlands an. Zudem geht es oft nicht nur um die rechtliche Passung, sondern darüber hinaus um die konkrete Lebenssituation im Licht der BRK: «Vor allem jedoch fehlt es an der Darstellung, inwiefern die gesellschaftlichen Teilhabechancen und Lebenslagen von Menschen mit Behinderungen in den verschiedenen Lebensbereichen im Licht der BRK zu bewerten sind und verbesserungswürdig erscheinen» (Nicklas-Faust, 2011, S. 103). Es gilt also, die Konvention zu nutzen und sich nach der Bedeutung der einzelnen Menschenrechte für die konkrete Lebenssituation von Menschen mit Behinderungen zu erkundigen. Es geht auch um die Frage, was notwendig ist, damit diese Rechte tatsächlich in Anspruch genommen werden können. Allerdings muss auch gesagt werden, dass mit dem Bezug auf die BRK nicht automatisch alle Fronten geklärt sind. Gerade jene Artikel, welche mit den angesprochenen Forderungen nach schulischer Inklusion oder selbstbestimmter Lebensführung verknüpft werden, sind selbst nicht ganz frei von Widersprüchen. Folgende zwei Beispiele zeigen, dass die Ziele der BRK selbst nicht unmissverständlich klar sind: 1. Der Bezug auf die Konvention schafft keine einheitlichen Pro- oder Kontra-Positionen, wie am Beispiel der Debatte ersichtlich ist, ob eine Ratiﬁzierung der Konvention automatisch die Abschaffung aller Sonderschulen bedeute. Neben den Befürwortern dieser Aussage gibt es eine Reihe von kritischen Stimmen. So sind manche der Meinung, dass das «blosse» Vorhandensein eines inklusiven Angebotes schon genüge, um der Konvention Rechnung zu tragen. Oder man benutzt andere Argumente gegen die inklusive Beschulung, wie beispielsweise die (andernorts besseren) Förderungsmöglichkeiten oder das Wohl und die Wünsche der Kinder und Eltern (Wohlgensinger, 2014, S. 95f.). Schweizerische Zeitschrift für Heilpädagogik, Jg. 21, 10 / 2015 BEHINDERTENRECHTSKONVENTION – TEILHABE UND BETEILIGUNG 2. Das Ziel «der unabhängigen Lebensführung» 1 ist bisher in keinem der Menschenrechtsverträge so explizit zur Geltung gekommen wie in der BRK. Allerdings ist die «unabhängige Lebensführung» scheinbar selbstredend mit dem Bild des Menschen als grundsätzlich autonomes Wesen verknüpft, was im Kontext von geistiger und psychischer Behinderung eine verkürzte Sichtweise darstellt und für manche BRK-Artikel zu Erklärungsbedarf führt (siehe auch den Artikel von Schriber und Wolﬁsberg in dieser Zeitschrift). Enge Schranken für die Heilpädagogik Der Meinung von Bürli (2015), dass die Normen der BRK nicht sakrosankt seien und man den tatsächlichen Vorgaben genau nachgehen sollte, um Potentiale und Herausforderungen realisitisch einschätzen zu können, ist beizupﬂichten: «Analog zu anderen normativen Aussagen mit heil- / sonderpädagogischem Bezug verläuft auch die Umsetzung der BRK nicht zwangsläuﬁg, sondern sie muss bewusst und gezielt gestaltet werden» (Bürli, 2015, S. 55). Welche Rolle will die Heilpädagogik im Gestaltungsprozess spielen? Laut Degener (2009) «besteht die grosse Gefahr wie üblich darin, sich dem Trend der Zeit anzuschliessen, ohne sich ernsthaft mit dem Menschenrechtsmodell von Behinderung auseinander zu setzen. Es ist einfach, der Bundesregierung die rote Karte zu zeigen, ohne sich um die Exklusionsmuster und -strukturen in den eigenen Reihen zu kümmern» (S. 283). 1 Einer der Kritikpunkte der deutschen Übersetzung der Konvention betraf den Begriff des «independently Living», was mit «unabhängiger Lebensführung» anstelle «selbstbestimmter Lebensführung» übersetzt wurde. Schweizerische Zeitschrift für Heilpädagogik, Jg. 21, 10 / 2015 Wenn wir uns der Gewährleistung der Menschenrechte aller Menschen mit Behinderung verschreiben, bedeutet das noch einmal vermehrt, sich von Aspekten der Fürsorglichkeit oder gar Fremdbestimmung zu verabschieden. Es gilt, den Blick auf die Möglichkeit der Umsetzung von Rechten zu lenken. Das bedeutet wiederum, dass der Fokus vermehrt auf Barrieren fällt – welche für das Verständnis von Behinderung eine zentrale Rolle spielen. Es geht vor allem darum, bauliche, ﬁnanzielle, soziale, technologische, rechtliche und andere Hindernisse auszuräumen, welche es erschweren oder verunmöglichen, die eigenen Rechte wahrzunehmen. Die Aufgaben der Heilpädagogik beﬁnden sich dann in relativ engen Schranken und haben Dienstleistungs- oder allenfalls Bildungscharakter. Im Auftrag von Menschen mit Behinderungen haben sie eventuell auch eine öffentliche Funktion, um auf die «Barrieren in den Köpfen, die ebenfalls zu den behindernden Faktoren gehören» (Aichele, 2015, S. 90) einzuwirken. Es gilt, den Blick auf die Möglichkeit der Umsetzung von Rechten zu lenken. Nun wäre es allerdings eine verkürzte Sichtweise, würde man behaupten, mit dem Abbau von Barrieren allein sei es generell getan. Das mag vor allem für Menschen mit einer Körperbehinderung gelten. Bei Menschen mit einer kognitiven oder psychischen Beeinträchtigung genügt die Konzentration auf umweltbedingte Schwierigkeiten nicht immer. Genau hier greift die BRK zu kurz. Es ist dem Umstand Beachtung zu schenken, «dass das traditionelle Bild der Menschenrechte nicht für alle Gültigkeit besitzt und manche Menschen einen Unterstützungs- 9 10 BEHINDERTENRECHTSKONVENTION – TEILHABE UND BETEILIGUNG bedarf aufweisen, wenn es darum geht, Rechte zu beanspruchen. Die Unterstützung kann sowohl in der Veränderung der Umwelt bestehen (zum Beispiel wenn es darum geht, Inhalte verständlich zu formulieren oder Assistenz zur Verfügung zu stellen), aber auch eine befähigende oder wo nötig gar stellvertretende Funktion haben» (Wohlgensinger, 2014, S. 78). Gefragt sind auch Fähigkeiten, ein Recht in befriedigender Art und Weise wahrzunehmen. Das bedeutet nicht, dass sich heilpädagogisches Handeln nicht auf Selbstbestimmung sowie zugleich Chancengleichheit und Partizipation als Zielperspektive ausrichten soll. Es gilt aber, den Umstand anzuerkennen, dass nicht alle Menschen in gleichem Umfang (oder zu jeder Zeit oder bezüglich jeder Sache) zu Autonomie und Selbstverantwortung fähig sind und daher das blosse Recht bezüglich Zugang oder Ausübung von Menschenrechten zu kurz greift. Gefragt sind auch Fähigkeiten, ein Recht (in befriedigender Art und Weise) wahrzunehmen. Diese müssen bisweilen vermittelt werden. Stellvertretende Entscheidungen lassen sich schwer und nur in begründeten Ausnahmen mit der Ausrichtung an der BRK vereinbaren. Dies zeigte sich beispielsweise auch im Schattenbericht der deutschen BRK-Allianz, in dem im Zusammenhang mit Artikel 12 (Gleiche Anerkennung vor dem Recht) gefordert wurde, das Konzept der stellvertretenden Entscheidung («substituted decision making») durch die unterstützte Entscheidung («supported decision making») zu ersetzen. Die damit verknüpfte Forderung ist, die Weiterentwicklung des deutschen Betreuungsrechts: «Menschen mit Behinderungen ist ohne Eingriff in ihr Selbstbestimmungsrecht Zugang zur unterstützten Entscheidung zu verschaffen» (BRK-Allianz, 2013, S. 26). Zusammenfassend kann festgestellt werden, dass heilpädagogische Interventionen mit der Anerkennung von Menschen mit Behinderungen als Rechtssubjekte nur schwer vereinbar sind. Profession und Disziplin müssen sich daher wohl auch die Frage gefallen lassen, inwiefern sie und ihr System der speziellen Hilfen, Wohn- und Arbeitseinrichtungen nicht auch selbst eine Barriere darstellen, wenn es darum geht, Menschenrechte zu gewährleisten (Wohlgensinger, 2014, S. 147). Liefern, nicht lafern Auch wenn der Inhalt der BRK zuweilen unterschiedlich interpretiert wird und auch wenn hinsichtlich deren Anwendung auf die Gruppe von Menschen mit einer geistigen oder psychischen Beeinträchtigung noch ein ganzes Stück Arbeit geleistet werden muss: Menschenrechte können universelle Geltung beanspruchen und auf breite Akzeptanz bauen. Sie sind «moralische Leitplanke» und haben auch das Potential zum politischen Durchsetzungsinstrument. Sie erwecken einen Anspruch der Verbindlichkeit und sind damit «eine verlässlichere, auf jeden Fall aber anschlussfähigere interindividuelle Basis als normative sonderpädagogische Eigenkreationen» (Liesen, Wolﬁsberg & Wohlgensinger, 2012, S. 23). Die Heilpädagogik ist in der Ermöglichung der Inanspruchnahme von (Menschen-)Rechten und damit von Selbstbestimmung und Partizipation natürlich nicht nur potentielles Hindernis, sondern auch förderlicher Faktor. Beispielsweise dann, wenn es um die Inanspruchnahme von Hilfsmitteln, Assistenz- und DolmetscherdiensSchweizerische Zeitschrift für Heilpädagogik, Jg. 21, 10 / 2015 BEHINDERTENRECHTSKONVENTION – TEILHABE UND BETEILIGUNG ten oder um das Erlernen von behinderungsspeziﬁschen Kompetenzen oder das Erhalten von Informationen in Leichter Sprache geht. Auch angepasste Bauten, Arbeitsplätze sowie Kommunikations- und Informationssysteme können in diesen Aufgabenbereich gehören, genauso wie die Sensibilisierung und Aufklärung der verschiedenen Systeme und Dienstleister etc. Nun heisst es, sich die Grundlage, die sich durch die BRK bietet, zu eigen zu machen und etwas von der anfänglich vorhandenen Starteuphorie am Leben zu erhalten. Gerade dies ist schwierig geworden, wie der Nationalrat Christian Lohr (2015) feststellt: Die BRK sei zwar ratiﬁziert, aber er «[…] habe den Eindruck gewonnen, dass man sich der Dimension des ganzen Projekts zuvor gar nicht so richtig bewusst gewesen ist. Dabei war bereits im Vorfeld klar, dass es um ganz konkrete Grundhaltungen und Massnahmen zur verbesserten gesellschaftlichen Teilhabe von Menschen mit Beeinträchtigungen gehen würde. Von theoretischen Ansätzen hiess es jetzt auf konkrete Ebenen zu wechseln und entsprechend rasch zu handeln. Oder um es ein wenig deutlicher zu formulieren: Es geht nicht mehr darum, was gewünscht wird, sondern was gemacht wird» (S. 4). Den Worten müssen nun also Taten folgen. Die BRK ist als Chance für die Heilpädagogik zu verstehen: Sie eignet sich hervorragend als normative Vorlage für die Reﬂexion von Profession und Disziplin sowie auch als Richtungsweiserin. Zugegebenermassen geht aus dem Konventionstext manchmal nicht mehr hervor als eben die Richtung, in welche die Reise gehen soll. Aufzuzeigen, welche Wege dabei eingeschlagen werden können, auch das gehört zu den Aufgaben einer Heilpädagogik, welche für die Menschenrechte von Menschen mit Behinderungen einsteht. Schweizerische Zeitschrift für Heilpädagogik, Jg. 21, 10 / 2015 Literatur Aichele, V. (2015). Unabhängig und kritisch: die Monitoring-Stelle zur UN-BRK. In T. Degener (Hrsg.), Handbuch Behindertenrechtskonvention. Teilhabe als Menschenrecht – Inklusion als gesellschaftliche Aufgabe (S. 85–92). Bonn: Bundeszentrale für politische Bildung. BRK-Allianz (2013). Für Selbstbestimmung, gleiche Rechte, Barrierefreiheit, Inklusion! Erster Bericht der Zivilgesellschaft zur Umsetzung der UN-Behindertenrechtskonvention in Deutschland. http://www.brkallianz.de/index.php/parallel-bericht.html [Zugriff am 20.08.2015]. Bürli, A. (2015). Zur Umsetzung der UN-Behindertenrechtskonvention. In A. Leonhardt, K. Müller & T. Truckenbrodt (Hrsg.), Die UN-Behindertenrechtskonvention und ihre Umsetzung. Beiträge zur Interkulturellen und International vergleichenden Heil- und Sonderpädagogik (S. 55–66). Bad Heilbrunn: Klinkhardt. Degener, T. (2009). Die neue UN Behindertenrechtskonvention aus der Perspektive der Disability Studies. Behindertenpädagogik, 3, 263–283. Kahle, U. (2012). Übergänge in der Beruﬂichen Rehabilitation – Probleme und Chancen. Gelingensbedingungen für eine Inklusive Bildung in Baden-Württemberg – auf dem Weg zur inklusiven Bildung. Behindertenpädagogik, 2, 143–155. Liesen, C., Wolﬁsberg, C. & Wohlgensinger, C. (2012). Heilpädagogik und Menschenrechte? Schweizerische Zeitschrift für Heilpädagogik, 7–8, 19–24. Lohr, C. (2015). Ist die Schweiz schon reif für die Behindertenrechtskonvention? Magazin Cerebral, 2, 4–5. http://www.vereinigung-cerebral.ch/uploads/media/Magazin_Cerebral_2_2015_low.pdf [Zugriff am 20.08.2015]. 11 12 BEHINDERTENRECHTSKONVENTION – TEILHABE UND BETEILIGUNG Nicklas-Faust, J. (2011). Erster Staatenbericht zur Umsetzung der Behindertenrechtskonvention beschlossen. Rechtsdienst, 3, 103–108. Übereinkommen über die Rechte von Menschen mit Behinderungen vom 13. Dezember 2006, durch die Schweiz ratiﬁziert am 15. April 2014, in Kraft seit dem 15. Mai 2014 (Behindertenrechtskonvention, BRK), SR 0.109. Wacker, E.(2011). Inklusion von Menschen mit Behinderung im Alter – noch Zukunftsmusik für die Behindertenhilfe und ihre Fachkräfte? Vierteljahresschrift für Heilpädagogik und ihre Nachbargebiete, 3, 235–241. Wocken, H. (2010). Restauration der Stigmatisierung! Kritik der «diagnosegeleiteten Integration». Behindertenpädagogik, 2, 117–134. Wohlgensinger, C. (2014). Behinderung und Menschenrechte. Ein Verhältnis auf dem Prüfstand. Opladen: Budrich UniPress. Impressum Schweizerische Zeitschrift für Heilpädagogik, 21. Jahrgang, 10 / 2015 ISSN 1420-1607 Herausgeber Stiftung Schweizer Zentrum für Heil- und Sonderpädagogik (SZH) Haus der Kantone Speichergasse 6, CH-3000 Bern 7 Tel. +41 31 320 16 60, Fax +41 31 320 16 61 szh@szh.ch, www.szh.ch Redaktion und Herstellung redaktion@szh.ch Verantwortlich: Beatrice Kronenberg, Lea Blatter Redaktion und Koordination: Silvia Schnyder, Silvia Brunner Amoser, David Bisang, Lea Blatter, Fabian Leuthold Rundschau und Dokumentation: Thomas Wetter Inserate: Remo Lizzi, Florian Schär Layout: Monika Feller Erscheinungsweise 9 Ausgaben pro Jahr, jeweils in der ersten Woche des Monats Inserate inserate@szh.ch Annahmeschluss: 10. des Vormonats; Preise: ab CHF 220.– exkl. MwSt.; Mediadaten unter www.szh.ch / zeitschrift Auﬂage 2633 Exemplare (WEMF / SW-beglaubigt) Druck Ediprim AG, Biel Jahresabonnement Schweiz CHF 76.90 (inkl. MwSt.); Ausland CHF 84.00 Preis Studierende mit Legi: CHF 53.85 (inkl. 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https://openalex.org/W2972263662	https://peerj.com/articles/7349.pdf	English	null	Reduced gray matter volume in male adolescent violent offenders	PeerJ	2,019	cc-by	7,484	ABSTRACT Background. Previous studies reported that reduced gray matter volume (GMV) was associated with violent-related behaviors. However, the previous studies were conducted on adults and no study has studied the association between GMV and violent behaviors on adolescents. The purpose of the study was to investigate GMV’s effects in adolescent violent offenders based on a Chinese Han population, which can address the problem of possible confounding factors in adult studies. Methods. We recruited 30 male adolescent violent offenders and 29 age- and sex- matched healthy controls (HCs). Differences in both whole-brain and GMV were Methods. We recruited 30 male adolescent violent offenders and 29 age- and sex- matched healthy controls (HCs). Differences in both whole-brain and GMV were evaluated using voxel-based morphometry (VBM). We assessed the accuracy of VBM Methods. We recruited 30 male adolescent violent offenders and 29 age- and sex- matched healthy controls (HCs). Differences in both whole-brain and GMV were evaluated using voxel-based morphometry (VBM). We assessed the accuracy of VBM using the receiver operating characteristic curve (ROC) and discriminant analysis. y ( ) evaluated using voxel-based morphometry (VBM). We assessed the accuracy of VBM using the receiver operating characteristic curve (ROC) and discriminant analysis. evaluated using voxel-based morphometry (VBM). We assessed the accuracy of VBM using the receiver operating characteristic curve (ROC) and discriminant analysis. Results. Compared with HCs, the male adolescent offenders showed significantly reduced GMV in five cortical and subcortical brain regions, including the olfactory cortex, amygdala, middle temporal gyrus and inferior parietal lobe in the left hemi- sphere, as well as the right superior temporal gyrus. Both ROC curve and discriminate analyses showed that these regions had relatively high sensitivities (58.6%–89.7%) and specificities (58.1%–74.2%) with 76.7% classification accuracy. Conclusions. Our results indicated that reduced volume in the frontal-temporal- parietal-subcortical circuit may be closely related to violent behaviors in male ado- lescents, which might be an important biomarker for detecting violent behaviors in male adolescents. Submitted 7 May 2019 Accepted 25 June 2019 Published 3 September 2019 Corresponding author Xiao-Ping Wang, xiaop6@csu.edu.cn Academic editor Tifei Yuan Additional Information and Declarations can be found on page 10 DOI 10.7717/peerj.7349 Copyright 2019 Zhang et al Submitted 7 May 2019 Accepted 25 June 2019 Published 3 September 2019 Subjects Neuroscience, Population Biology Keywords Brain structural abnormalities, Discriminate analyses, Violence, ROC, VBM Reduced gray matter volume in male adolescent violent offenders ing-Dong Zhang1,2, Jian-Song Zhou1,2, Feng-Mei Lu3,4 and Xiao-Ping Wang1,2 1 Department of Psychiatry & Mental Health Institute of the Second Xiangya Hospital, Central South University, Changsha, Hunan, China 1 Department of Psychiatry & Mental Health Institute of the Second Xiangya Hospital, Central South University, Changsha, Hunan, China 2 National Clinical Research Center on Mental Disorders & National Technology Institute on Mental Disorders, Hunan Key Laboratory of Psychiatry and Mental Health, Changsha, Hunan, China 3 The Clinical Hospital of Chengdu Brain Science Institute, MOE Key Lab for Neuroinformation, University of Electronic Science and Technology of China, Chengdu, Sichuan, China 2 National Clinical Research Center on Mental Disorders & National Technology Institute on Mental Disorders, Hunan Key Laboratory of Psychiatry and Mental Health, Changsha, Hunan, China 3 The Clinical Hospital of Chengdu Brain Science Institute, MOE Key Lab for Neuroinformation, University of Electronic Science and Technology of China, Chengdu, Sichuan, China 4 School of Life Science and Technology, Center for Information in Medicine, University of Electronic Science and Technology of China, Chengdu, Cichuan, China How to cite this article Zhang Y-D, Zhou J-S, Lu F-M, Wang X-P. 2019. Reduced gray matter volume in male adolescent violent offend- ers. PeerJ 7:e7349 http://doi.org/10.7717/peerj.7349 INTRODUCTION Violence is a global public health problem that causes great personal sufferings as well as social problems (Krug et al., 2002). The World Health Organization report shows that the global homicide rate in males is significantly higher than in females (13.6 versus 4.0 per 100,000 population) in all age groups (Krug et al., 2002). Among different age groups, adolescents and young adults (15–29 years old) have the highest homicide rates: 19.4 for males and 4.4 for female per 100,000 population Distributed under Creative Commons CC-BY 4.0 OPEN ACCESS (Krug et al., 2002). These reports suggest that the violence problem is more severe in adolescents and young adults, especially males. Moreover, the youth violence has an extremely negative impact on communities and social stability (Greenwood, 2008). Therefore, early identification and management of violent behavior among adolescents not only can save their lives, but also can prevent the development of criminal behaviors in adulthood, thus reducing the burden of crime on society (Greenwood, 2008). The relationship between abnormal brain structures and violent behaviors has long been noted. It was reported that damage to certain brain areas was related to aggression and violence (Grafman et al., 1996). A longitudinal brain study also revealed that abnormal brain structure was associated with an increased amount of criminal behaviors (McKinlay et al., 2014). In addition, it was reported that the abnormal brain structure and function were key biological risk factors for violence (Rosell & Siever, 2015). Previous identification of persons who were prone to violent behavior was primarily based on personality assessments and electroencephalography. In recent years, the development of noninvasive imaging tools, such as magnetic resonance imaging (MRI), single photon emission computed tomography, and positron emission tomography, has offered new ways for identifying individuals who are at risk of engaging in violent behavior (Leutgeb et al., 2016; Puri et al., 2008). Evidence from limited structural neuroimaging studies has indicated that reduction of the gray matter volume (GMV) in several brain regions that subserve both emotion processing and behavior regulation (Shi et al., 2016), such as the orbitofrontal cortex (OFC), prefrontal cortex, medial temporal cortex, amygdala, basal ganglia, anterior cingulate cortex, was closely related to violent-related behaviors (Leutgeb et al., 2016; Rosell & Siever, 2015). The brain develops steadily in terms of both structure and function throughout childhood and early adulthood. The developmental trajectory of GMV is complicated and heterogeneous between different brain regions. Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 Participants A total of 30 male adolescent offenders (aged from 15 to 18 years), who had been convicted of aggressive behavior in court (four murder or manslaughter, 15 intentional injury, and 11 robbery with injury), and 29 age- and gender-matched healthy controls (HCs) were recruited. Male adolescent offenders were recruited from the Youth Detention Centre (YDC), Hunan province, China. All of them had been convicted of either homicide or assault. Male HCs were students, recruited from a middle school in Changsha and further screened by research psychiatrists to exclude subjects with past mental disorders. All participants were right-handed, and had no history of neurological impairments. The substance abusers were excluded based on their urine analysis, self-report and family informant report within the last 3 months. All interviews were conducted by research psychiatrists. The Chinese version of the Schedule for Affective Disorders and Schizophrenia for School-Age Children Present and Lifetime version (K-SADS-PL) (Shanee, Apter & Weizman, 1997) was used for detecting current and past psychiatric problems according to Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV criteria. We excluded 2 adolescent offenders who were diagnosed with either current or past psychiatric disorders. In addition, we also collected the information of criminal history, psychosocial history, alcohol or other drug use, family history, history of psychiatric, and other medical treatments for each participant. Signed written informed consent was obtained from all participants and all study procedures were approved by Institutional Review Board of the second Xiangya Hospital, Central South University. INTRODUCTION For example, the GMV in cortex and some subcortical structures (such as the caudate) follows an inverted U-shape developmental course (Giedd & Denker, 2015). In comparison, this developmental trajectory has not been found in other subcortical structures (such as amygdala and hippocampus) (Giedd & Denker, 2015). Moreover, most of the previous studies regarding violence-related structural abnormality were based on adults (Leutgeb et al., 2016; Rosell & Siever, 2015). Only a few studies in children and adolescents have indicated that decreased GMV of several brain regions may be a constant marker for violence-related behaviors (Sterzer & Stadler, 2009; Vloet et al., 2008). For example, lower amygdala volume was reported to be associated with increased violent behaviors from childhood to adulthood and even predicted future violence (Pardini et al., 2014). More importantly, an adolescents based study can avoid possible confounding factors that are commonly seen in adult based studies, such as the shorter duration of illness, medication naïve and lower rates of comorbidities. Thus, studying structural neuroimaging in adolescents has advantages over that in adults. Moreover, most previous studies on violent behaviors usually were conducted in patients with psychiatric disorders such as conduct disorder (CD), antisocial personality disorder (APD), borderline personality disorder (BPD), attention-deficit hyperactivity disorder (ADHD) or schizophrenia, with aggression, impulsivity, psychopathy or callous- unemotional (CU) traits (Laakso et al., 2002; Schiffer et al., 2011). Only a few previous Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 2/15 studies investigated the neuroimaging characteristics of impulsive or aggressive behaviors in healthy subjects (Boes et al., 2009; Boes et al., 2008; Ducharme et al., 2011; Matsuo et al., 2009a; Matthies et al., 2012; Pardini et al., 2014). Furthermore, most previous violence- related brain studies were based on males (Boes et al., 2009; Boes et al., 2008; De Brito et al., 2009; Ducharme et al., 2011; Ermer et al., 2013; Fairchild et al., 2011; Huebner et al., 2008; Sterzer et al., 2007). The aforementionedviolence-brainstudiesarelimited eitherbyusingadultsorbylacking emphasis on healthy subjects. To fill the gap of the aforementioned GMV-violence studies, we investigated the effects of abnormal brain cerebral structures on violent behaviors and focused the study on healthy male adolescents. More specifically, using voxel-based morphometry (VBM) (Xue, 2016) and region of interest (ROI)-based methods, we analyzed the differences in GMV and white matter volume (WMV) between violent offenders and controls. Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 Data processing and statistical analysis Demographic and clinical characteristics analysis: Two-sample t-tests, chi-square test, and the Fisher exact test were used to analyze the sociodemographic and clinical characteristics of the participants. A p-value less than 0.05 was considered as statistically significant. All analyses were performed using the Statistical Package for Social Sciences (SPSS) version 16.0 (SPSS, Chicago, Ill., USA). VBM: For the morphometric analysis, the VBM8 (http://dbm.neuro.uni-jena.de/ vbm8/), a toolbox of SPM8 (https://www.fil.ion.ucl.ac.uk/spm/software/spm8/) software, was applied with the DARTEL algorithm. For each participant, the origin of the images was first manually aligned to the anterior commissure for a better registration. The structural images were then segmented into gray matter (WM), white matter (WM) and cerebrospinal fluid (CSF) using a standard unified segmentation model in SPM8. Figure 1 shows the segmentation results of one subject. After that, the DARTEL approach was performed for registration, normalization and modulation in the DARTEL space. In details, a new DARTEL template was constructed based on the deformation fields from the segmentation procedure, and all the individual deformation filed map was registered to the new template. The GM images were then normalized to a study-specific template in Montreal Neurological Institute (MNI) space. To preserve the volume of GM, Jacobian determinants are used to modulate the voxel values of GM. After that, the images were smoothed with the Gaussian kernel of 88 mm. After spatial preprocessing, the normalized, modulated and smoothed GM and WM images were used for further statistical analyses. In our study, both whole-brain level and region-of-interest (ROI) level VBM analyses were performed. The whole-brain level analysis is automated and unbiased, making no assumptions of about any regions of particular interest. However, this technique requires a great number of subjects to achieve a statistical significance and therefore changes in smaller structures may be difficult to identify due to the small sample size. To address this problem, a secondary ROI level analysis is often used to test the differences only in the voxels that are deemed of interest by an a priori hypothesis. The ROI level analysis can be used to corroborate the findings of previous whole-brain level studies, or those obtained during the whole-brain analysis. This is of special importance in studies with a small sample size (Husain et al., 2011). Whole brain analysis: Two sample T-tests were performed to evaluate the significance of the data using the initial threshold of P = 0.001. MRI data acquisition The structural three-dimensional (3D) T1-weighted MRI images were acquired using a 3.0 T Siemens Vision scanner at the Magnetic Resonance Center of the Hunan Provincial People’s Hospital. A 3D magnetization-prepared rapid-acquisition gradient echo (3D MPRAGE) sequence was used with the following parameter settings: Repetition Time (TR) 3/15 3/15 Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 = 2,000 ms; Echo Time (TE) = 3.36 ms; flip angle = 9◦; pixel matrix = 256 × 256, Field of view (FOV) = 256 × 256 mm2, voxel size = 1 × 1 × 1 mm3, and number of slices = 144. Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 Data processing and statistical analysis The p-values were then adjusted using false discovery rate (FDR) correction (with cluster size >100) to correct multiple testing. A FDR-corrected p-value < 0.05 was considered as statistically significant. ROI analysis: Brain regions that showed significant differences in GMV between two groups in the whole brain analysis were identified as significant ROIs. The main advantage of using a priori ROI test over a whole brain analysis is that it reduces type I errors by narrowing down the statistical tests to only a few ROIs (Husain et al., 2011). For each 4/15 Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 Figure 1 Segmentation results of one typical subject. These results are obtained in the original space of the images. (A) segmented grey matter (c1X.img); (B) segmented white matter (c2X.img); (C) segmented CSF (c3X.img); (D) segmented skull (c4X.img). CSF, cerebrospinal fluid. Full-size DOI: 10.7717/peerj.7349/fig-1 participant, the mean GMV of each ROI was then extracted using an approach described Figure 1 Segmentation results of one typical subject. These results are obtained in the original space of the images. (A) segmented grey matter (c1X.img); (B) segmented white matter (c2X.img); (C) segmented CSF (c3X.img); (D) segmented skull (c4X.img). CSF, cerebrospinal fluid. Full-size DOI: 10.7717/peerj.7349/fig-1 participant, the mean GMV of each ROI was then extracted using an approach described previously (Santillo et al., 2013). We then used the receiver operating characteristic curve (ROC) analyses to investigate the sensitivity and specificity of the detected abnormalities in GMV, which enabled us to evaluate the discriminative power of these abnormalities in identifying the violent adolescent offenders. In addition, as suggested in one previous study (Jednorog et al., 2014), a further Fisher discriminant analysis was performed to predict 5/15 Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 Table 1 Sociodemographic and clinical characteristics (mean ± S.D.) in adolescent offenders and healthy controls (two-sample t-tests). HC (N = 29) AO (N = 30) Statistic P-value Age, years 17.6 ± 0.5 17.7 ± 0.8 t = 0.45 0.66 Literacy, years 10.0 ± 0.0 7.5 ± 2.4 t = −5.10 0.00 TIV, mm3 1335 ± 86 1348 ± 107 t = 0.45 0.65 GMV, mm3 633 ± 39 629 ± 46 t = −0.36 0.72 WMV, mm3 462 ± 38 473 ± 50 t = 0.84 0.40 Notes. Abbreviations: S.D., standard deviation; HC, healthy controls; AO, adolescent offenders; TIV, total intracranial volume; GMV, gray matter volume; WMV, white matter volume. RESULTS Demographic and clinical characteristics Demographic and clinical characteristics The results of two-sample T-tests were presented in Table 1. As shown in Table 1, there is no significant difference between the adolescent offenders and HCs in terms of age, total intracranial volume, GMV, and WMV (all p-values > 0.05). In comparison, the educational level of the adolescent offenders was significantly lower than that of the HCs (7.5 ± 2.4 vs 10.0 ± 0.0 years; t = −5.10; p-value < 0.01), which was adjusted in the following analyses. The educational difference is due to that most of the adolescent offenders had dropped out from the junior high school, while all HCs were students from senior schools. Whole brain analysis The result of whole-brain analysis was presented in Table 2. Compared to the HCs, the adolescent offenders showed significantly lower GMV (p < 0.05, FDR corrected) in the olfactory cortex, amygdala, middle temporal gyrus and inferior parietal lobe in the left hemisphere, as well as the right superior temporal gyrus (Fig. 2). In comparison, there was no significant difference in GMV of the whole brain between the offenders and HCs. Similarly, no significant difference in WMV was observed between the two groups. Data processing and statistical analysis Table 1 Sociodemographic and clinical characteristics (mean ± S.D.) in adolescent offenders and healthy controls (two-sample t-tests). Notes. Abbreviations: S.D., standard deviation; HC, healthy controls; AO, adolescent offenders; TIV, total intracranial volume; GMV, gray matter volume; WMV, white matter volume. whether a participant was a violent offender according to the individual’s GMV for those ROIs which exhibited significant differences (p < 0.001) in GMV between the two groups. whether a participant was a violent offender according to the individual’s GMV for those ROIs which exhibited significant differences (p < 0.001) in GMV between the two groups. whether a participant was a violent offender according to the individual’s GMV for those ROIs which exhibited significant differences (p < 0.001) in GMV between the two groups. Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 ROI Analysis These five ROIs were then used for a discriminant analysis The overall classification Table 2 Regions with smaller gray matter volume in adolescent offenders (N = 31) compared with healthy controls (N = 29), measured using voxel-based morphometry (corrected false discovery rate at P < 0.05). Table 2 Regions with smaller gray matter volume in adolescent offenders (N = 31) compared with healthy controls (N = 29), measured using voxel-based morphometry (corrected false discovery rate at P < 0.05).* Notes. Abbreviations: BA, Brodmann’s area; T, intensity; L, left; R, right; OC, olfactory cortex; AMG, amygdala; MTG, middle temporal gyrus; IPL, inferior parietal lobe; STG, superior temporal gyrus. The duration of education was select as the covariant. Uncorrected false discovery rate. The duration of education was select as the covariant. *Uncorrected false discovery rate. Figure 2 Brain voxel-based morphometry showing lower gray matter volume in male violent adoles- cent offenders superimposed on a T1-weightedtemplate (violent adolescent offenders vs. controls; two- sample t-tests). These brain areas include the left olfactory cortex (A, B), the left amygdala (C, D), the left middle temporal gyrus (C, D), the left inferior parietal lobe (E), and the right superior temporal gyrus (F). Figure 2 Brain voxel-based morphometry showing lower gray matter volume in male violent adoles- cent offenders superimposed on a T1-weightedtemplate (violent adolescent offenders vs. controls; two- sample t-tests). These brain areas include the left olfactory cortex (A, B), the left amygdala (C, D), the left middle temporal gyrus (C, D), the left inferior parietal lobe (E), and the right superior temporal gyrus (F). The color bar indicates the t value of the between-group analysis. Full-size DOI: 10.7717/peerj.7349/fig-2 Figure 2 Brain voxel-based morphometry showing lower gray matter volume in male violent adoles- cent offenders superimposed on a T1-weightedtemplate (violent adolescent offenders vs. controls; two- sample t-tests). These brain areas include the left olfactory cortex (A, B), the left amygdala (C, D), the left middle temporal gyrus (C, D), the left inferior parietal lobe (E), and the right superior temporal gyrus (F). The color bar indicates the t value of the between-group analysis. Full-size DOI: 10.7717/peerj.7349/fig-2 Figure 2 Brain voxel-based morphometry showing lower gray matter volume in male violent adoles- cent offenders superimposed on a T1-weightedtemplate (violent adolescent offenders vs. controls; two- sample t-tests). ROI Analysis Five ROIs were identified as significant based on the whole-brain analysis, including the left olfactory cortex, left amygdala, left middle temporal gyrus, left inferior parietal lobe, and the right superior temporal gyrus. A further ROC analysis was conducted on these ROIs and the results were presented in Table 3. The sensitivity, specificity and area under the curve (AUC) values were 58.6%, 67.7% and 0.642 for the left olfactory cortex; 86.2%, 74.2% and 0.834 for the left amygdala; 79.3%, 58.1% and 0.721 for the left middle temporal gyrus; 86.2%, 74.2% and 0.834 for the left inferior parietal lobe; and 89.7%, 64.5% and 0.790 for the right superior temporal gyrus. It was suggested that the AUC values can be interpreted as excellent if AUC ≥0.90; good if 0.90 > AUC ≥0.80; fair if 0.80 > AUC ≥0.70; poor if 0.70 > AUC ≥0.60; and no effect if AUC < 0.60 (Linden, 2006). Hence, our Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 6/15 Table 2 Regions with smaller gray matter volume in adolescent offenders (N = 31) compared with healthy controls (N = 29), measured using voxel-based morphometry (corrected false discovery rate at P < 0.05). Regions BA Stereotactic coordinates (mm) Cluster size (mm3) T P-value x y z L OC 11 −17 12 −14 232 −4.68 0.000 L AMG 34 −26 3 −15 191 −6.50 0.014 L MTG 21 −65 −1 −11 103 −5.12 0.047 L IPL 39 −39 −52 54 130 −4.43 0.000 R STG 21 40 0 −15 104 −4.72 0.000** Notes. Abbreviations: BA, Brodmann’s area; T, intensity; L, left; R, right; OC, olfactory cortex; AMG, amygdala; MTG, middle temporal gyrus; IPL, inferior parietal lobe; STG, superior temporal gyrus. The duration of education was select as the covariant. *Uncorrected false discovery rate. Figure 2 Brain voxel-based morphometry showing lower gray matter volume in male violent adoles- cent offenders superimposed on a T1-weightedtemplate (violent adolescent offenders vs. controls; two- sample t-tests). These brain areas include the left olfactory cortex (A, B), the left amygdala (C, D), the left middle temporal gyrus (C, D), the left inferior parietal lobe (E), and the right superior temporal gyrus (F). The color bar indicates the t value of the between-group analysis. Full-size DOI: 10.7717/peerj.7349/fig-2 results suggested that except for the left olfactory cortex, GMV reduction of each of the other four ROIs represented a fair or good biomarker for violence in juveniles. DISCUSSION To our best knowledge, this study represented the first attempt to use the VBM method for identifying brain structural abnormalities in a cohort of male violent adolescents with no history of mental disorders. We found that male adolescent offenders had a reduced GMV in the olfactory cortex, amygdala, middle temporal gyrus and inferior parietal lobe in the left hemisphere, and in the right superior temporal gyrus than male HCS. These findings provide evidence that male violent adolescent offenders exhibit reductions of GMV in several brain regions. The pathophysiology of violence-related behaviors has not been understood clearly. Several lines of studies showed that violent behaviors were related with structural or functional abnormalities in several brain regions that subserve both emotion processing and behavior regulation (Leutgeb et al., 2016; Rosell & Siever, 2015). The amygdala is a subcortical structure of limbic system that plays an essential role in the integration of a wide range of sensory and motivationally salient stimuli, as well as in transmission of this information to various cortical and subcortical regions. These processes underlie the amygdala’s essential role in fear mediating, defensive reactions, emotional learning, and motivation. The amygdala has long been considered as the most important neural region for violence-related behaviors (Lane, Kjome & Moeller, 2011; Perathoner, Cordero-Maldonado & Crawford, 2016). Similar to the finding in our current study, several previous studies found a decreased amygdala volume in juvenile subjects prone to violence than their healthy peers (Fairchild et al., 2011; Huebner et al., 2008; Sterzer et al., 2007; Stevens & Haney-Caron, 2012). One longitudinal study reported that a lower amygdala volume was a significant factor for detecting increased violent behaviors from childhood to adulthood (Pardini et al., 2014). Further, the frontal cortex, especially the OFC, exhibits the most robust reciprocal anatomical connections with the amygdala, which receives limbic inputs from amygdala and other medial temporal areas as well as sensory inputs (Leutgeb et al., 2016). Thus, frontal cortex may integrate sensory information with affective signals, inhibitory control signals from other areas and is considered as the trigger point of violence-related behaviors (Lane, Kjome & Moeller, 2011; Rosell & Siever, 2015). ROI Analysis These brain areas include the left olfactory cortex (A, B), the left amygdala (C, D), the left middle temporal gyrus (C, D), the left inferior parietal lobe (E), and the right superior temporal gyrus (F). The color bar indicates the t value of the between-group analysis. Full-size DOI: 10.7717/peerj.7349/fig-2 results suggested that except for the left olfactory cortex, GMV reduction of each of the other four ROIs represented a fair or good biomarker for violence in juveniles. These five ROIs were then used for a discriminant analysis. The overall classification accuracy was 76.7%: 75.9% accuracy for the HC group versus 77.4% accuracy for violent adolescent offenders. The validity of using GMV to distinguish two groups was supported by Wilk’s lambda = 0.57 (df = 4, p < 0.001). These five ROIs were then used for a discriminant analysis. The overall classification accuracy was 76.7%: 75.9% accuracy for the HC group versus 77.4% accuracy for violent adolescent offenders. The validity of using GMV to distinguish two groups was supported by Wilk’s lambda = 0.57 (df = 4, p < 0.001). Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 7/15 Table 3 Area under the curve (AUC) details for the five brain regions of interest showing significant differences in gray matter volume between male adolescent offenders and male age-matched controls. L OC L AMG L MTG L IPL R STG Sensitivity% 58.6 86.2 79.3 86.2 89.7 Specificity% 67.7 74.2 58.1 74.2 64.5 AUC 0.642 0.834 0.721 0.834 0.790 Notes. Abbreviations: L, left; R, right; OC, olfactory cortex; AMG, amygdala; MTG, middle temporal gyrus; IPL, inferior pari- etal lobe; STG, superior temporal gyrus; AUC, area under the curve. L OC L AMG L MTG L IPL R STG Sensitivity% 58.6 86.2 79.3 86.2 89.7 Specificity% 67.7 74.2 58.1 74.2 64.5 AUC 0.642 0.834 0.721 0.834 0.790 Notes. Abbreviations: L, left; R, right; OC, olfactory cortex; AMG, amygdala; MTG, middle temporal gyrus; IPL, inferior pari- etal lobe; STG, superior temporal gyrus; AUC, area under the curve. tes. Abbreviations: L, left; R, right; OC, olfactory cortex; AMG, amygdala; MTG, middle temporal gyrus; IPL, inferior pari- etal lobe; STG, superior temporal gyrus; AUC, area under the curve. Notes. Abbreviations: L, left; R, right; OC, olfactory cortex; AMG, amygdala; MTG, middle temporal gyrus; IPL, inferior pari- etal lobe; STG, superior temporal gyrus; AUC, area under the curve. Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 DISCUSSION Several previous studies reported decreased GMV of OFC in subjects prone to violence (Bertsch et al., 2013; Boes et al., 2009; De Oliveira-Souza et al., 2008; Huebner et al., 2008; Laakso et al., 2002; Tiihonen et al., 2008), negative correlation between GMV of OFC and impulsive (Kumari et al., 2009; Matsuo et al., 2009a), aggressive (Gansler et al., 2009) or psychopathic Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 8/15 (Ermer et al., 2012; Ermer et al., 2013) level. These results support our finding of decreased GMV of olfactory cortex, as part of the OFC, in violent adolescents. In addition, the temporal cortex and the parietal cortex, both of which are closely connected to the limbic system, are important for regulating the affective nature of interpersonal experiences and play a pivotal role in the development of emotional behavior (Soderstrom et al., 2002). Previous studies showed decreased GMV of several regions of the temporal or the parietal lobe, such as superior temporal cortex (Bertsch et al., 2013; De Oliveira-Souza et al., 2008; Muller et al., 2008; Stevens & Haney-Caron, 2012), middle temporal cortex (Bertsch et al., 2013), inferior temporal cortex (Bertsch et al., 2013; Gregory et al., 2012; Stevens & Haney-Caron, 2012), hippocampus (Barkataki et al., 2006; Huebner et al., 2008; Stevens & Haney-Caron, 2012; Yang et al., 2010), parahippocampal gyrus (Bertsch et al., 2013; Stevens & Haney-Caron, 2012; Yang et al., 2010), temporal pole (Bertsch et al., 2013), inferior parietal cortex (Tiihonen et al., 2008), postcentral cortex (Bertsch et al., 2013; Stevens & Haney-Caron, 2012; Tiihonen et al., 2008), angular gyrus (Puri et al., 2008) and supramarginal gyrus (Puri et al., 2008; Stevens & Haney-Caron, 2012) in subjects with a tendency toward violence. Also, we found low GMV of three regions in the temporal or parietal lobe, the middle temporal gyrus, the superior temporal gyrus and the inferior parietal lobe in adolescent violent offenders, which were consistent with these previous studies. Our results and previous literatures indicate that reduced GMV in the frontal- temporal-parietal-subcortical circuit may lead to difficulties in suppressing expressions of emotion, which may further lead to inappropriate, or even violent behaviors. DISCUSSION Low GMV of several areas in the frontal-temporal-parietal-subcortical circuit was found to be a marker for violence-related behaviors both in juveniles (Boes et al., 2009; Boes et al., 2008; Bussing et al., 2002; Ducharme et al., 2011; Ermer et al., 2013; Fairchild et al., 2011; Huebner et al., 2008; Kruesi et al., 2004; Sterzer et al., 2007) and in adults (Barkataki et al., 2006; Bertsch et al., 2013; De Oliveira-Souza et al., 2008; Ermer et al., 2012; Gansler et al., 2009; Gregory et al., 2012; Kumari et al., 2009; Laakso et al., 2002; Matsuo et al., 2009a; Matsuo et al., 2009b; Matthies et al., 2012; Muller et al., 2008; Pardini et al., 2014; Puri et al., 2008; Raine et al., 2000; Tiihonen et al., 2008; Yang et al., 2010; Yang et al., 2005; Zhang et al., 2013). Low GMV of several areas in the frontal-temporal-parietal-subcortical circuit was found to be a marker for violence-related behaviors both in juveniles (Boes et al., 2009; Boes We evaluated the discriminative power of both GMV and the identified brain regions in classifying violent offenders and controls.The ROC analysis with VBM on each region revealed that reduced GMV in four regions (left amygdala, left middle temporal lobe, left inferior parietal lobe, and right superior temporal gyrus) represented either a fair or a good biomarker of violence in juveniles. Further discriminant analysis indicated that reduced GMVs of the five regions could predict whether a subject was a violent offender with 76.7% accuracy, suggesting that reduced GMV of the five regions could be regarded as a biomarker of violent behavior in male adolescents. The high classifyication accuracy demonstrates that abnormal brain structure may be a partial cause of violent behaviors. Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 Grant Disclosures The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: National Natural Science Foundation of China: 30800368, 81371500, 81571341, 81501637. The MOE (Ministry of Education in China) Project of Humanities and Social Sciences: 13YJC190033. National Natural Science Foundation of China: 30800368, 81371500, 81571341, 81501637. The MOE (Ministry of Education in China) Project of Humanities and Social Sciences: 13YJC190033. CONCLUSIONS We found a reduced GMV in five different brain regions in male adolescent offenders compared to that in male adolescent HCS. Moreover, our analyses verified the validity and practicality of using structural neuroimaging analyses to distinguish violent adolescent offenders and non-violent adolescents in males. Specifically, VBM technique is helpful for characterizing violent male adolescents. In addition, the findings in this study suggest that reduced volume in the frontal-temporal-parietal-subcortical circuit may be closely associated with violent behaviors in male adolescents, and thus could represent an important potential biomarker for detecting violent behavioral tendencies in male adolescents. Limitations There are several limitations in this study. (1) This study was conducted solely in male adolescents. Thus, our findings cannot be generalized to females, adults, and children Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 9/15 younger than 14 years old. (2) In our study setting, we only have two classes: adolescents with violent behaviors versus adolescents without violent behaviors. That is to say, different types of violent behaviors are treated as the same class, i.e., violent behavior. As a result, only classification experiment analysis was conducted. Nevertheless, it is feasible to quantify different types of violent behaviors by assigning different scores according to the severity. Further correlation analysis based on the scores may help uncover more findings. (3) The study is based on a small cohort of 59 participants. Additional experiments on other independent samples/cohort can help further validate our findings. However, as mentioned in our introduction, little work has been done to investigate the association of abnormal brain with violence tendency in healthy male adolescents. Therefore, at the moment we are not able to find an independent cohort to validate the findings. (4) Several other factors, e.g., stress and personality traits, are also related to violence tendency. However, the information of these personality traits were not available in the current cohort, which limited the conclusion of our work. Funding This study was supported by National Natural Science Foundation of China (NO. 30800368, 81371500, 81571341 and 81501637), and the MOE (Ministry of Education in China) Project of Humanities and Social Sciences (Project No. 13YJC190033). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.7349#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.7349#supplemental-information. Author Contributions Author Contributions • Ying-Dong Zhang performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft. • Jian-Song Zhou and Feng-Mei Lu authored or reviewed drafts of the paper, approved the final draft. • Xiao-Ping Wang conceived and designed the experiments, contributed reagents/materi- als/analysis tools, authored or reviewed drafts of the paper, approved the final draft. Human Ethics Human Ethics The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): All study procedures were approved by Institutional Review Board of the second Xiangya Hospital, Central South University. All study procedures were approved by Institutional Review Board of the second Xiangya Hospital, Central South University. Data Availability The following information was supplied regarding data availability: The following information was supplied regarding data availability: The raw data is available as a Supplemental File. The raw data is available as a Supplemental File. Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 Competing Interests The authors declare there are no competing interests. 10/15 Zhang et al. (2019), PeerJ, DOI 10.7717/peerj.7349 REFERENCES Barkataki I, Kumari V, Das M, Taylor P, Sharma T. 2006. Volumetric structural brain abnormalities in men with schizophrenia or antisocial personality disorder. Behavioural Brain Research 169:239–247 DOI 10.1016/j.bbr.2006.01.009. Barkataki I, Kumari V, Das M, Taylor P, Sharma T. 2006. Volumetric structural brain abnormalities in men with schizophrenia or antisocial personality disorder. Behavioural Brain Research 169:239–247 DOI 10.1016/j.bbr.2006.01.009. 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https://openalex.org/W2915009601	https://www.scielo.br/j/brag/a/nPcmrkpLJfvY73FmHNt3MXp/?lang=en&format=pdf	English	null	Agronomic performance and sweet corn quality as a function of inoculant doses (Azospirillum brasilense) and nitrogen fertilization management in summer harvest	Bragantia	2,019	cc-by	9,782	Agronomic performance and sweet corn quality as a function of inoculant doses (Azospirillum brasilense) and nitrogen fertilization management in summer harvest Agronomic performance and sweet corn quality as a function of inoculant doses (Azospirillum brasilense) and nitrogen fertilization management in summer harvest Alberto Yuji Numoto1, Pedro Soares Vidigal Filho1, Carlos Alberto Scapim1, Antônio Augusto Nogueira Franco2, Alex Henrique Tiene Ortiz1, Odair José Marques3, Murilo Fuentes Pelloso1 1.Universidade Estadual de Maringá - Departamento de Agronomia - Maringá (PR), Brazil. 2.Instituto Federal do Pará - Departamento de Agronomia - Paragominas (PA), Brazil. 3.Universidade Federal de Uberlândia - Departamento de Agronomia - Monte Carmelo (MG), Brazil. ABSTRACT: The inoculation of sweet corn seeds with Azospirillum brasilense in association to nitrogen fertilizer may be an agronomic alternative for increasing the crop yield and net income of plant growers. Therefore, the aim of this study was to investigate the effect of the different doses of inoculant (Azospirillum brasilense) associated to the nitrogen fertilization management on the phenotypic traits of one sweet corn hybrid in summer growing periods, under supplemental irrigation, in the Northwestern Paraná state, Brazil. The experiment followed the complete randomized block design with four replications. The treatments were: i) five inoculant doses (0.0, 50, 100, 150 and 200 mL∙ha–1) containing Azospirillum brasilense; ii) two N doses (0.0 and ABSTRACT: The inoculation of sweet corn seeds with Azospirillum brasilense in association to nitrogen fertilizer may be an agronomic alternative for increasing the crop yield and net income of plant growers. Therefore, the aim of this study was to investigate the effect of the different doses of inoculant (Azospirillum brasilense) associated to the nitrogen fertilization management on the phenotypic traits of one sweet corn hybrid in summer growing periods, under supplemental irrigation, in the Northwestern Paraná state, Brazil. The experiment followed the complete randomized block design with four replications. The treatments were: i) five inoculant doses (0.0, 50, 100, 150 and 200 mL∙ha–1) containing Azospirillum brasilense; ii) two N doses (0.0 and 30.0 kg∙ha–1) applied at sowing time; and iii) two topdressing doses of N (0.0 and 110.0 kg∙ha–1) applied at the V4 stage. The sweet corn hybrid RB 6324 was evaluated in 2012/2013, 2013/2014 and 2014/2015. The traits plant height (ranged from 2.11 to 2.26 m), leaf area index (3.33 to 4.32), crop yield (7.21 to 10.43 Mg.ha–1), and the sugar kernel contents (38.46 to 43.31%) and protein (12 to 12.81%) were positively influenced by the seed inoculation with A. brasilense, and the nitrogen fertilizer increased all the traits except the kernel total sugars. https://doi.org/10.1590/1678-4499.2018044 https://doi.org/10.1590/1678-4499.2018044 A. Y. Numoto et al. BASIC AREAS - Article Bragantia, Campinas, v. 78, n. 1, p.26-37, 2019 INTRODUCTION The method stimulates the seed germination and accelerates the seedling growth under field conditions (Cassán et al. 2009). Furthermore, the A. brasilense can increase tolerance of sweet corn to water deficits and increase the biomass production as reported by Rodríguez-Salazar et al. (2009). INTRODUCTION 2006), volatilization (Carvalho et al. 2006), denitrification and immobilization by microorganisms (Silva et al. 2006) justify the efforts for improving the crop management. We know that N is absorbed in great quantities by sweet corn plants (Silva et al. 2005), thereby affecting In Brazil, sweet corn plantations are stretching from Rio Grande do Sul to Goiás through São Paulo and Minas Gerais, where Goiás has the largest hectarage. Currently, the national production has supplied the industry of processed food (Barbieri et al. 2005). In contrast with the common maize, sweet corn has been enlarging in the national market because of its higher net return to plant growers (Canianto et al. 2007). The highest added value is the natural sweet flavor from the kernels because of the expressive quantity of sugar stored into the seed endosperm (Tracy 2010). This characteristic in the super sweet group is the effect from the mutant recessive gene Shrunken-2 (Sh2) that impedes the conversion of sugar into starch. Sweet corn is also considered a vegetable (Tracy 2001) for consuming in natura or canned (Luz et al. 2014). Other crop possibilities are the production of baby corn (Santos et al. 2014) or silage (Idikut et al. 2009). Currently, studies related to N management involving diazotrophic bacteria, specifically from the genus Azospirillum and sweet corn plants are scarce, and scientific studies at regional level for making correct recommendations to plant growers have been necessary. Therefore, the purpose of the current experiment was to evaluate the effect of the different doses of inoculant (Azospirillum brasilense) associated to the nitrogen fertilization management on the phenotypic traits of one sweet corn hybrid in summer growing periods applying supplementary irrigation. Despite many technologies and technical information available in the maize literature, little attention has been paid to the management of sweet corn under cropping systems. This fact may result in improper management of nitrogen fertilizers (Okumura et al. 2014), thereby reducing the potential of crop production. Otherwise, the application of Azospirillum brasilense has been useful for promoting plant growth (Piccinin et al. 2015; Spolaor et al. 2016). Many studies indicate the seed inoculation with A. brasilense in conjunction with nitrogen fertilizer for improving the vegetative traits of sweet corn, thereby increasing the crop yield (Hungria et al. 2010; Braccini et al. 2012; Brum et al. 2016). Agronomic performance and sweet corn quality as a function of inoculant doses (Azospirillum brasilense) and nitrogen fertilization management in summer harvest The dose of inoculant that provided the best agronomic result was 100 mL∙ha–1 in conjunction with the application of N either at sowing or topdressing. Key words: Zea mays, shrunken-2, diazotrophic bacteria, ear yield. Corresponding author: numotoay@gmail.com Received: Feb. 5, 2018 – Accepted: Mar. 26, 2018 Corresponding author: numotoay@gmail.com Received: Feb. 5, 2018 – Accepted: Mar. 26, 2018 26 26 26 Bragantia, Campinas, v. 78, n. 1, p.26-37, 2019 Agronomic responses from sweet corn INTRODUCTION along growing periods (Machado et al. 1998). Thus, seed inoculation with diazotrophic bacteria can reduce these costs, increasing the sweet corn yield as verified with soybean crops (Alves et al. 2006) and common maize that had increases of 29% (Ferreira et al. 2013), 11% (Piccinin et al. 2015) and 4.3% (Brum et al. 2016) when the A. brasilense was applied to the seeds. In Brazil, sweet corn plantations are stretching from Rio Grande do Sul to Goiás through São Paulo and Minas Gerais, where Goiás has the largest hectarage. Currently, the national production has supplied the industry of processed food (Barbieri et al. 2005). In contrast with the common maize, sweet corn has been enlarging in the national market because of its higher net return to plant growers (Canianto et al. 2007). The highest added value is the natural sweet flavor from the kernels because of the expressive quantity of sugar stored into the seed endosperm (Tracy 2010). This characteristic in the super sweet group is the effect from the mutant recessive gene Shrunken-2 (Sh2) that impedes the conversion of sugar into starch. Sweet corn is also considered a vegetable (Tracy 2001) for consuming in natura or canned (Luz et al. 2014). Other crop possibilities are the production of baby corn (Santos et al. 2014) or silage (Idikut et al. 2009). Despite many technologies and technical information available in the maize literature, little attention has been paid to the management of sweet corn under cropping systems. This fact may result in improper management of nitrogen fertilizers (Okumura et al. 2014), thereby reducing the potential of crop production. Otherwise, the application of Azospirillum brasilense has been useful for promoting plant growth (Piccinin et al. 2015; Spolaor et al. 2016). Many studies indicate the seed inoculation with A. brasilense in conjunction with nitrogen fertilizer for improving the vegetative traits of sweet corn, thereby increasing the crop yield (Hungria et al. 2010; Braccini et al. 2012; Brum et al. 2016). The method stimulates the seed germination and accelerates the seedling growth under field conditions (Cassán et al. 2009). Furthermore, the A. brasilense can increase tolerance of sweet corn to water deficits and increase the biomass production as reported by Rodríguez-Salazar et al. (2009). Nitrogen use efficiency (NUE) in cropping systems has been affected by the levels of soil moisture, organic matter, texture and fertility. Nitrogen losses by lixiviation (Aita et al. MATERIAL AND METHODS Rainfall Maximum temperature Minimum temperature 2012/2013 2012/2013 2012/2013 2013/2014 2013/2014 2013/2014 2014/2015 2014/2015 2014/2015 Days aer sowing Temperature (oC) Rainfall (mm) 0 0 5 10 15 20 25 30 35 40 7 14 21 28 35 42 49 56 63 70 77 84 91 0 20 40 60 80 100 120 Figure 1. Rainfall, maximum and minimum temperatures during the summer growing seasons 2012/2013, 2013/2014 and 2014/2015, in Maringá, Northwestern Paraná, Brazil. Azospirillum brasilense (strains AbV5 and AbV6) with minimum 2 × 108 colony forming unit (CFU) applied to the seeds; two N doses applied at the sowing time (0.0 and 30.0 kg∙ha–1); two topdressing doses of N applied at the V4 stage (0.0 and 110.0 kg∙ha–1) (Ritchie et al. 1993), to fertilize the soil for growing the single hybrid RB 6324 from the super sweet group. Each plot had fi ve rows with 6 m in length and 0.9 m apart where the total area had 27.0 m2 and the useful area had 13.5 m2. at 0.24 L∙ha–1 both amended with 1.0 L∙ha–1 of methylated soybean oil as adjuvant before the V4 stage. The insects Dichelops melacanthus, Agrotis ípsilon and Elasmopalpus lignosellus were controlled by seed treatment with imidacloprid + thiodicarb at 0.3 L∙ha–1. Th e fall armyworm (Spodoptera frugiperda) and the corn earworm (Helicoverpa zea) were controlled by methomyl at 0.5 L∙ha–1 and lufenuron at 0.3 L∙ha–1 when the insects reached the threshold level for sweet corn crops tolerance (Gallo et al. 2002).l At full fl owering (VT) (Ritchie et al. 1993) we evaluated the vegetative traits plant height and leaf area index. Plant height (PH) was taken by the stem length (m) from the ground up to the base of the panicle by choosing ten randomly plants from each plot (Lana et al. 2009). Th e leaf area index (LAI) was fi rst calculated by the expression LA = C × L × 0.74, where C and L are the length and width taken from leaves with green area larger than 50% by choosing fi ve random plants in each plot. Next, the leaf area index was calculated by the equation LAI = LA / (e1 × e2), where e1 and e2 are the space in meters between plants in the row and sowing rows, respectively (Francis et al. 1969). Aft er harvesting the ears at the phenotypic stage R3 (Ritchie et al. MATERIAL AND METHODS The experiments were conducted in the summer growing seasons 2012/2013, 2013/2014 and 2014/2015, on Fazenda Experimental de Iguatemi (23°20’48” SL, 52°04’17” WL, and altitude of 550 m), Universidade Estadual de Maringá, Northwestern Paraná, Brazil. The soil in the experimental area is classified as Distroferric Red Nitosol with clay texture (clay: 520 g∙kg–1; silt: 140 g∙kg–1; sand: 340 g∙kg–1) (Embrapa 2013). The chemical characteristics in uppermost soil layer from 0.0 to 0. 20 m, collected in three growing seasons were: in 2012/2013, pH (CaCl2) = 4.10, H+ + Al3+ = 3.97 cmolc∙dm–3, K+ = 0.29 cmolc∙dm–3, Ca2+ = 4.05 cmolc∙dm–3, Mg2+ = 1.50 cmolc∙dm–3, V = 59.53 %, P = 22.4 mg∙dm–3, C = 13.81 g∙kg–1; in 2013/2014, pH (CaCl2) = 4.68, H+ + Al3+ = 2.32 cmolc ∙dm–3, K+ = 0.44 cmolc∙dm–3, Ca2+ = 4.75 cmolc∙dm–3, Mg2+ = 1.95 cmolc∙dm–3, V = 77.82 %, P = 24.2 mg∙dm–3, C = 13.63 g∙kg–1; 2014/2015, pH (CaCl2) = 4.39, H+ + Al3+ = 2.42 cmolc∙dm–3, K+ = 0.58 cmolc∙dm–3, Ca2+ = 4.20 cmolc∙dm–3, Mg2+ = 2.95 cmolc∙dm–3, V = 78.30 %, P = 18.25 mg∙dm–3, C = 17.92 g∙kg–1. The regional climate is the Cfa based on the Köppen classification (IAPAR 1994). Data set of rainfall, maximum and minimum temperatures collected daily by Seed Science and Technology Laboratory are in Fig. 1 for the three summer growing seasons. Nitrogen use efficiency (NUE) in cropping systems has been affected by the levels of soil moisture, organic matter, texture and fertility. Nitrogen losses by lixiviation (Aita et al. 2006), volatilization (Carvalho et al. 2006), denitrification and immobilization by microorganisms (Silva et al. 2006) justify the efforts for improving the crop management. We know that N is absorbed in great quantities by sweet corn plants (Silva et al. 2005), thereby affecting significantly their physiological activities (Fernandes et al. 2005). Nitrogen application represents 75% of the costs with sweet corn fertilization, which means about 40% of the inputs The treatments follow the combination of five doses (0.0, 50, 100, 150 and 200 mL∙ha–1) of inoculant containing Bragantia, Campinas, v. 78, n. 1, p.26-37, 2019 27 A. Y. Numoto et al. Figure 1. Rainfall, maximum and minimum temperatures during the summer growing seasons 2012/2013, 2013/2014 and 2014/2015, in Maringá, Northwestern Paraná, Brazil. RESULTS AND DISCUSSION The joint analysis of variance indicated the seed inoculation as the significant factor (p < 0.05) affecting the LAI and the kernel PROT (Table 1). Table 1. Summary of the variance analysis for the phenotypic traits: plant height (PH), leaf area index (LAI), yield of marketable ear (YME), protein content (PROT) and total sugar (TS) in the kernels of sweet corn cultivate in three summer growing seasons. Table 1. Summary of the variance analysis for the phenotypic traits: plant height (PH), leaf area index (LAI), yield of marketable ear (YME), protein content (PROT) and total sugar (TS) in the kernels of sweet corn cultivate in three summer growing seasons. Table 1. Summary of the variance analysis for the phenotypic traits: plant height (PH), leaf area index (LAI), yield of marketable ear (YME), protein content (PROT) and total sugar (TS) in the kernels of sweet corn cultivate in three summer growing seasons. Table 1. Summary of the variance analysis for the phenotypic traits: plant height (PH), leaf area index (LAI), yield of marketable ear (YME), protein content (PROT) and total sugar (TS) in the kernels of sweet corn cultivate in three summer growing seasons. Source of variation DF1 Mean squares PH (m) LAI (m2∙m–2) YME (Mg∙ha–1) PROT (%) TS (%) Inoculant (I) 4 0.005ns 0.546 0.403ns 6.157* 8.174ns N Sowing (NS) 1 0.292* 2.765* 8.303* 17.822* 0.048ns N Topdressing (NT) 1 0.185* 8.543* 96.698* 29.260* 191.602* Growing Season (GS) 2 0.215* 6.916* 153.223* 4.174* 65.182ns NS × NT 1 0.053* 1.837* 3.626ns 0.265ns 17.281ns I × NS 4 0.004ns 0.271ns 1.290ns 0.441ns 9.395ns I × NT 4 0.010ns 0.090ns 0.109ns 0.332ns 16.068ns I × NS × NT 4 0.000ns 0.134ns 0.833ns 1.382ns 64.892ns I × GS 8 0.010ns 0.446* 2.145* 0.897ns 47.441ns NS × GS 2 0.042* 0.447ns 5.837* 0.964ns 38.196ns NT × GS 2 0.039* 1.072* 45.480* 0.368ns 72.210ns NS × NT × GS 2 0.011ns 0.135ns 0.381ns 0.082ns 43.877ns I × NS × GS 8 0.005ns 0.131ns 1.681ns 0.591ns 60.324ns I × NT × GS 8 0.011ns 0.174ns 1.278ns 0.804ns 70.565ns I × NS × NT × GS 8 0.005ns 0.218ns 1.143ns 1.353ns 36.497ns Blocks/GS 9 0.022 0.358 3.084 4.132 76.793 Residues 171 0.007 0.213 1.015 0.935 36.173 Averages 2.18 3.71 8.41 12.56 40.08 CVs (%)2 3.81 12.45 11.98 7.70 15.00 * Significant (p < 0.05), and ns not significant (p > 0.05) by F test. MATERIAL AND METHODS 1993), we assessed the following traits: yield of marketable ear (YME) (diameter larger than 3 cm and longer than 15 cm, free from insect damage and disease symptoms following the recommendations from Albuquerque et al. (2008) and Rocha et al. (2011)); protein content (PROT); and total sugars (TS) in the kernels (Brasil 2005). In October, 20 days before seed sowing, we controlled the weeds applying glyphosate (480 g∙L–1 a.i.) at 2.5 L∙ha–1. Th e crops before the sweet corn were black oats (Avena strigosa Schreb.) and fodder turnip (Raphanus sativus L.) in 2012/2013, sweet corn (Zea mays L.) in 2013/2014, and millet (Pennisetum glaucum L.) in 2014/2015. At the sowing time, we applied 50 kg∙ha–1 of P2O5 using triple superphosphate and 40 kg∙ha–1 of K2O using potassium chloride at 0.10 m into the soil for the treatments in the three agricultural years. Th e N dose of 30.0 kg∙ha–1 was applied only onto the plots. Nitrogen fertilization at 110.0 kg∙ha–1 using urea (46% N) as the topdressing N source was carried out at the V4 stage (Ritchie et al. 1993) and applied by handy workers (Okumura et al. 2014). Supplementary spray irrigation was provided during critical periods of the plant development as the fl owering stage and beginning of the grain fi lling. Th e experiments were carried out under no-till system for achieving the population of 55.500 plants∙ha–1 or 5.0 plants∙m–1. Aft er the crop establishment, the weeds were controlled by atrazine at 3.0 L∙ha–1 associated to the tembotrione herbicide First, the experimental data from every growing season were submitted to the Shapiro-Wilk to verify the error normality Bragantia, Campinas, v. 78, n. 1, p.26-37, 2019 28 Agronomic responses from sweet corn and the Levene tests (p > 0.05) for the homocedasticity of variances. Thus, data were submitted to individual analysis to verify whether the ratios between the residual mean squares were lower than 7:1 (Banzatto and Kronka 2006). Next, the data sets were submitted to joint analysis of variance with the pertinent partitions when necessary (Perecin and Cargnelutti Filho 2008; Barbin 2013). The effects of seed inoculation were evaluated by polynomial regression. The effects of the N doses applied at sowing and topdressing were studied by the F test (p < 0.05) and the effects of growing seasons were studied by the t test (LSD) (p < 0.05). MATERIAL AND METHODS All these statistical analyses were carried out using the Sisvar Software (Ferreira 2011). The N factor at sowing time was significant for all the traits except TS, similarly to the factor growing season (p > 0.05). The N factor at topdressing was non-significant (p > 0.05) only for the LAI (Table 1). The partition of the first order interaction was significant (p < 0.05) for PH, LAI and YME. Finally, the results of the joint analysis did not show significant effects from second or third order interaction for all the phenotypic traits (Table 1). The partitions of the first (I × GS) and second order (NS × NT × GS) interactions were significant (p < 0.05) for PH. We found that in the first growing season the seed inoculation in conjunction with the mean of N doses applied at sowing time and topdressing stage had positive influence on the vegetative development of the plants (Fig. 2a). In 2012/2013, the quadratic model had the best goodness of fit, and the maximum PH was 2.26 m, achieved after applying the inoculant at 116.90 mL∙ha–1. The explanation may rest on the fact that A. brasilense makes the N fixed from the atmosphere partially available to the plants. Furthermore, these diazotrophic bacteria can influence the growth and Bragantia, Campinas, v. 78, n. 1, p.26-37, 2019 * Significant (p < 0.05), and ns not significant (p > 0.05) by F test. 1DF = Degrees of freedom, and 2CV = Coefficient of variation. RESULTS AND DISCUSSION 1DF = Degrees of freedom, and 2CV = Coefficient of variation. 29 A. Y. Numoto et al. Figure 2. (a) Plant height and (b) leaf area index of sweet corn (RB 6324) based on the inoculant doses in the average of the nitrogen fertilizations in the summer growing seasons: 2012/2013, 2013/2014 and 2014/2015. Plant height (m) Leaf area index Doses of inoculant (mL·ha-1) Ŷ = - 0.000005X2 + 0.001169X + 2.194 Ŷ = Y = 2.17 m R2 = 0.93 _ _ Ŷ = Y = 2.14 m Ŷ = - 0.002805X + 3.7665 Ŷ = Y = 3.54 R2 = 0.99 R2 = 0.97 _ Ŷ = - 0.000025X2 + 0.003941X + 3.513 Doses of inoculant (mL·ha-1) 0 100 50 150 200 0 100 50 150 200 2.30 2.25 2.20 2.15 2.10 4.5 4.0 3.5 3.0 (a) (b) Leaf area index 0 100 50 150 200 4.5 4.0 3.5 3.0 (b) Plant height (m) f ( 1) 0 100 50 150 200 2.30 2.25 2.20 2.15 2.10 (a) Figure 2. (a) Plant height and (b) leaf area index of sweet corn (RB 6324) based on the inoculant doses in the average of the nitrogen fertilizations in the summer growing seasons: 2012/2013, 2013/2014 and 2014/2015. development of the sweet corn roots through the synthesis of plant hormones such as the auxins (indol-3-acetic acid and indol-3-butiric acid), gibberellins (GA1, GA3, GA9, GA19 and GA20) and cytokinin (Cassán et al. 2014) that justify greater absorption of mineral nutrients and water from the soil. Probably, Avena strigosa Schreb (with high C/N ratio in the straw) and Raphanus sativus L. (with low C/N ratio) that preceded the sweet corn have collaborated to make the N from other mineral nutrients signifi cantly available during the sweet corn sub-stages. Th is possibility may explain the detrimental responses from last two growing seasons 2013/2014 with Ȳ = 2.17 m and 2014/2015 with Ȳ = 2.14 m. Furthermore, high temperatures (> 30 °C) observed in the vegetative period probably favored the physiological plant mechanisms. 0.028 for each 10 mL of inoculant applied to the seeds. In 2014/2015, the best fi t was achieved with the quadratic model from which the maximum LAI was estimated at 3.67 for the dose 78.82 mL∙ha–1. In 2013/2014, no signifi cant eff ect was observed from the regression model (Ȳ = 3.54). Marini et al. Bragantia, Campinas, v. 78, n. 1, p.26-37, 2019 RESULTS AND DISCUSSION (2015) also reported signifi cant responses (p < 0.05) in the leaf area of hybrids of common sweet corn treated with A. brasilense and the increase was 11.0 % higher than estimates from non-inoculated plants.h Th e crops Avena strigosa Schreb and Raphanus sativus L. cultivated before the sweet corn in 2012/2013, as verifi ed in the plant height characteristic, probably made large quantities of N (mainly) available through the mineralization process during the vegetative stages (Fontoura and Bayer 2009). This association with the N biologically fixed by A. brasilense justifi es the better leaf area in the growing seasons 2013/2014 and 2014/2015. Plants well supplied with N have better leaf areas and root systems because this nutrient participates directly in the cell division and expansion along the photosynthesis process (Eft himiadou et al. 2009). In plants, the N available either through nitrogen fertilizer or associated to biological fi xation tends to increase the synthesis of hormones. Th is allows the cell division and expansion in the leaves based on leaf lengthening and expansion rate (Sivasankar et al. 1993; Taiz and Zeiger 2014). Kappes et al. (2013) reported higher plant height and ear insertion when sweet corn seeds were inoculated with A. brasilense (strains AbV5 and AbV6). Th ey based on the averages of the topdressing N doses and the urea applied onto the leaves. Th ese authors suggested that their results were the responses from growth substances from the bacteria. In contrast, Lana et al. (2012) and Piccinin et al. (2015) did not fi nd signifi cant increases in plant height of common corn when they inoculated the seeds with the A. brasilense. Th is response could be attributed to the genetic characteristics of the hybrids assessed in their experiments. Th e LAI of the sweet corn plants varied with inoculant doses and growing seasons (Fig. 2b), and the responses were positive to the nitrogen fertilizer. In 2012/2013, the responses were linear. Th e increase estimated by the angular coeﬃ cient was No matter the growing season, non-signifi cant diff erences were found when we did not inoculate the seeds, but the plants from 2012/2013 were taller than plants from the treatment with seed inoculation (Table 2). Th ese responses are likely associated to environmental factors that have Bragantia, Campinas, v. 78, n. 1, p.26-37, 2019 30 Agronomic responses from sweet corn Table 2. RESULTS AND DISCUSSION Plant height (m) in relation to the N doses applied at sowing and topdressing, in three summer growing seasons. N at sowing1 N at topdressing 2012/2013 2013/2014 2014/2015 Abs. Pres. Abs. Pres. Abs. Pres. Abs. 2.22Aa 2.23Aa 2.09Bb 2.18Aa 2.00Bb 2.15Ba Pres. 2.25Aa 2.26Aa 2.19Aa 2.21Aa 2.17Aa 2.22Aa Growing seasons2 NS NT NS NT NS NT NS NT Abs. Abs. Abs. Pres. Pres. Abs. Pres. Pres. 2012/2013 2.22a 2.23a 2.25a 2.26a 2013/2014 2.09b 2.19ab 2.19b 2.22a 2014/2015 2.00c 2.15b 2.17b 2.21a Means followed by distinct letters in the column (upper case) and in the line (lower case) are different at a 5% probability level by 1F test and by 2t-test (LSD), LSD = 0.052 m. NS = N applied at sowing and NT = N applied at topdressing. Abs. = Absence and Pres. = Presence. 2. Plant height (m) in relation to the N doses applied at sowing and topdressing, in three summer growing seasons Means followed by distinct letters in the column (upper case) and in the line (lower case) are different at a 5% probability level by 1F test and by 2t-test (LSD), LSD = 0.052 m. NS = N applied at sowing and NT = N applied at topdressing. Abs. = Absence and Pres. = Presence. from the combination no application of N at sowing with the topdressing application did not differ (p > 0.05) from 2013/2014. Finally, when the nitrogen fertilizer was applied at both times, no difference for growing season was found (p > 0.05). Piccinin et al. (2015) also found significant increases with N doses applied at topdressing in two growing seasons, in Northwestern Paraná state, Brazil. direct influence on the performance of the diazotrophic bacteria during the fixation of the atmospheric N. These environmental factors are high temperature and high soil moisture (Bashan et al. 2004; Malhotra and Srivastava 2009) usually found in subtropical and tropical regions. Concerning the LAI, the responses from the growing season 2012/2013 was better than the responses from 2014/2015 (p < 0.05) at all the doses of inoculant; in 2013/2014 the LAI estimates under no inoculation and at 50 mL∙ha–1 did not differ from the growing season 2012/2013 (p > 0.05). The A. brasilense provided better leaf area development and likely a higher number of leaves photosynthetically active per plant (Müller et al. 2016) taking into account the average of the N doses applied at sowing and topdressing. RESULTS AND DISCUSSION The enzymes associated to photosynthesis (phosphoenolpyruvate carboxylase and ribulose 1.5-biphosphate carboxylase/oxidase) generally increase their activities in plants inoculated with A. brasilense (Stancheva et al. 1992). Nitrogen fertilizer significantly increased the LAI regardless the application period. N application in both periods did not differ from just one application (p > 0.05) either at sowing or topdressing (Table 3) based on the average from the growing seasons. Veloso et al. (2009) found positive LAI responses when they increased the N doses at topdressing application in the R5 stage of the common maize growing in marsh drained soil. We highlighted LAI dependency on crop development stages as reported by França et al. (2011) when the maximum LAI (4.41) was observed at the R1 stage with the highest N available. The LAI decreased after this period because of the leaf senescence.hi Table 2 has the results from the partition NS × NT × GS. In 2013/2014 and 2014/2015 we find significant differences when the N was applied only at sowing time. The average increase in 2013/2014 was 0.10 m (4.8%), and 0.17 m (8.5%) in 2014/2015. The combination of the N doses applied at sowing (30.0 kg∙ha–1) and topdressing (110.0 kg∙ha–1) were different and the estimates were higher than the dose of 110.0 kg∙ha–1 applied only one time when the increase was only 0.07 m. The topdressing nitrogen fertilizer significantly increased the PH when N was not applied at sowing time in 2013/2014 (0.09 m) and 2014/2015 (0.15 m). Gomes et al. (2007) also found linear increase in the plant height of common corn with the estimate 2.22 m at 150 kg∙ha–1. In 2012/2013, the plants were taller (Table 2), but the estimates The partition of the first order interaction (I × GS) for YME indicated different performances based on the linear (2012/2013) and quadratic model (2014/2015), in the mean of the N doses applied at sowing and topdressing (Fig. 3). Table 3. Leaf area index in relation to the N doses applied at sowing and topdressing. Table 3. Leaf area index in relation to the N doses applied at sowing and topdressing. N at sowing N at topdressing Absence Presence Absence 3.32Bb 3.88Aa Presence 3.71Ab 3.92Aa Means followed by distinct letter in the line (lower case) and in the column (upper case) are different (p < 0.05) by F test. RESULTS AND DISCUSSION Th e YME in 2014/2015 was higher than in 2012/2013 and 2013/2014 (p < 0.05) in all the combinations (NS + NT), except the fi rst (with no N application). Okumura et al. (2014) also found positive results from sweet corn YME with the increased doses of topdressing N. The highest crop productivity of 10.41 Mg∙ha–1 yields marketable ears at the N dose of 110.84 kg∙ha–1, in the Northwestern Paraná state, Brazil. Economically, based on the quote from the green sweet corn (US$0.32 = R$1.15∙kg–1 green ears) in the wholesale national market (CEASA/PR; Curitiba County) in April 20, 2017, the price of inoculant, and the hybrid seeds of the RB 6324, the gain with the seed inoculation in the 2014/2015 The joint analysis of variance for TS from sweet corn kernels had significant principal effect (p < 0.05) for the N factor topdressing (Table 1). Although the factors inoculant × growing seasons (p > 0.05) were independent after the partition, we found significant regression model (Fig. 4a). In this case, the first order interaction was chosen by the principal factor (N at topdressing). In 2014/2015, the quadratic model was the best (Fig. 4a) and the maximum TS in the kernels of the RB 6324 hybrid was 42.23% estimated after inoculating 106.38 mL∙ha–1. Thus, the sugar increase in the endosperm was 3.94% higher than the control (no inoculation). However, the increase in TS from the dose 200 mL∙ha–1 may be related to decreases in photo- assimilates in the sweet corn plants. This happens when the carbohydrates reserves are used to supply the energy demand to maintain the association with the diazotrophic bacteria (Schubert and Evans 1976; Taiz and Zeiger 2014). Expressive sugar accumulation in the kernels is blocked by the sugar conversion into starch in the endosperm, specifically in the super sweet genotype group (sh2) (Tracy 2010). The small presence of the ADP-glucose pyrophosphorylase enzyme in the endosperm catalyzes the reaction between glycose-1-phosphate and adenosine Figure 3. Yield of marketable ear of the RB 6324 sweet corn hybrid in relation to the inoculant doses in the summer growing seasons 2012/2013, 2013/2014 and 2014/2015. RESULTS AND DISCUSSION Means followed by distinct letter in the line (lower case) and in the column (upper case) are different (p < 0.05) by F test. 31 Bragantia, Campinas, v. 78, n. 1, p.26-37, 2019 A. Y. Numoto et al. In 2013/2014, the regression models were non-signifi cant (p > 0.05). was about R$920.00∙ha–1 for the maximum crop yield of 10.38 Mg∙ha–1 based on the model Ŷ = –0.000075X2 + 0.014922X + 9.636. Th erefore, cropping sweet corn on small farms is more profi table than common corn (Canianto et al. 2007) because of the high unitary price of the ear (Camilo et al. 2015). The increase in the YME estimated by the angular coeﬃ cient was approximately 39.04 kg∙ha–1 for each 10.0 mL of inoculant. We found that the maximum yield estimated by the model was 10.38 Mg∙ha–1 for 99.48 mL∙ha–1, and this increase was 740 kg∙ha–1 higher than the control. We highlight that the commercial ears should have the length higher than 14.0 cm, diameter larger than 3.0 cm, and free from insect damages and diseases symptoms (Albuquerque et al. 2008; Rocha et al. 2011). High production of commercial ears in the fi eld is also desirable because they will be commercialized (Okumura et al. 2014). We inferred that the biological activity of A. brasilense in conjunction with the nitrogen fertilizer did not only result in gain of kernel mass but improved the chemical/physical quality of the ears. Th e diff erent YME responses from growing seasons (Fig. 3) may be associated to weather and environmental eff ects. Furthermore, yield is also infl uenced by participation and supply of carbohydrates and organic matter acids produced by plants to attend the requirements from the diazotrophic bacteria (Hartman and Baldani 2006) and carbohydrates accumulation into the kernels (Magalhães and Jones 1990). Regardless the dose of the inoculant, the growing season 2014/2015 was better than 2012/2013 and 2013/2014 (p < 0.05). Th is result suggests that both the climatic conditions (temperature and rainfall) (Fig. 1) and soil fertility favored the agronomic performance of the sweet corn hybrid in the last growing season. Despite the moment we applied the N, the fertilizer increased the YME, and the higher increment was verifi ed aft er N applications either at seed sowing or topdressing (Table 4). RESULTS AND DISCUSSION (a) Total sugars in kernels based on inoculant doses and summer growing seasons; (b) protein contents in the grain of sweet corn hybrid RB 6324. Total sugars (%) Protein (%) Doses of inoculant (mL·ha-1) Ŷ = - 0.000352X2 + 0.074469X + 38.289 Ŷ = Y = 38.86% _ _ Ŷ = Y = 40.74% R2 = 0.77 R2 = 0.64 Ŷ = - 0.000030X2 + 0.009500X + 12.054 Doses of inoculant (mL·ha-1) 0 100 50 150 200 0 100 50 150 200 44 42 40 38 36 13.0 12.5 12.0 11.5 (a) (b) Means followed by distinct lett er in the column (upper case) and in the line (lower case) are diff erent at the 5 % probability level by 1F-test and by 2t-test (LSD), LSD = 0.225 Mg∙ha–1. NS = N applied at sowing and NT = N applied in topdressing. Abs. = Absence and Pres. = Presence. Figure 4. (a) Total sugars in kernels based on inoculant doses and summer growing seasons; (b) protein contents in the grain of sweet corn hybrid RB 6324. Total sugars (%) Protein (%) Doses of inoculant (mL·ha-1) Ŷ = - 0.000352X2 + 0.074469X + 38.289 Ŷ = Y = 38.86% _ _ Ŷ = Y = 40.74% R2 = 0.77 R2 = 0.64 Ŷ = - 0.000030X2 + 0.009500X + 12.054 Doses of inoculant (mL·ha-1) 0 100 50 150 200 0 100 50 150 200 44 42 40 38 36 13.0 12.5 12.0 11.5 (a) (b) Total sugars (%) Doses of inoculant (mL·ha-1) 0 100 50 150 200 44 42 40 38 36 (a) Protein (%) Doses of inoculant (mL·ha-1) 0 100 50 150 200 13.0 12.5 12.0 11.5 (b) Figure 4. (a) Total sugars in kernels based on inoculant doses and summer growing seasons; (b) protein contents in the grain of sweet corn hybrid RB 6324 in kernels based on inoculant doses and summer growing seasons; (b) protein contents in the grain of sweet corn Figure 4. (a) Total sugars in kernels based on inoculant doses and summer growing seasons; (b) protein contents in the grain of sweet corn hybrid RB 6324. triphosphate and drastically reduces the starch synthesis (Tracy 2001; Souza et al. 2013). marketable ear (Uribelarrea et al. 2004; Khodarahmpour 2011). Th e topdressed N dose of 110.0 kg∙ha–1 reduced the TS in the kernels by –1.79% as we reported in Table 5. RESULTS AND DISCUSSION Ŷ = 0.003904X + 7.279 Ŷ = Y = 7.55 Mg ha-1 R2 = 0.74 R2 = 0.94 2012/2013 2013/2014 2014/2015 _ Ŷ = - 0.000075X2 + 0.014922X + 9.636 Doses of inoculant (mL·ha-1) Vield of marketable ear (Mg·ha-1) 0 100 50 150 200 11 10 9 8 7 6 5 Vield of marketable ear (Mg·ha-1) Figure 3. Yield of marketable ear of the RB 6324 sweet corn hybrid in relation to the inoculant doses in the summer growing seasons 2012/2013, 2013/2014 and 2014/2015. Bragantia, Campinas, v. 78, n. 1, p.26-37, 2019 32 Agronomic responses from sweet corn ble ear (Mg∙ha–1) in relation to the N doses applied at sowing and topdressing, in three summer growing seasons. 4. Yield of marketable ear (Mg∙ha–1) in relation to the N doses applied at sowing and topdressing, in three summer Table 4. Yield of marketable ear (Mg∙ha–1) in relation to the N doses applied at sowing and topdressing, in three summer growing seasons. N at sowing1 2012/2013 2013/2014 2014/2015 N at topdressing Abs. Pres. Abs. Pres. Abs. Pres. Absence 7.65Aa 7.80Aa 6.81Bb 8.04Aa 7.94Bb 11.11Ba Presence 7.67Aa 7.57Aa 7.46Aa 7.91Aa 9.12Ab 11.85Aa Growing seasons2 NS NT NS NT NS NT NS NT Abs. Abs. Abs. Pres. Pres. Abs. Pres. Pres. 2012/2013 7.65a 7.80b 7.67b 7.57b 2013/2014 6.81b 8.04b 7.46b 7.91b 2014/2015 7.94a 11.11a 9.12a 11.85a Means followed by distinct lett er in the column (upper case) and in the line (lower case) are diff erent at the 5 % probability level by 1F-test and by 2t-test (LSD), LSD = 0.225 Mg∙ha–1. NS = N applied at sowing and NT = N applied in topdressing. Abs. = Absence and Pres. = Presence. N at sowing1 2012/2013 2013/2014 2014/2015 N at topdressing Abs. Pres. Abs. Pres. Abs. Pres. Absence 7.65Aa 7.80Aa 6.81Bb 8.04Aa 7.94Bb 11.11Ba Presence 7.67Aa 7.57Aa 7.46Aa 7.91Aa 9.12Ab 11.85Aa Growing seasons2 NS NT NS NT NS NT NS NT Abs. Abs. Abs. Pres. Pres. Abs. Pres. Pres. 2012/2013 7.65a 7.80b 7.67b 7.57b 2013/2014 6.81b 8.04b 7.46b 7.91b 2014/2015 7.94a 11.11a 9.12a 11.85a Means followed by distinct lett er in the column (upper case) and in the line (lower case) are diff erent at the 5 % probability level by 1F-test and by 2t-test (LSD), LSD = 0.225 Mg∙ha–1. NS = N applied at sowing and NT = N applied in topdressing. Abs. = Absence and Pres. = Presence. Figure 4. ORCID IDs A. Y. Numoto https://orcid.org/0000-0002-5671-7882 P. S. Vidigal Filho https://orcid.org/0000-0003-3196-8631 C. A. Scapim http://orcid.org/0000-0002-7047-9606 A. A. N. Franco https://orcid.org/0000-0002-4174-8598 A. H. T. Ortiz https://orcid.org/0000-0003-0556-8503 O. J. Marques https://orcid.org/0000-0003-0946-6272 M. F. Pelloso https://orcid.org/0000-0001-5627-8886 A. Y. Numoto https://orcid.org/0000-0002-5671-7882 P. S. Vidigal Filho https://orcid.org/0000-0003-3196-8631 C. A. Scapim http://orcid.org/0000-0002-7047-9606 A. A. N. Franco https://orcid.org/0000-0002-4174-8598 A. H. T. Ortiz https://orcid.org/0000-0003-0556-8503 O. J. Marques https://orcid.org/0000-0003-0946-6272 M. F. Pelloso https://orcid.org/0000-0001-5627-8886 A. Y. Numoto https://orcid.org/0000-0002-5671-7882 RESULTS AND DISCUSSION Protein content (%) from the N doses applied at sowing time and topdressing, based on the mean dose of inoculant and growing seasons. N at sowing N at topdressing Absence Presence Absence 11.90Bb 12.67Ba Presence 12.52Ab 13.15Aa Means followed by distinct lett er in the line (lower case) and in the column (upper case) are diff erent (p < 0.05) by F test. 99.48 mL applied to the seeds in the summer growing seasons 2013/2014 and 2014/2015, respectively. Th e application of nitrogen fertilizer increased the vegetative growth traits, ear yield and kernel protein content. Th e dose of inoculant that provided the best agronomic result was 100 mL∙ha–1 in conjunction with the application of N either at sowing or topdressing. Table 5. Total sugar contents in sweet corn kernel in relation to the N doses applied at topdressing in summer growing seasons. Table 5. Total sugar contents in sweet corn kernel in relation to the N doses applied at topdressing in summer growing seasons. Table 6. Protein content (%) from the N doses applied at sowing time and topdressing, based on the mean dose of inoculant and growing seasons. CONCLUSION Inoculation of the hybrid sweet corn seeds RB 6324 with Azospirillum brasilense aff ected positively the vegetative growth traits, ear yield, protein, and total sugars contents in kernels. Th e increase in ear yield was 39.04 kg∙ha–1 for each 10.0 mL of inoculant and 740 kg∙ha–1 from the dose M. F. Pelloso https://orcid.org/0000-0001-5627-8886 ACKNOWLEDGEMENTS N at sowing N at topdressing Absence Presence Absence 11.90Bb 12.67Ba Presence 12.52Ab 13.15Aa Means followed by distinct lett er in the line (lower case) and in the column (upper case) are diff erent (p < 0.05) by F test. We thank the Conselho Nacional de Desenvolvimento Científi co e Tecnológico (CNPq/Brazil) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/ Brazil) for the Productivity Research and Doctor Scholarship, respectively, and the Fundação Araucária by fi nancial support to this research work. Means followed by distinct lett er in the line (lower case) and in the column (upper case) are diff erent (p < 0.05) by F test. (2014). Okumura et al. (2014) obtained signifi cant increases in PROT with increasing doses of N applied at topdressing in three summer growing seasons, in Northwestern Paraná, Brazil. Th e N in the sweet corn plant is important not only for the vegetative and reproductive traits, but also for kernel chemical composition (Tables 5 and 6). Th erefore, appropriate N management during the sweet corn cycle is fundamental to raise the nutritional quality of the kernel (Oikeh et al. 1998; Okumura et al. 2014). RESULTS AND DISCUSSION In plants, increase in N concentration results in decreases of carbohydrates in the system because much of the nutrient is used for synthesizing amino acid, protein, and other nitrogen metabolites (Singletary and Below 1989; Kusano et al. 2011). The PROT in the sweet corn kernels was described (p < 0.05) by the quadratic model for the inoculant main factor (Fig. 4b). Th us, the maximum PROT given by the model was 12.81% estimated aft er 158.33 mL∙ha–1 or 4.9% higher than the control (no inoculation). Th ese results are in agreement with reports from Naserirad et al. (2011) who found positive eff ects on the protein contents in kernels of three common maize genotypes inoculated with Azospirillum and Azotobacter. Although the joint analysis of variance showed interdependence between the N factors at sowing and topdressing, we verifi ed aft er partitioning NS × NT signifi cant eff ects on the PROT contents in the kernels. In this case we chose the fi rst order interaction (Table 6) because we found that the N applied at sowing and topdressing resulted in a signifi cant increase in the kernel PROT (Table 6). Th e greatest protein content was found when the N was applied at both times (13.15%), and also alone at topdressing (12.67%). Positive eff ects of nitrogen fertilizer on the kernel PROT were obtained by Bueno et al. (2009) and Okumura et al. Th e total sugars in the sweet corn grain aft er the seed inoculation with 100 mL∙ha–1 was 43.31% in 2014/2015, and higher than 4.61% (p < 0.05) in 2012/2013. Th e values from the current study are similar to reports from Okumura et al. (2014) (an average of 34.0%). In 2012/2013, the PROT in the kernels was higher (p < 0.05) than in the other growing seasons, but this result was negatively correlated with the 33 Bragantia, Campinas, v. 78, n. 1, p.26-37, 2019 A. Y. Numoto et al. Table 5. Total sugar contents in sweet corn kernel in relation to the N doses applied at topdressing in summer growing seasons. N at topdressing Total sugar contents (%) Absence 40.98a Presence 39.19b Means followed by distinct lett er in the column are diff erent at the 5% probability level by F test. Table 6. e comerciais de milho para produção de milho verde. Ciência e Agrotecnologia, 32, 768-775. Alves, B. J., Zotarelli, L., Fernandes, F. M., Heckler, J. C., Macedo, R. A. T., Boddey, R. M., Jantalia, C. P. and Urquiaga, S. (2006). Fixação biológica de nitrogênio e fertilizantes nitrogenados no balanço de nitrogênio em soja, milho e algodão. Pesquisa Agropecuária Brasileira, 41, 449-456. e comerciais de milho para produção de milho verde. Ciência e Agrotecnologia, 32, 768-775. REFERENCES Aita, C., Port, O. and Giacomini, S. J. (2006). Dinâmica do nitrogênio no solo e produção de fi tomassa por plantas de cobertura no outono/inverno com o uso de dejetos de suínos. 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https://openalex.org/W4303712350	https://ria.ua.pt/bitstream/10773/33759/1/Documento_Ana_Filipa_Ribeiro.pdf	English	null	Circulation and Transport Processes during an Extreme Freshwater Discharge Event at the Tagus Estuary	Journal of marine science and engineering	2,022	cc-by	23,740	Universidade de Aveiro Universidade de Aveiro Circulação e processos de transporte durante um evento extremo de descarga fluvial no Estuário do Tejo Circulation and transport processes during an extreme freshwater discharge event at the Tagus Estuary Ana Filipa Ferreira Ribeiro Universidade de Aveiro 2021 Universidade de Aveiro 2021 Ana Filipa Ferreira Ribeiro And other waves will come. Circulação e processos de transporte durante um evento extremo de descarga fluvial no Estuário do Tejo Circulation and transport processes during an extreme freshwater discharge event at the Tagus Estuary Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Ciências do Mar e da Atmosfera, realizada sob a orientação científica do Doutor Nuno Alexandre Firmino Vaz, Pro- fessor Auxiliar do Departamento de Física da Universidade de Aveiro, e da Doutora Magda Catarina Ferreira de Sousa, Investigadora Doutorada do Departamento de Física da Universidade de Aveiro. o júri / the jury presidente / president Prof. Doutor José Manuel Henriques Castanheira Professor Auxiliar do Departamento de Física da Universidade de Aveiro Prof. Doutor Nuno Alexandre Firmino Vaz Professor Auxiliar do Departamento de Física da Universidade de Aveiro Prof. Doutor Nuno Alexandre Firmino Vaz Professor Auxiliar do Departamento de Física da Universidade de Aveiro vogais / examiners committee Doutora Magda Catarina Ferreira de Sousa Investigadora Doutorada do Departamento de Física da Universidade de Aveiro Resumo Os modelos numéricos desenvolvidos ao longo dos últimos anos são ferramentas úteis na análise multidisciplinar dos aspetos estuarinos, nomeadamente de processos hidrodinâmicos e de transporte, induzidos por eventos de forçamentos específicos. De 20 de março a 11 de abril de 2013, a região centro de Portugal, que incluí o rio Tejo e as zonas costeiras próximas, esteve sob influência de ventos intensos de sul e elevado caudal fluvial (quase 9000 m3/s). Estes forçamentos atuaram na alteração da hidrodinâmica e transporte de calor e sal durante o período referido. Este e íodo foi sado co o efe ê cia a a ca acte i a a iações es ecificas e Os modelos numéricos desenvolvidos ao longo dos últimos anos são ferramentas úteis na análise multidisciplinar dos aspetos estuarinos, nomeadamente de processos hidrodinâmicos e de transporte, induzidos por eventos de forçamentos específicos. De 20 de março a 11 de abril de 2013, a região centro de Portugal, que incluí o rio Tejo e as zonas costeiras próximas, esteve sob influência de ventos intensos de sul e elevado caudal fluvial (quase 9000 m3/s). Estes forçamentos atuaram na alteração da hidrodinâmica e transporte de calor e sal durante o período referido. Este período foi usado como referência para caracterizar variações especificas e, além disso, nesta dissertação, foi estudado o impacto de potenciais alterações futuras na temperatura do ar e na subida do nível do mar em eventos extremos como o que ocorreu no inverno de 2013. A abordagem seguida foi o uso do modelo hidrodinâmico Delft3D. Para tal, seis cenários idealizados foram desenvolvidos e aplicados, considerando os novos resultados do relatório do CMIP6. Este período foi usado como referência para caracterizar variações especificas e, além disso, nesta dissertação, foi estudado o impacto de potenciais alterações futuras na temperatura do ar e na subida do nível do mar em eventos extremos como o que ocorreu no inverno de 2013. A abordagem seguida foi o uso do modelo hidrodinâmico Delft3D. Para tal, seis cenários idealizados foram desenvolvidos e aplicados, considerando os novos resultados do relatório do CMIP6. Antes do evento, o estuário do Tejo apresentava valores normais de temperatura da água (cerca de 16ºC), consistente com a época do ano. A zona de mistura do estuário apresentava condições de mistura na coluna de água (valores baixos de frequência de Brunt-Väisälä). Palavras Chave Delft3D, Estratificação, Caudal, CMIP6, Temperatura da Água, Subida do Nível do Mar. agradecimentos / acknowledgements Aos meus orientadores: Professor Nuno, pelas sugestões e pelo acompanhamento desde a minha licenciatura, e Doutora Magda pelo incentivo e toda a disponibilidade ao longo desta dissertação. A todos os professores que fizeram parte do meu percurso académico e que me deram as bases de conhecimento necessárias para o meu futuro. Aos colegas do Núcleo de Modelação Estuarina e Costeira - Ana, Américo, Carina, Humberto e João - por toda a ajuda nos obstáculos que surgiram e pela motivação diária, tornaram este caminho mais leve. Aos meus amigos e colegas de curso, por todo o companheirismo, apoio e por terem feito de Aveiro a minha casa, nos últimos anos. Ao Nuno, por estar sempre presente em todas as conquistas e, principalmente, pelo encorajamento nos momentos mais exigentes deste percurso. À minha família. Pai, Daniel e David, obrigada por todo o amor e por acreditarem nas minhas capacidades mais do que eu. Palavras Chave Abstract Numerical models developed over the past years are useful tools to analyze multi- disciplinary aspects of estuarine environments, namely hydrodynamic and transport processes, induced by specific forcing events. From March 20 to April 11 of 2013, the central region of Portugal, which includes the Tagus estuary and near coastal waters, was under the influence of intense southern winds and extreme freshwater discharge (almost 9000 m3/s). These forcings acted to change the hydrodynamics and heat and salt transport during the referred period. This period was used as a reference to characterize specific variations and, moreover, in this dissertation, the impact of potential future changes in air temperature and sea level rise (SLR) in extreme events like the occurred in winter of 2013 was studied. The followed approach was the use of the Delft3D hydrodynamic model. For that, six idealized scenarios were developed and applied, considering the new results of the CMIP6 report. Before the event, the Tagus estuary presents normal values of water temperature (about 16ºC), consistent with the year season. The mixing area of the estuary pre- sented well-mixed conditions (low Brunt-Väisälä frequency values). In the marine area of the estuary, some stratification was present, with a vertical gradient of 2 km−1 in this region. During the event, which was characterized by a large increase in the freshwater inflow, the estuary was filled with freshwater (with salinity values lower than usual), and the mixing region was advected toward the coast. This leads to the estuary water temperature being lower than the conditions previously to the freshwater event. Nine days later, on April 11, some event effects (as low salinity) remaining inside of the estuary, but some of the original features started to appear again (e.g., well-mixed conditions in the mixing region of the estuary). SLR has a more intense impact than the air temperature increase, in the salinity and Brunt-Väisälä frequency patterns. In opposition, the response of water temperature is directly related to the increase of the air temperature.i In the first 20 km from the Tagus river mouth, the three study variables will not change their values, due to the influence of the extreme freshwater discharge. From 20 km to the estuary mouth, the increase of air temperature and the SLR will increase salinity, water temperature, and water column mixing. Keywords Delft3D, Stratification, Flow, CMIP6, Water Temperature, Sea Level Rise. Resumo A região marinha do estuário apresentava alguma estratificação, com um gradiente vertical de 2 km−1 nesta região.l Durante o evento, que foi caracterizado pelo elevado aumento do fluxo de água doce, o estuário estava cheio de água doce (com valores de salinidade abaixo do habitual), e a região de mistura foi advectada em direção à costa. Isto levou a que a temperatura da água do estuário se tornasse mais baixa do que nas condições que antecederam o evento de descarga fluvial. Nove dias mais tarde, no dia 11 de abril, alguns efeitos do evento (como a baixa salinidade) permaneciam na região dentro do estuário, mas algumas características iniciais voltaram a aparecer (por exemplo, condições de mistura, na região de mistura do estuário). A subida do nível do mar tem impactos mais intensos do que o aumento da tem- peratura do ar, nos padrões de salinidade e de frequência de Brunt-Väisälä. Por outro lado, a resposta da temperatura da água está diretamente relacionada com o aumento da temperatura do ar. Nos primeiros 20 km desde a embocadura do rio Tejo, as três variáveis em estudo não mudarão os seus valores, devido à influência da descarga fluvial extrema. A partir dos 20 km até à embocadura do estuário, o aumento da temperatura do ar e a subida do nível do mar aumentarão a salinidade, temperatura da água e mistura da coluna de água. A embocadura do estuário e as regiões pouco profundas serão mais propensas a alterações do que as regiões a montante, em termos de salinidade, temperatura da água e estratificação da coluna de água. Conforme esperado, as alterações projetadas são maiores para o cenário SSP5-8.5 (cenário de emissões elevadas de gases com efeito de estufa (GEE)) e considera- velmente menores para o SSP1-2.6 (cenário de baixas emissões de GEE). A implementação do modelo desenvolvida nesta dissertação contribui para a com- preensão da hidrodinâmica do estuário do Tejo e a região costeira adjacente, assim como o impacto dos cenários de alterações futuras previstas na salinidade, tempe- ratura da água e estratificação, em condições de elevada descarga fluvial. Contents Contents i List of Figures iii List of Tables v List of Acronyms vii 1 Introduction 1 1.1 Motivation and Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2.1 Numerical Modelling at Tagus Estuary . . . . . . . . . . . . . . . . . . . . . . 3 1.2.2 Predicted Changes in Estuaries . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.3 Work Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2 Study Area 7 3 Data 11 3.1 Bathymetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.2 Tides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.3 Rivers Discharge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3.4 Hydrographic Variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3.5 Meteorological Conditions . . . . . . . . . . . . . . . Abstract The estuary mouth and the shallow regions will be more prone to changes than the upstream region, in terms of salinity, water temperature, and water column stratification. As expected, the projected changes are greater for the scenario SSP5-8.5 (high greenhouse gas (GHG) emissions scenario) and considerably smaller for the SSP1- As expected, the projected changes are greater for the scenario SSP5-8.5 (high greenhouse gas (GHG) emissions scenario) and considerably smaller for the SSP1- 2.6 (low GHG emissions scenario). The model implementation developed in this dissertation contributed to the un- derstanding of the hydrodynamics of the Tagus estuary and the coastal adjacent region, as well the impact of predicted future change scenarios on salinity, water temperature, and stratification, in conditions of high freshwater discharge. Contents . . . . . . . . . . . . . . . . . . 13 4 Numerical Model 15 4.1 Delft3D-FLOW Numerical Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 4.1.1 Grid Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 4.1.2 Numerical Model Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4.2 Model Implementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 4.3 Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Contents Contents 1 Introduction 1 1.1 Motivation and Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2.1 Numerical Modelling at Tagus Estuary . . . . . . . . . . . . . . . . . . . . . . 3 1.2.2 Predicted Changes in Estuaries . . . . . . . . . . . . . . . . . . . . . . . . . . 5 1.3 Work Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2 Study Area 7 3 Data 11 3.1 Bathymetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.2 Tides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.3 Rivers Discharge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4 Numerical Model 4 Numerical Model 15 4.1 Delft3D-FLOW Numerical Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 4.1.1 Grid Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 4.1.2 Numerical Model Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4.2 Model Implementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 4.3 Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 i 4.4 Model Scenarios . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 5 Results and Discussion 31 5.1 2013 Event Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 5.2 Predicted changes for the Tagus Estuary . . . . . . . . . . . . . . . . . . . . . . . . . 39 5.2.1 #Sc1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 5.2.2 #Sc2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of Figures 2.1 Study Area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.2 Water surface distribution at high tide areas. . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.1 Wind Patterns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 4.1 Numerical Grid and Bathymetry of Tagus estuary . . . . . . . . . . . . . . . . . . . . . . 17 4.2 Schematized horizontal numerical grid of Delft3D-FLOW . . . . . . . . . . . . . . . . . . 18 4.3 Schematized vertical numerical grid of Delft3D-FLOW . . . . . . . . . . . . . . . . . . . . 18 4.4 Sea Surface Elevation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 4.5 Historical Greenhouse Gas Emissions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 5.1 2013 March/April Event Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 5.2 Daily average salinity along the longitudinal section - #ScRef . . . . . . . . . . . . . . . 33 5.3 Daily average water temperature along the longitudinal section - #ScRef . . . . . . . . . 34 5.4 Daily average Brunt-Väisälä frequency along the longitudinal section - #ScRef . . . . . . 35 5.5 Daily Mean salinity for the Tagus Estuary - #ScRef . 4 Numerical Model 42 5.2.3 #Sc3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 5.2.4 #Sc4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 6 Conclusions 51 55 ii ii List of Figures . . . . . . . . . . . . . . . . . . . . 37 5.6 Daily Mean water temperature for the Tagus Estuary - #ScRef . . . . . . . . . . . . . . 38 5.7 Vertical patterns under an increase of the air temperature, in the longitudinal section . . 40 5.8 Horizontal patterns under an increase of the air temperature . . . . . . . . . . . . . . . . 41 5.9 Vertical patterns under a SLR, in the longitudinal section. . . . . . . . . . . . . . . . . . 43 5.10 Horizontal patterns under a SLR scenario . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 5.11 Vertical patterns under an increase of the air temperature and SLR (scenario #Sc3), in the longitudinal section. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 5.12 Horizontal patterns under an increase of water temperature and SLR (scenario #Sc3). . . 47 5.13 Vertical patterns under an increase of the air temperature and SLR (scenario #Sc4), in the longitudinal section. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 5.14 Horizontal patterns under an increase of water temperature and SLR (scenario #Sc4). . . 50 iii List of Tables 3.1 Location of tidal gauges used in the validation of the model. . . . . . . . . . . . . . . . . 12 4.1 Layer Thickness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4.2 Bottom friction coefficients - Manning Values. . . . . . . . . . . . . . . . . . . . . . . . . 21 4.3 Parameters and Specifications of model implementation . . . . . . . . . . . . . . . . . . . 22 4.4 Error values for tidal water levels - RMSE (m), its Error (%) and Skill . . . . . . . . . . 25 4.5 Model accuracy in reproducing the main tidal constituents (M2, S2, O1, K1) . . . . . . . 26 4.6 Error values for water temperature and salinity - RMSE and Skill, relative to the buoy measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.7 Idealized scenarios used in the model simulations . . . . . . . . . . . . . . . . . . . . . . . 29 v v 1.1 Motivation and Objectives The Tagus Estuary is a coastal plain estuary that is located in the central region of Portugal, near the metropolitan area of Lisbon. It is densely populated region subject to an enormous natural and human stress. It is situated in the Eastern North Atlantic system, one of the main coastal upwelling systems. In this region, northerly winds promote upwelling events which push the nutrient-rich Eastern Atlantic Central Water (ENACW) to the surface. It is responsible to holding high productivity and biodiversity, as the form of nurseries for fish species, sheltering for vessels and harbour industries (Rodrigues, 2015; Des, 2020). Due to their ecological and economic value, it is necessary to study the current behavior to forecast future conditions and possible effects due to climate change. Besides anthropogenic pressure in estuaries and coastal systems, a set of other forcings such as wind and freshwater discharge, can impact in the estuary (Vaz and Dias, 2014), turning these regions into vulnerable systems, and requesting frequent monitorization, to control the risks and damage associated. In the period from March 20 to April 11 of 2013, the region surrounding the Tagus river was under the influence of intense southern winds (Vaz et al., 2018). In the evening of March 27, the authority for civil protection of Santarém (Comando Distrital de Operações de Socorro (CDOS)) communicated that was under a ”expectable gradual rise of the Tagus river, in the next hours”, putting the region with the yellow warning. This alert was given due to high precipitation and discharges from Iberian dams, which increase the Tagus river flow (Diário de Notícias, 2013c). At 10 am on the March 28, it was already registered the submersion of parking lots, near Zêzere river (Tagus tributary), roads and agricultural fields. Furthermore, it was expected the flood of other places (Jornal de Notícias, 2013a). On the March 29, some villages were confined. The local firefighter companies had to distribute the supplies of essentials in the population. Two days later, at March 31, the transport started to be done by boat, due to rise of water level. In this day, 14 of 18 portuguese mainland districts were in risk situation due to the intense rain predictions and wind gusts, according to the portuguese meteorology agency (Instituto Português do Mar e da Atmosfera (IPMA)) (Diário de Notícias, 2013a,b). List of Acronyms 2DH Two-Dimensional Horizontal 2DV Two-Dimensional Vertical ADCP Acoustic Doppler Current Profiler APA Agência Portuguesa do Ambiente BoM Bureau of Meteorology CDOS Comando Distrital de Operações de Socorro CFL Courant-Friedrichs-Lewy number CMIP Coupled Model Intercomparison Project CMIP5 Fifth Coupled Model Intercomparison Project CMIP6 Sixth Coupled Model Intercomparison Project CNES Centre National d’Études Spatiales ECMWF European Centre for Medium-Range Weather Forecasts ENACW Eastern Atlantic Central Water GEBCO General Bathymetric Chart of the Oceans GHG Greenhouse Gas IH Instituto Hidrográfico IPCC Intergovernmental Panel on Climate Change IPMA Instituto Português do Mar e da Atmosfera JEEP-92 Joint European Estuarine Project LIDAR Light Detection and Ranging N Brunt-Väisälä frequency NASA National Aeronautics and Space Administration RMSE Root Mean Square Error SLR Sea Level Rise SNIRH Sistema Nacional de Informação de Recursos Hídricos SSE Sea Surface Elevation SSP Shared Socioeconomic Pathways SST Sea Surface Temperature WCRP World Climate Research Programme WGCM Working Group on Coupled Modeling vii 1 1 1 1.1 Motivation and Objectives Just after April 8 the emergency plan for floods in the Tagus basin named ”Plano Especial de Emergência para Cheias na Bacia do Tejo” and applied since 1 March 27 was disabled. Nonetheless, a considerable number of roads were inactive due to the floods (Jornal de Notícias, 2013c,b). According to de Pablo et al. (2019), during the event, the river flow reached 8711 m3/s, and the mean daily over three consecutive days attained 7500 m3/s. This has led an abrupt decrease of salinity values within the estuary. As said by Vaz et al. (2018), when the river inflow is high (higher than 1000 m3/s), the strong input of freshwater enhances estuarine ebb velocities, promoting the propagation of low salinity estuarine water toward the shelf. Also, the influence of intense southern winds induce a strong alongshore propagation of the Tagus plume and intensifying the poleward current (Vaz et al., 2018). In the huge scale of future change, a higher frequency of extreme events - such as intense precipitation, heat and cold waves and storms - is projected, turning the study of its behavior to improve the global understanding of coastal lagoons response to global change so critical (IPCC, 2013, 2021; Rodrigues et al., 2021). Climate change is also expected to have a significant environmental impact affecting primary production, and consequently, the economy and population. Sea Level Rise (SLR), water temperature changes, variations in circulation patterns and frequency, and severity of extreme events will impact the marine ecosystems (Des, 2020). The analysis of the possible vulnerabilities of these systems, in a scenario of change, allows establishing mitigation and adaptation procedures to obtain the correct management and protection of such vulnerable ecosystems. The results generated by numerical models are very useful to increase the knowledge of the predicted climate change impacts and to support decision making. Numerical models are one of the best tools to reproduce the tendency of change with enough accuracy and to provide data that would be rather difficult to obtain through surveys, such as future projections (Des, 2020). However, their authenticity depends on the appropriate selection of the numerical model, as well it physical processes. Also, space and time scales must be correctly parameterized (Ribeiro, 2015). 1.2 Literature Review The ocean is our planet’s largest ecosystem. It balances the climate, stores carbon, sustains enormous biodiversity, and supports human well-being through food, energy resources and recreational services (UNESCO, nd). On a smaller scale, the productive waters and the facility of navigation lead to human growth in estuarine zones. Nowadays, several of the principal cities are located near estuaries. Consequently, industry and commerce expansion can bring changes in estuarine natural balance due to bathymetric adjustments, to facilitate navigation. These bathymetric changes can lead to current velocity variations and, consequently, to changes in other variables concentration (e.g. suspended sediments, salinity and other). These changes can cause variations in ecological variables, which can be related to events of pollution or algae blooms (for example). These anthropogenic pressures in estuarine regions may contribute to an increase of harmful impacts, which can be add to those from climate change. Over the last decades, several ocean studies, particularly in coastal areas have been implemented, due to the high importance in terms of economic, ecological, and social aspects of the estuarine region. The Tagus estuary, one of the largest estuaries in Europe, has been studied since the 1980s, with a publication of modeling studies on water quality management, which combine algorithms for physical transport processes and biochemical reactions (Câmara et al., 1985; Vaz et al., 2020). The Tagus coastal hydrography is influenced by a complex interaction between the land-water-atmosphere compartments. As many other fundamental systems, estuaries and coastal areas are facing climate change effects that will modify the physical parameters in the ecosystems. In order to take the best decisions to the future management, it is necessary to improve knowledge about these areas (Rodrigues, 2015; Des, 2020). 1.1 Motivation and Objectives The main objective of this dissertation is to study the impact of possible future changes in the air temperature and SLR in extreme events like the occurred in winter of 2013, at Tagus Estuary. Vaz et al. (2018) studied the effects of this event, on the estuarine plume. This work aims to study the impacts on inner estuary stratification and circulation patterns, as a way to complement the previous research done. To achieve this goal, it is necessary the implementation of a three-dimensional baroclinic model which allows to simulate the hydrodynamic conditions of the study area. This work should also allows to achieve specific goals: • Develop a numerical model application to reproduce the propagation of the Tagus estuary, including the validation of the numerical model Delft3D-Flow; • Characterize the hydrodynamics of the Tagus estuary and coastal adjacent region; • Evaluate the impact of predicted future change scenarios on sal and stratification, in conditions of high freshwater discharge. • Evaluate the impact of predicted future change scenarios on salinity, water temperature and stratification, in conditions of high freshwater discharge. 2 1.2 Literature Review 1.2.1 Numerical Modelling at Tagus Estuary During the last years, the innovation and development of estuarine hydrodynamics models was unquestionable, including in the Tagus estuary. The estuary has been a natural and numerical laboratory for a great number of multidisciplinary studies since the 1980s, with a publication of modeling study about water quality management (Câmara et al., 1985). The more studies were done, the greater was the evolution of the models, because the increase of computational resources and the development of sophisticated assimilation techniques.i Vaz et al. (2020) show that 40 years later, we can find a large number of studies using numerical models, focusing on Tagus Estuary dynamics. In fact, the first modeling study published in the 1990s, simulates suspended sediment transport in two tidal estuaries (Scheldt and Tagus), in a 2-dimensional and 1-dimensional combined grid (Portela and Neves, 1994). Later, the Joint European Estuarine Project (JEEP-92), where a description of the use of different hydrodynamic and ecological models to study major microbiological processes in European estuaries was developed (Heip and Herman, 1995). Still in this decade, other study regarding sediment dynamics was developed (Barros, 1996). It was also published the first 3 work on estuarine hydrodynamics, with the aim to investigate the complex circulation at the mouth of the Tagus Estuary to understand the tidal dynamics in the estuary and build a prognostic water quality model to support estuarine management (Fortunato et al., 1997). Later, the same model was used to study the effect of tidal flats (Fortunato et al., 1999). In the beginning of the 2000s decade, one study about the evaluation of the impact of climate change in the Tagus estuary was published, where risk maps were developed (Simas et al., 2001). Two relevant numerical studies about eutrophication were also published with the aim to tackle the role of seaweeds on the estuary eutrophication, and, later, a study with aim to reduce the risk of eutrophication caused by nitrates from agricultural sources (Alvera-Azcárate et al., 2003; Saraiva et al., 2007), respectively. Braunschweig et al. (2003) developed another study with the aim to determine the water renewal time in the estuary and a model application was used to evaluate the role of nutrient and light limitation in primary production by Mateus and Neves (2008). Another study was developed to evaluate the impact of estuarine waters on the coastal area, revealing the competition between estuary discharge and wind propagation (Vaz et al., 2009). 1.2.1 Numerical Modelling at Tagus Estuary At the end of this decade, the importance of this areas on sedimentation processes was also investigated (Silva et al., 2009). Three modeling studies focused on the hydrodynamics of the estuary were published, destined to study the influence of currents and waves on the circulation at the entrance of the estuary (Rusu et al., 2011), evaluate the semidiurnal and spring-neap tide-induced variation on estuarine biochemistry (Vaz et al., 2011), and analyze the complex tidal dynamics of the estuary (Dias and Valentim, 2011). It was concluded that tidal dissipation is higher in deeper areas, where tidal currents are higher, than in shallow regions. A study to evaluate the relevance of having variable C:Chla ratios (Mateus et al., 2012), and another one, considering the link between coastal hydrodynamics and heat transport and chlorophyll patterns, reveals an increase in Sea Surface Temperature (SST) and CHL-A as consequence of an increase in concentration of such variables inside the estuary were developed (Vaz et al., 2015). The projected SLR in Tagus estuary and nearby coastal waters was also studied, revealing significant differences in the residual circulation in the upper reaches of the estuary as a response to different sea level rise scenarios (Valentim et al., 2013a), and the impacts on the salt marsh dynamics also reveals differences on the current velocity (Valentim et al., 2013b). About sediment dynamics in estuary, a study reveals the importance of wind and waves on cohesive sediment erosion (Franz et al., 2014). Another study concludes that the SLR will lead to an amplification of both semi-diurnal and quarter-diurnal tidal constituents, along the mixing region of the estuary (Guerreiro et al., 2015). MOHID was also used in a decision support system that combines water quality and hydrodynamic modeling to simulate the impact of the sewage system (Póvoa et al., 2015). The importance of the use of approaches for managing the estuary was also analyzed (França and Cabral, 2016). Fortunato et al. (2017) developed one study to analyze the impact on water level of an extreme natural event induced by a storm. One more study identifies the main modes of variability of the region, and sets the main horizontal distribution patterns of salinity and water temperature in response to different wind conditions (Vaz et al., 2018). 1.2.2 Predicted Changes in Estuaries Sue Barrell, chief-scientist at the Bureau of Meteorology (BoM) - the Australian Meteorol- ogy Agency until 2018, refers in ONU (2019), the importance of oceans in climate regulation and the natural risks related to meteorological conditions. The author conclude the deeper are the knowledge and studies about ocean science, the better are the advances about the earth global system, benefiting all people around the world.i Climate change means a significant alteration in the statistical values of meteoceanographic variables, along periods ranging from decades to longer time scales. These modifications may induce changes in average conditions (for example, in the number of extreme events). Future changes in climate are caused by different aspects, including ocean processes as oceanic circulation, solar radiation received on earth, volcanic eruptions or large-scale movements of tectonic plates. Moreover, the ecosystem alterations that are human-induced are generating a large number of impacts such as a rise in air temperature. Once estuaries are one of the marine systems more subject to human action, the connection between estuarine systems dynamics and future change is clear (Chapman, 2012; Statham, 2012; Rodrigues et al., 2021). The anthropogenic forcing causing global warming will produce the global mean sea level to rise over the 21st century (IPCC, 2013; Rodrigues et al., 2015; Ferrero et al., 2021; IPCC, 2021; Rodrigues et al., 2021). Also, future projections by IPCC (2021) express that air temperature is getting higher and changes in water column depths, water temperature and salinity in coastal regions (De Pascalis et al., 2012; Shalby et al., 2021), can affect nutrient fluxes and the phytoplankton dynamics (Brito et al., 2012; Lloret et al., 2008; Pesce et al., 2019). Studies about future changes in estuaries are increasing over the last years. Statham (2012) studied the impact of climate change in estuarine nutrients; Shalby et al. (2021) determining the impacts of future climatic conditions in an Egyptian lagoon, while Rodrigues et al. (2021), Vargas et al. (2017) and Lopes et al. (2019) focus on future changes on the ecology of the Ria Formosa and Ria de Aveiro, respectively. Ruela et al. (2020) see the evolution of SST under climate change. Moreover, Lobanova et al. (2016) and Rodrigues et al. (2016) are also good examples of the work about the impact of climate change on the Tagus Estuary. 1.2.1 Numerical Modelling at Tagus Estuary A study with the aim to perform simulation 4 4 of the estuary continuum, providing hydrodynamics and water quality parameters was also published (de Pablo et al., 2019). In the end of this decade, studies about saltwater intrusion at the upper reaches in the estuary showed that the impact of a moderate sea level rise on salinity intrusion is weak when compared with the impact of low river discharges (Rodrigues et al., 2019). 1.2.2 Predicted Changes in Estuaries This dissertation intends to developed the knowledge on how predicted changes in meteo- ceanographic variables like mean sea level or air temperature can alter (or not) stratification and circulation inside the Tagus estuary under extreme events of freshwater discharge, con- tributing to the study of this topic. Since there are no studies in this region, considering the new projections of Intergovernmental Panel on Climate Change (IPCC), it will be studied with the aim to add some information to the actual state of the art. 5 1.3 Work Structure This dissertation is structured in six chapters. Chapter 1 introduces the present work, including the motivation and aims as well the literature review of important aspects to the study. In Chapter 2, it is given a detailed characterization of the study area, with special focus to the general overview and hydrodynamic of Tagus River and the adjacent coastal area. The Chapter 3 includes specifications about the data used to perform this work. The next one, Chapter 4, shows all the methodology followed during this dissertation, with special attention to the the numerical model - Delft3D - and its application. In this chapter, also it will be presented the scenarios applied. The Chapter 5, is dedicated to show all the results obtained and respective discussion. Finally, in Chapter 6, the main final conclusions from the researches presented in the previous chapter and some suggestions of future work will also be showed. 6 6 2 2 Study Area The Tagus estuary (Figure 2.1) is located on the south-central region of continental Portugal’s west coast and it is the largest estuarine system in the Iberian Peninsula (Vaz et al., 2020). This estuary has been used for a great number of numerical tests bed over the last four decades, once it’s subject to expressive natural and, predominantly, human pressures due to its location within the heavily populated metropolitan area of Lisbon. The Tagus estuary includes three parts: the Tagus river mouth, the inner estuarine area and the adjacent coastal zone. These regions are connected and interact between them as one balanced system of river-estuary-coast. The complex geomorphology of the estuary is composed by a long and narrow inlet which connects the Atlantic coast to a shallow inner region with tidal flats and salt marsh areas (Vaz et al., 2020). The width varies between 2 km in the estuary channel and 17 km in the mixing zone (Franz et al., 2014). Figure 2.1: Study Area: a) Iberian Peninsula with Tagus Estuary highlighted in the red rectangle; b) the Tagus Estuary. The red line corresponds to the section, white dot to the buoy and triangles to the stations (P - Cascais, Q - Paço de Arcos, R - Lisboa, S - Alcochete and T - Vila Franca de Xira) are also marked. : Study Area: a) Iberian Peninsula with Tagus Estuary highlighted in the red rectangle; b) the Tagus Estuary. The red line corresponds to the section, white dot to the buoy and triangles to the stations (P - Cascais, Q - Paço de Arcos, R - Lisboa, S - Alcochete and T - Vila Franca de Xira) are also marked. 7 7 The central area of the estuary (its mixing region) presents depths from 0 m to 12 m. The deeper zones (with depths of about 25-30 m) belong to the channel that connects the estuary and the coastal sea. The upper areas have depths lower than 1 m, presenting intertidal and salt marsh regions of around 43% of the 320 km2 estuarine area (Figure 2.2) (Castanheiro, 1986; Vaz et al., 2020). The Tagus estuary is also a nature reserve placed in the Lisbon district, the largest urban center of the Atlantic European Coast, with near by 3002 km2, which have about 3 million people (INE, 2021), representing a population density of about 940 inhabitants per km2 (Pinto and Kondolf, 2016). Study Area Infrastructures, sewage system, dredging, and human activities in general, as anthropogenic forcing, and tide, wind patterns and river discharge, as natural forcing, are just some examples of issues that cause tensions in the estuarine system. Their impacts cause changes that should be anticipated to have better management options. A large number of numerical models have the capacity to combine hydrology, morphology, habitat, water and sediment quality, and meteorological parameters (Vaz et al., 2020). Thus, the knowledge about estuarine system dynamics, given by numerical models, is an important management tool to the decisions makers and government authority. Tides, wind stress, and freshwater discharge controls the water circulation of the region (Vaz et al., 2009). However, other drivers, like atmospheric pressure and surface waves, may also play an important role in the estuarine dynamic (Fortunato et al., 2017).fl The Tagus Estuary act as the interface between the coastal sea and the main affluent of the estuarine system - the Tagus river - that has a total length of approximately 1000 km and discharge an average flow of about 12 500 hm3/year (about 300 m3/s) (Portela and Neves, 1994; Lobanova et al., 2016). Although in a smaller scale, the Sorraia river and the Trancão river are also relevant to the freshwater inflow in the estuarine system (Rodrigues et al., 2009). Figure 2.2: Water surface distribution at high tide areas at Tagus Estuary (Vaz et al., 2020). Figure 2.2: Water surface distribution at high tide areas at Tagus Estuary (Vaz et al., 2020). 8 Moreover, the Tagus river basin contributes to the estuary turbidity with a high sediment influx of about 1 −5x106 metric ton/year (Pinto and Kondolf, 2016).ll Moreover, the Tagus river basin contributes to the estuary turbidity with a high sediment influx of about 1 −5x106 metric ton/year (Pinto and Kondolf, 2016).ll The main limitation at this stage (regarding river inflow), was the lack in flow data of the Trancão and Sorraia rivers. However, the portuguese National System of Information of Hydric Resources (Sistema Nacional de Informação de Recursos Hídricos (SNIRH) - http://snirh.apambiente.pt/) data revels that the hydrographic basin of Sorraia river, about 5847.16 km2, is 8.66% of Tagus river basin, with about 67 482.31 km2. Study Area Besides that, when analyzing the flow between October of 2012 and September of 2013, it is possible to observe the same ratio: the Tagus river flow is more than 12 times higher than the Sorraia river flow. During this period, Sorraia river had a discharge of 736 510 dam3, whereas Tagus river has values of 9 181 180 dam3. Following the same rule, Trancão river basin has an area of 293 km2, i.e., 0.4% of Tagus river basin (Lúcio, 2014). The tide in the estuary is predominantly semidiurnal and the system is mesotidal, with tidal ranges at the coast oscillating between 0.55 m and 3.86 m (Guerreiro et al., 2015), being intensified along the estuary as a result of resonance effects, as a consequence of depth reduction and high current velocities (Fortunato et al., 1997, 1999; Franz et al., 2014). The tidal range increases by 12% at 14 km upstream and 30% at 36 km upstream. The maximum tidal range occurs near Alverca (40 km upstream) (Portela and Neves, 1994). The mean tidal prism is about 7.5x108 m3/s and the average freshwater volume entering the estuary, during a tidal cycle, is 2 % of the tidal prism in normal conditions (Franz et al., 2014; Vaz et al., 2020), revealing the importance of tidal propagation in the estuary circulation.i Globally, the estuary is considered well-mixed, although it is possible to identify stratifica- tion at different tidal cycles stages, especially in high river flow and low tidal ranges scenarios (Rodrigues and Fortunato, 2017; Neves, 2010). Either way, vertical stratification is induced by salinity advection, caused by the interaction between tides and river inflow and heat exchange (Vaz et al., 2020). Local wind is an essential forcing of circulation inside of the estuary. Along with freshwater discharge, the wind contributes to control the residual currents which can lead to distinct transport pathways (Vaz and Dias, 2014). During winter, the wind circulation has predominant directions from the northwest, north and southeast, and the predominant wind ranges from 1.5 m/s to 14 m/s. On the other hand, during spring, the predominant winds are mainly from the north or the northwest and range from 1 m/s to 15 m/s (Vaz et al., 2018). Data The main source of error in the the numerical simulations is the quality of the data inputs (Lencart e Silva, 2007; Des, 2020). To achieve the goals of this work, the accuracy of the model inputs is crucial. A suitable correspondence between data and model results will determine the best representation of the physical processes to simulate real conditions with the most adequate spatial and temporal resolution. 3.1 Bathymetry The hydrodynamic model’s performance is mostly influenced by the accuracy of the bathymetry used (Lencart e Silva, 2007). The numerical bathymetry used was an extension of the bathymetry implemented by Ribeiro (2015) and Ribeiro et al. (2016). It was generated based on a combination of bathymetric data provided by different sources: the General Bathymetric Chart of the Oceans (GEBCO) - https://www.gebco.net/; Light Detection and Ranging (LIDAR) data, and samples obtained in Portuguese Hydrographic Institute (Instituto Hidrográfico (IH) http://www.hidrografico.pt/) in shapefile format (compilation of data surveys collected between 1964 and 2009). Study Area Further than 40 km upstream of the mouth, the water level depends mostly on the river discharge, although in the lower estuary, it depends essentially on tides and storm surges (Rodrigues et al., 2019). 9 9 3 3.3 Rivers Discharge In this work, daily and hourly mean data of freshwater discharge from SNIRH were used. In this work, daily and hourly mean data of freshwater discharge from SNIRH were used. The Portuguese Agency of Environment (Agência Portuguesa do Ambiente (APA) - http://www.apambiente.pt/) manage a large number of flow monitoring stations, with records since the early 1930s. According to SNIRH, there are 177 stream gauge stations in Tagus River Basin, of which only 65 are presently active. At Sorraia and Trancão rivers, most of the monitoring stations are presently deactivated, which represents a substantial lack of data in these tributaries. Herewith, it was just possible to ensure monitoring data values for Tagus river. The freshwater inflow values used in this work are daily values from October 2 of 1973 to March 2 of 2021. This data was validated and imposed in the simulations. For the analysis of the extreme event, a shorter time series of hourly data was used, at the interval from 00h of March’s 1 and 00h of April’s 13 of 2013. However, between the 3 pm of March’s 31 and the 1 am of April’s 3 there is no data available. The Sorraia river basin is near 8% of Tagus river basin then, for this study period, it was established that the Sorraia river discharge is 8% of the freshwater discharge values of Tagus river. The same proceeding was imposed in Trancão river. In this case, the values adopted are 1% of Tagus river freshwater discharge. 3.2 Tides Two types of tidal data were used in this work. The first one as the open boundary forcing and the second one as observational data for the validation. The tidal propagation imposed on the model as boundary condition was obtained from the TOPEX/POSEIDON. TOPEX/POSEIDON is a joint satellite altimeter mission between National Aeronautics and Space Administration (NASA), of the United States of America space agency, and Centre National d’Études Spatiales (CNES), of the French space agency, to map ocean surface topography and it is the first space mission specifically designed for studying circulation in the world’s oceans (Fu et al., 1994). The data is based on a global ocean tidal model - the NAO.99b model (Matsumoto et al., 2000) - representing the major 13 tidal harmonic constants (M2, S2, N2, K2, K1, O1, P1, Q1, MF, MM, M4, MS4, MN4) with a spatial resolution of 0.083°. 11 To the validation, water level data were obtained from five tidal stations placed along the estuary - Vila Franca de Xira, Alcochete, Lisboa, Paço de Arcos and Cascais (Figure 2.1). This data, downloaded by the Delft Dashboard tool, was given to tide gauges located in the estuary, and it was used to verify the model accuracy. The position of each station is presented in Table 3.1. Table 3.1: Location of tidal gauges used in the validation of the model. Table 3.1: Location of tidal gauges used in the validation of the model. Latitude (N) Longitude (W) Vila Franca de Xira 38° 56’ 59" 8° 59’ 17" Alcochete 38° 45’ 22" 8° 57’ 57" Lisboa 38° 41’ 38" 9° 14’ 6" Paço de Arcos 38° 41’ 24" 9° 17’ 28" Cascais 38° 41’ 38" 9° 24’ 57" 3.4 Hydrographic Variables The salinity and water temperature data used in the validation of the model are obtained from a buoy located in the central region of the estuary (38.747°N 9.05°W - see Figure 2.1). This buoy has incorporated an Acoustic Doppler Current Profiler (ADCP), at a depth of 1.8 m, and multiparametric probes at a depth of 1 m. It records marine and atmospheric variables 12 as air and water temperature, wind speed and direction, atmospheric pressure, salinity and radiation, every 20 minutes (Franz et al., 2014). These data were used to evaluate the model accuracy in reproducing transport conditions inside of the estuary. 3.5 Meteorological Conditions The meteorological data used in the simulations are the same used by Ribeiro (2015) and Ribeiro et al. (2016) which was Era-Interim. The meteorological data used in the simulations are the same used by Ribeiro (2015) and Ribeiro et al. (2016) which was Era-Interim. According to the European Centre for Medium-Range Weather Forecasts (ECMWF) (https://www.ecmwf.int/), ERA-Interim is a global atmospheric reanalysis at 80 km spatial resolution on 60 levels in the vertical from the surface up to 0.1 hPa. The data assimilation system used to produce ERA-Interim is based on a system that includes a 4-dimensional variational analysis with a 12-hour analysis window. 10 meter zonal and meridional wind components, 2 meter temperature and dew-point temperature, surface pressure, surface net solar radiation, and surface thermal radiation downwards were downloaded. The data was obtained from the ECMWF, a research institute that provides operational services, producing and disseminating numerical weather predictions. It was between January’s 1st of 2012 and December’s 31st of 2014, with a temporal resolution of 3 hours and a grid of 0.125x0.125 in a domain between 41°N and 37°N and 11°W and 7°W. The Figure 3.1 represents the data wind patterns between January 1 of 2012 and December 31 of 2014 and the wind for the period between March 1 and April 14 of 2013. In this study area, the wind direction is frequently from north whereas, during the event, the wind direction tends to be southward, with a mean speed of 5.78 m/s. Figure 3.1: Wind Patterns: a) between January 1 of 2012 and December 31 of 2014; b) during the event - between March 1 and April 14 of 2013 at the Tagus Estuary. Figure 3.1: Wind Patterns: a) between January 1 of 2012 and December 31 of 2014; b) during the event - between March 1 and April 14 of 2013 at the Tagus Estuary. 13 4 4 4 Numerical Model A mathematical model may be treated as an approximate reconstruction of a real phe- nomenon (Vaz et al., 2007). Modelling can permit simulate scenarios with known parameters, in order to understand the system dynamic and to predict future evolution in estuarine systems, with the complexity and accuracy required (Vaz et al., 2020).i Implementation range from highly detailed models of a specific process, to more idealized global climate system models. They are applied in a large type of applications like studies of processes, data assimilation and analysis, attribution, historical and paleoclimate simulation, seasonal to interannual climate prediction, future climate projections, and regional downscaling (WCRP, nd-b). Over the past decades, the model implementation improve and, with that, better versions with innovative/sharp algorithms and more complex domains are available, increasing the accuracy between the reality and model results. That evolution is the consequence of the development of the computational resources. 4.1 Delft3D-FLOW Numerical Model The model used in this study is the Delft3D-FLOW (Deltares, 2021). Delft3D is an open source software, developed by WL\|Delft Hydraulics in cooperation with Delft University of Technology. It is used to study hydrodynamics, waves, water quality, sediment transport, and morphology for fluvial, estuarine and coastal environments to reproduce realistic conditions. The Delft3D program is composed by a set of modules, covering a distinct range of features, and can be executed independently or in combination with other modules (Deltares, 2021; Des, 2020). The FLOW module is a multi-dimensional (Two-Dimensional Horizontal (2DH) - depth averaged, or three-dimensional - 3D) hydrodynamic model that calculates unsteady flow and transport processes, like salinity and heat, resulting from tidal and meteorological forcing on a curvilinear, boundary fitted grid or spherical coordinates. This module simulates many processes as wave and tidal forces, wind shear, density driven flows and stratification due to salinity and water temperature gradients or atmospheric pressure changes. It is also important to simulate drying/flooding of intertidal flats and to analyze the transport of properties where 15 the horizontal flow field shows significant variation in the vertical direction, (e.g. estuarine circulation) even in well-mixed estuaries (Deltares, 2021; Des, 2020; Ribeiro, 2015).i In 3D simulations, the vertical grid is defined following a σ coordinate approach or Z-layer approach (Deltares, 2021). In this work, it will be used the 3D version of this module, with a σ coordinate grid. In 3D simulations, the vertical grid is defined following a σ coordinate approach or Z-layer approach (Deltares, 2021). In this work, it will be used the 3D version of this module, with a σ coordinate grid. 4.1.1 Grid Aspects The model used here is capable of handling curvilinear grids in Cartesian coordinate systems, where the coordinates are in meters, and spherical coordinate system, with the coordinates in decimal degrees. This provides good performance in terms of computational velocity and accuracy, improving the model adaptability. The numerical grid was constructed, expanded, and orthogonalized based on the Ribeiro (2015) and Ribeiro et al. (2016) work, using the Delft boundary fitted grid generation tool (RGFGRID). An unique domain approach to the grid design was used, once it avoids numerical dis- crepancies at the domain frontiers (Des, 2020). This grid covers an area from approximately 38°N to 39°N and from 8.8°W to 9.5°W. The spatial resolution ranges from 118 meters, in the inner estuary, to 1298 meters, near the ocean boundary, with a mean resolution value of 377 meters. This creates a total number of grid cells of 9069. The representation of the grid and bathymetry used in this work is presented in Figure 4.1. Here, we can distinguish the intertidal zone (with depths of about 0m), the path taken by the rivers towards the central zone of the estuary, and the navigation channel with depths higher than 25 meters, and the adjacent coastal zone. This numerical model solves the continuity equation, the horizontal equations of motion and the transport equations for conservative constituents. The equations are formulated in orthogonal curvilinear coordinates or in spherical coordinates on the globe. For this work, the spherical coordinates where the reference plane follows the Earth’s curvature to solve the equations of motion was chosen (Deltares, 2021). 16 Figure 4.1: Numerical Grid and Bathymetry (color) of Tagus estuary and adjacent coastal zones, used in the implementation of the model Figure 4.1: Numerical Grid and Bathymetry (color) of Tagus estuary and adjacent coastal zones, used in the implementation of the model In the numerical grid, a few parameters such as water level, salinity, and water temperature were defined in different locations of the grid as shown in Figure 4.2 (Ribeiro, 2015; Deltares, 2021).i All variables are organized in a specific way on the grid to permit the discretization of the 3D shallow water equations. Staggered grids are particular interesting once, for shallow water, they restrict spatial oscillations in water levels (Stelling, 1983) and the boundary conditions can be implemented in a rather simple way. 4.1.2 Numerical Model Formulation Delft3D solves a set of mathematical equations to better represent physical processes (subsection 4.1.1). This numerical hydrodynamic model solves the Navier Stokes equations for an incompressible fluid, under the shallow water and the Boussinesq assumptions. The hydrostatic pressure equation is used once the vertical accelerations are neglected, in the vertical momentum equation. In 3D models, vertical velocities are computed from the continuity equation. A finite difference grid solve a combination of the initial and boundary conditions with a proper group of partial differential equations. All equations used in Delft3D model may be consulted in Deltares (2021). The contour of the model domain is constituted by two parts: “land-water” lines (coast- lines), which are called closed boundaries, and a part across the flow field which is called open boundary. Closed boundaries are natural boundaries, while open boundaries are always artificial “water-water” boundaries. In the numerical model, open boundaries are introduced to restrict the computational area and, consequently, the computational effort. The conditions of flow and transport imposed at the open boundaries represent the influence of the non-modelled area surrounding the modeled area (Deltares, 2021).i A set of initial boundary conditions for water level and horizontal velocities was specified, to get a well-posed mathematical problem with a unique solution. In addition to the water level and flow velocity components, the transport conditions as salinity and heat (e.g., water temperature) were specified per layer to start computation. In this work, the open boundary was divided in 6 sections, each one with their own imposed conditions. The type of water level with hydrodynamic forcing was introduced as astronomic components. For large-scale simulations, the influence of the shear-stresses along the closed boundary is neglected, so it was applied free slip condition (zero tangential shear stress) in all closed boundaries. Finally, rivers discharge was prescribed as boundary condition of type discharge per cell. In the Delft3D-FLOW, the numerical grid is usually too rough and the time step too large to resolve the turbulent scales of motion. In this way, the turbulent processes had to be solved in a “sub-grid” scale. In this numerical model, four turbulence closure models are implemented to determine νV , eddy viscosity coefficient, and DV , eddy diffusivity coefficient:fi 1. Constant coefficient; 2. Algebraic Eddy viscosity closure Model (AEM); 3. k −L turbulence closure model; 4. k −ε turbulence closure model. 4.1.1 Grid Aspects Furthermore, it is possible to use a smaller number of discrete state variables in comparison with discretizations on non-staggered grids, to obtain the same accuracy (Deltares, 2021). This exact staggered grid is defined as an Arakawa C-grid. The water level points, i.e. pressure points, are defined in the centre of a continuity cell. On the other hand, the velocity components are perpendicular to the grid cell faces where they are computed (Deltares, 2021). 17 Figure 4.2: Schematized horizontal numerical grid of Delft3D-FLOW. Source: Deltares (2021) Figure 4.2: Schematized horizontal numerical grid of Delft3D-FLOW. Source: Deltares (2021) Figure 4.2: Schematized horizontal numerical grid of Delft3D-FLOW. Source: Deltares (2021) In the vertical direction it was used a σ-coordinate, defined as: σ = z −ζ H , where H = d + ζ (4.1) (4.1) with z being the vertical coordinate in physical space, ζ the free surface elevation above the reference plane (at z = 0) and d the depth below the reference plane. The H represents the total water depth, given by d + ζ. The σ-grid was introduced by Phillips (1957) for atmospheric models. This vertical grid consists of layers bounded by two σ-planes, which follow the bottom topography and the free surface. Here, the grid is boundary-fitted and the number of layers is constant, varying only the relative layer thickness, as it is possible to compare in Figure 4.3 (Des, 2020; Deltares, 2021). As referred before, the number of layers over the horizontal computational area is constant, independent of the local water depth. In this work 15 σ-layers are used, with the first ones thinner than the bottom layers, to improve the vertical mixing perception by the model, based on Ribeiro (2015) (see Table 4.1). Figure 4.3: Schematized vertical numerical grid of Delft3D-FLOW. Source: Deltares (2021) Figure 4.3: Schematized vertical numerical grid of Delft3D-FLOW. Source: Deltares (2021) 18 Table 4.1: Layer Thickness Table 4.1: Layer Thickness Layer 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Layer Thickness (%) 2 3 4 5 6 8 8 8 8 8 8 8 8 8 8 4.1.2 Numerical Model Formulation The turbulence closure models differ in their turbulent kinetic energy, dissipation rate of turbulent kinetic energy and the mixing length. 19 The formulation of the first turbulence closure model (1. Constant coefficient) is simpler than the others once it is based on a constant value previously specified, while the other three turbulence closure models are based on the eddy viscosity concept of Kolmogorov (1941) and Prandtl and Wiegkardt (1945), respectively. However, in this work, it will be explored a k −ε turbulence closure. In the k −ε model, the transport equations must be solved for both the turbulent kinetic energy k and for the energy dissipation ε. The details of the other options can be consulted in Deltares (2021). Sun radiation reaches the earth in the form of an electromagnetic wave. Then, in the atmosphere, the radiation is diffused, reflected, and absorbed by air, clouds, dust, and other particles. Here, the heat exchange at the free surface is modelled by taking into account the independent effects of solar (short wave) and atmospheric (long wave) radiation, and heat loss due to back processes as radiation, evaporation and convection.lf The total heat flux through the free surface is a sum of different radiation contributors (equation 4.2). (4.2) Qtot = Qsn + Qan −Qbr −Qev −Qcv (4.2) where: ■Qsn is the net incident solar radiation (short wave) ■Qsn is the net incident solar radiation (short wave) ■Qsn is the net incident solar radiation (short wave) ■Qan is the net incident atmospheric radiation (long wave) ■Qbr is the back radiation (long wave) ■Qev is the evaporative heat flux (latent heat) ■Qcv is the convective heat flux (sensible heat) ■Qan is the net incident atmospheric radiation (long wave) ■Qbr is the back radiation (long wave)l ■Qev is the evaporative heat flux (latent heat)l ■Qcv is the convective heat flux (sensible heat) In the Delft3D-Flow, five heat flux models are available: heat flux model 1, heat flux model 2, excess temperature model, Murakami heat flux model, and ocean heat flux model. In this work, the numerical model implementation used the Heat Flux model 2. This specific model is based on formulations given by Octavio (1977) and combine the net solar (short wave) with the net atmospheric (long wave) radiation. 4.1.2 Numerical Model Formulation The source of data for the combined net incoming radiation terms (Qsn + Qan) must come from direct measurements, once the solar radiation has to be specified by the user. The remaining terms related to heat losses due to evaporation, back radiation and convection are computed by the model and may be consulted in Deltares (2021). 4.2 Model Implementation There are a lot of models, with different features that can be really useful if chosen according the type of phenomena and environment characteristics in study.l All the parameters reflect the uncertainty on how to model the physical process and provide the opportunity to adjust models using measured data (Ribeiro, 2015). According to the implementation adopted in this dissertation, the simulations were set for the period between July 25 of 2012 and July 31 of 2013, but only between March 22 and 20 April 12 were used. The spin-up period needs to be, at least, 2 times the estuary’s residence time, according with Braunschweig et al. (2003). The modelling time step is 30 seconds, to be in accordance with the Courant-Friedrichs-Lewy number (CFL), as recommended by Deltares (2021). If the time step of the model is too large when related to the grid cell size and the water depth, instability in the the tidal wave propagation can occur. The tidal propagation was calibrated through the adjustment of bottom roughness. Once again, the bottom roughness coefficient used is the same of Ribeiro (2015).fi This parameter assumes the depth-dependent Manning coefficient, with the formulation of the Chezy coefficient (Deltares, 2021). The Manning coefficients were adjusted considering that an increase in the bottom friction will produce a decrease in the tidal wave amplitude in that zone of the channel and in the channel’s upstream. Moreover, an increase in the bottom roughness will produce an increase in the phase lag for high tide and a decrease for low tide (Fry and Aubrey, 1990). The values are represented in the Table 4.2. Table 4.2: Bottom friction coefficients - Manning Values. Table 4.2: Bottom friction coefficients - Manning Values. 4.2 Model Implementation Depth (m) Manning’s Values -5.0 ≤h < -2.5 0.028 -2.5 ≤h < -2.0 0.025 -2.0 ≤h < -1.5 0.020 -1.5 ≤h < -1.0 0.016 -1.0 ≤h < 0.0 0.015 0.0 ≤h < 0.5 0.022 0.5 ≤h < 1.0 0.023 1.0 ≤h < 3.0 0.022 3.0 ≤h < 10.0 0.020 10.0 ≤h < 5000.0 0.019 The background horizontal eddy viscosity and diffusivity were set to 10 m2/s and the background vertical eddy viscosity and diffusivity as the value of 10−3 m2/s.l A Heat flux model 2 was applied, taking into account air temperature, relative humidity, and net solar radiation to calculate heat losses from convection, evaporation, and back radiation (see section 3.5).il Three freshwater points were also defined as outflows representing the Tagus, Sorraia, and Trancão rivers (see section 3.3).ii The final modelling configurations are listed in Table 4.3. 21 Table 4.3: Parameters and Specifications of model implementation pi p Parameter Specification Time Step 30 seconds Run Period 2012-07-25 to 2013-07-31 Process Constituents Salinity, Water temperature and Wind Wind Variable (Era-Interim) Discharge Variable Boundary Forcing Harmonic constituents Bottom Roughness Variable Manning coefficient Background horizontal eddy viscosity and diffusity 10 m2/s Background vertical eddy viscosity and diffusity 10−3 m2/s Air Temperature Heat Flux model Heat Flux model Absolute flux, net solar radiation Threshold depth 0.1 m Turbulence closure k −ε Wall slip condition Free Spin-up period 2 months Spin-up period 4.3 Validation Despite the data and model limitations, numerical models are the more suitable tools to represent the reality evolution tendency (Des, 2020). Even being aware of possible failures, the validation is fundamental to obtain the best results. Although this grid has already been calibrated and validated by Ribeiro (2015), it is necessary to do it again since it has been expanded for this dissertation. The validation data procedure consists in running the model, crosschecking the results of the measured and simulated results, and adjusting the parameters in the model. These steps should be repeated until the simulation results are accurate. A considerable number of model runs were executed to validate the numerical model, to reduce the differences between data and predictions. All the model runs were subject to a cold start, with all forcers included, as wind and rivers discharge, to give the model time to adjust to the initial conditions (Ribeiro, 2015; Des, 2020). The crosschecking of the results, was divided in two steps: the comparison between observed and predicted tidal amplitudes and phases of mean tidal constituents and the comparison between observed and predicted water temperature and salinity. The analysis of the relation between both parameters was established according to the next error equations. 22 The Root Mean Square Error (RMSE), was calculated, according to Stow et al. (2009), as: RMSE = " 1 N N X i=1 \|Xobs(ti) −Xmod(ti)\|2 #1/2 (4.3) (4.3) where, Xobs(ti) and Xmod(ti) are the Sea Surface Elevation (SSE) data and the predicted by the model at time ti, respectively. N is the number of observations. The closer the value to zero, the better is the model performance. where, Xobs(ti) and Xmod(ti) are the Sea Surface Elevation (SSE) data and the predicted by the model at time ti, respectively. N is the number of observations. The closer the value to zero, the better is the model performance. Dias et al. (2009) propose, that the RMSE must be lower than 5% of the local amplitude to represent an excellent approach between model and reality. Also, the RMSE error between 5% and 10% is suggested as a very good agreement. To calculate this relative error, ∆E, it was applied the equation 4.4. 4.3 Validation ∆E = RMSE 1 n Pn i=1 (Amaxobs(ci) −Aminobs(ci))x100 (4.4) (4.4) where, Amaxobs(ci) and Aminobs(ci) are the maximum and minimum, respectively, elevations for each tidal cycle and n is the number of tidal cycles.i On the other hand, it was calculated the Skill parameter, which quantifies the agreement between model prediction and observations (Willmott, 1981; Warner et al., 2005). This parameter is given by equation 4.5. Skill = 1 − PN i=1 \|Xmod(ti) −Xobs(ti)\|2 PN i=1 \|Xmod(ti) −Xobs(ti)\| + \|Xobs(ti) −Xobs(ti)\| 2 (4.5) (4.5) whew, Xobs represent a temporal mean of the observed data. A Skill value equals to one means a perfect agreement between model prediction and observations. Also Dias et al. (2009) refer that Skill values above of 0.95 represent excellent agreement. whew, Xobs represent a temporal mean of the observed data. A Skill value equals to one means a perfect agreement between model prediction and observations. Also Dias et al. (2009) refer that Skill values above of 0.95 represent excellent agreement. In Figure 4.4 results for the 5 stations used for the validation are presented as a comparison between observed and predicted SSE. The location of these stations is presented in Table 3.1, and the period chosen for comparison is December 2012. The visual comparison between observed and predicted SSE permits to distinguish that the model represents very well the SSE over the spring-neap cycle for the 5 stations. 23 ure 4.4: Comparison between observed and predicted sea surface elevation (black line: measurements; red line: model results), in December of 2013, for 5 stations Franca de Xira; b) Alcochete; c) Lisboa; d) Paço de Arcos and e) Cascais. Figure 4.4: Comparison between observed and predicted sea surface elevation (black line: observed measurements; red line: model results), in December of 2013, for 5 stations : a) Vila Franca de Xira; b) Alcochete; c) Lisboa; d) Paço de Arcos and e) Cascais. 24 After this first analysis, it was calculated the previous parameters - RMSE, ∆E and Skill (Table 4.4). After this first analysis, it was calculated the previous parameters - RMSE, ∆E and Skill (Table 4.4). After this first analysis, it was calculated the previous parameters - RMSE, ∆E and Skill (Table 4.4). 4.3 Validation Table 4.4: Error values for tidal water levels - RMSE (m), its Error (%) and Skill Table 4.4: Error values for tidal water levels - RMSE (m), its Error (%) and Skill Station RMSE (m) ∆E (%) SKILL Vila Franca de Xira 0.19 6.29 0.99 Alcochete 0.14 4.71 0.99 Lisboa 0.10 3.31 0.99 Paço de Arcos 0.42 16.04 0.92 Cascais 0.07 2.81 0.99 The stations of Alcochete, Lisboa, and Cascais present the best results, with RMSE near 0 and Skill of almost 1. Also, the ∆E is lower than 5%. Vila Franca de Xira presents values also good but with an higher ∆E. The station with the worst values is Paço de Arcos with a RMSE higher than of 40 cm and a ∆E lower than 16%. The results for error values for tidal water levels are in the same order than the previous studies (Ribeiro, 2015). The RMSE shows values under 20 cm, ∆E lower than 10% and Skill of 0.99 in all stations, except to the Paço de Arcos station. Therefore, it is possible to conclude that the agreement between data and predictions can be considered very good. The observed tide is a sum of a large number of partial tide constituents. To complement the previous table, the model accuracy in reproducing the main tidal constituents (M2, S2, O1 and K1) was analyzed (Table 4.5). Here it was studied the differences between amplitude and phase of the tidal constituents. For the diurnal constituents (O1 and K1), the agreement is good for all stations. The difference of amplitude for these tidal constituents is null. M2 and S2 are the most energetic in the region. The agreement for these constituents may be considered very good, except for Paço de Arcos station where it was found the highest difference of phase (about 32°). However, the average difference of amplitude for these semidiurnal tidal constituents is about 5 cm to M2 and 2 cm to S2. Average phase differences of 10° and 11° for M2 and S2 can be found, which represent a delay of about 21 minutes in the water propagation for both semidiurnal tidal constituents. Similar values of differences between amplitude and phase of the tidal constituents may be found in identical studies published by Ribeiro (2015) and Rodrigues (2015). 4.3 Validation 25 Table 4.5: Model accuracy in reproducing the main tidal constituents (M2, S2, O1, K1) M2 S2 O1 K1 Amplitude (m) Phase (°) Amplitude (m) Phase (°) Amplitude (m) Phase (°) Amplitude (m) Phase (°) Vila Franca de Xira Data 1.19 103.88 0.30 143.56 0.06 336.20 0.09 72.10 Model 1.11 113.03 0.27 154.53 0.06 68.00 0.08 78.33 Difference -0.08 9.15 -0.03 10.97 0.00 91.80 -0.01 6.23 Alcochete Data 1.26 83.72 0.35 116.50 0.07 321.08 0.10 62.56 Model 1.17 88.67 0.31 125.42 0.06 60.39 0.09 66.36 Difference -0.09 4.95 -0.04 8.92 -0.01 99.31 -0.01 3.80 Lisboa Data 1.15 76.63 0.32 108.31 0.06 320.08 0.09 55.34 Model 1.10 73.74 0.30 104.70 0.06 53.81 0.09 58.76 Difference -0.05 -2.89 -0.02 -3.61 0.00 93.73 0.00 3.42 Paço de Arcos Data 1.01 100.44 0.28 131.39 0.06 332.13 0.09 64.96 Model 1.00 68.80 0.28 98.99 0.06 51.12 0.09 56.63 Difference -0.01 -31.64 0.00 -32.40 0.00 78.99 0.00 -8.33 Cascais Data 0.98 65.61 0.28 92.65 0.06 313.36 0.10 46.82 Model 0.97 63.71 0.27 92.52 0.06 47.64 0.09 53.55 Difference -0.01 -1.90 -0.01 -0.13 0.00 94.28 -0.01 6.73 26 Dias and Lopes (2006) refer that the salinity distribution reflects the combined results of tidal current, density circulation, freshwater flows and turbulent mixing processes. Salinity is considered a natural tracer for the validation of transport processes in estuarine environments (Des, 2020).l The heat transport process totally depends on the heat flux model used. The validation of salinity and water temperature was done using the buoy measurements presented in the section 3.4 (see Table 4.6). The RMSE for the water temperature is lower than 0.5°C and for salinity is about 1.68. The water temperature value is lower than the value obtained by Ribeiro (2015) but the salinity value is higher. Although Skill values are not satisfactory as in the water level validation, these were the best values obtained within the series of tests developed. Finally, the agreement between both predicted and data shows that Delft3D-Flow imple- mentation, used in this work, can be accurately applied to simulate future conditions in the Tagus estuary. Table 4.6: Error values for water temperature and salinity - RMSE and Skill, relative to the buoy measurements Water Temperature (°C) Salinity Water Temperature (°C) Salinity RMSE 0.48 1.68 SKILL 0.77 0.61 4.4 Model Scenarios The world’s most complex climate models are currently running a range of experiments as part of the Sixth Coupled Model Intercomparison Project (CMIP6), which is composed by 33 participating model groups and 23 CMIP6-Endorsed MIPs (Nicholls et al., 2021; WCRP, nd-a). Coupled Model Intercomparison Project (CMIP) is a project of the World Climate Research Programme (WCRP)’s Working Group on Coupled Modeling (WGCM). Since 1995, CMIP has coordinated several climate model experiments involving multiple international modeling teams worldwide, to a better understanding of past, present and future climate change and variability in a multi-model framework. The goal of the WGCM is the development and review of coupled climate models, advancing the scientific understanding of the Earth system. CMIP model simulations have also been regularly assessed as part of the IPCC Climate Assessments Reports (WCRP, nd-a). The scenarios presented by IPCC were created to explore future evolution of the global environment, with special attention to the Greenhouse Gas (GHG) production and to the aerosols emissions. The scenarios have in consideration several driving forces of climate change, including the population growth and socioeconomic development. These driving 27 forces are highly unsettled, once recognize GHG sources and sinks as energy production and consumption systems, as well as land use changes. To better understand this concept, in Figure 4.5 it is possible to analyze the historical GHG emissions along the last years, given by Climate Watch (https://www.climatewatchdata.org/). As we can see, the biggest slice, about 75%, is appointed to the energy production. In the next years, if this parameter progress is different from the expected, the emissions may reduce or increase. With this uncertainty, different scenarios (Shared Socioeconomic Pathways (SSP)) were created. Note that, although all scientific efforts applied and for all previous reasons, future change effects must be considered as a possibility, but not necessarily probable. CMIP6 studies reveals four scenarios with more conclusive results, with a large number of model runs available: ■SSP1 – 2.6 considers the limit warming lower than 2°C, which follows the topic 1.a) of Article 2 in the Paris Agreement (United Nations Treaty Collection, 2015), where the goal is to limit global warming to 1.5°C, when compared to pre-industrial levels. Studies show that average warming of 2°C with the range between 1.3°C and 2.9°C for the lowest and highest warming among models for each scenario, respectively. 4.4 Model Scenarios ■SSP2 – 4.5 shows an average warming of 3.0°C in the range of [2.1°C – 4.3°C]. ■SSP3 – 7.0 is the new scenario which lies right in the middle of the range of baseline outcomes produced by energy system models. It was obtained with a mean warming of ■SSP3 – 7.0 is the new scenario which lies right in the middle of the range of baseline outcomes produced by energy system models. It was obtained with a mean warming of 4.1°C in the range between 3.0°C and 6.2°C. Scenario Sea Level Rise (m) Air Temperature (°C) #ScRef 0 0 #Sc1 0 + 1.0 #Sc2 + 0.40 0 #Sc3 + 0.40 + 1.0 #Sc4 + 0.70 + 4.0 Scenario Sea Level Rise (m) Air Temperature (°C) 29 5 5 5 Figure 4.5: Historical Greenhouse Gas Emissions. Source: Climate Watch Figure 4.5: Historical Greenhouse Gas Emissions. Source: Climate Watch Figure 4.5: Historical Greenhouse Gas Emissions. Source: Climate Watch 28 4.1°C in the range between 3.0°C and 6.2°C. 4.1°C in the range between 3.0°C and 6.2°C. 4.1°C in the range between 3.0°C and 6.2°C. ■SSP5 – 8.5, the modellers examine the worst case scenario, without any climate policies. Here, the studies showed an average warming of 5.0°C with the range between 3.8°C and 7.4°C. ■SSP5 – 8.5, the modellers examine the worst case scenario, without any climate policies. Here, the studies showed an average warming of 5.0°C with the range between 3.8°C and 7.4°C. Herewith, it is possible to observe CMIP6 variants showing around 0.4°C higher warming, comparably to Fifth Coupled Model Intercomparison Project (CMIP5) (IPCC, 2021). To reach the proposed goals, two scenarios from the four above were chosen. In this dissertation, it will be analyzed the SSP1-2.6 - low GHG emissions scenario, and SSP5-8.5 - high GHG emissions scenario, in order to include all the range of possibilities. From IPCC Interactive Atlas (https://interactive-atlas.ipcc.ch/), it was possible to obtain the specific values of SLR and Air Temperature, at point 38.57°N 9.15°W of the study area. All scenarios used in this work are detailed in the Table 4.7. The #ScRef is the Reference Scenario, with the same implementation of the validation and without any change in SLR and air temperature. In the next scenarios the values presented in Table 4.7 were added to the implementation. #Sc1 was developed to study the influence of an increase of 1°C in air temperature in events like the one occurred in 2013. #Sc2 has a similar role as the previous one, but to a SLR of 0.40 m. The last two scenarios, #Sc3 and #Sc4, correspond to SSP1 – 2.6 and SSP5 – 8.5 values, respectively. In this case, as the goal is to study an event of extreme freshwater discharge, the freshwater drivers will not change in the simulations. Table 4.7: Idealized scenarios used in the model simulations. Table 4.7: Idealized scenarios used in the model simulations. Table 4.7: Idealized scenarios used in the model simulations. 5.1 2013 Event Characterization The event under analysis in this dissertation refers to an extreme freshwater outflow from the Tagus Estuary. The event is characterized in Figure 5.1, where the freshwater outflow and wind data are depicted. In the figure, the freshwater discharge of the Tagus river, between March 1 and April 13 of 2013 is depicted to evaluate the flow evolution (Figure 5.1a). The period of March 22 to April 12 of 2013 was highlighted because it is when the river discharge reaches the highest value. As it can be observed, the Tagus river shows a typical flow pattern, with values lower than 2000 m3/s. When the maximum discharge occurs, on April 1, a value of 8711 m3/s is reached. Some days before the peak of freshwater, since March 27, the flow values started to be about 1000 m3/s higher. Furthermore, the high flow values remain high until April 5.i For the same period of the previous figure, the six-hourly wind speed in the estuary was represented (Figure 5.1b). The goal is to evaluate the wind evolution during this specific period. Although the wind direction is variable during these days, the maximum discharge occurs at the same time as the wind blows south-southwesterly. On April 5, in addition to the flow decrease, the wind blows from the north (changing its direction), with intensities of almost 12 m/s, changing the atmospheric conditions, and promoting the return to ”natural” conditions. Once the Tagus estuary is generally considered well mixed (Rodrigues and Fortunato, 2017), the mean salinity evolution (Figure 5.1c) of the longitudinal section represented in Figure 2.1 is depicted in the lower panel. The results were filtered with a cut-off frequency of 33h to remove tidal effects and inertial components (Otero et al., 2008). Note that the filter may smooth the signal and, consequently, the salinity values depicted. With this Two-Dimensional Vertical (2DV) representation, the salinity horizontal gradient is recognized. The upstream region reveals salinity values near 0, while the estuary mouth gets values higher than 30, except between March 31 and April 5. In this period, the central region of the estuary is filled with freshwater, and salinity values of 28 are reached outside the estuary. In normal conditions of freshwater and wind, the mixing region is located between 20 and 45 km from the upstream region of the estuary, near the river mouth. Since March 28, this region has been advected downstream. 5.1 2013 Event Characterization 31 Figure 5.1: 2013 March/April Event Characterization, in terms of: a) Freshwater Discharge of Tagus River; b) Wind Speed; and c) Mean Salinity over the section with beginning at the point T (Vila Franca de Xira). 32 Figure 5.1: 2013 March/April Event Characterization, in terms of: a) Freshwater Discharge of Tagus River; b) Wind Speed; and c) Mean Salinity over the section with beginning at the point T (Vila Franca de Xira). 32 The high-flow effects take some hours to reach the estuary, once the lowest values of salinity along the section only occur between 2 and 3 of April. On April 2, the mixing zone was located between 30 and 60 km from the beginning of the section at Vila Franca de Xira, covering a region from the middle of the estuary until the coast outside. On April 9, the section salinity pattern returns gradually to the previous state, with an increased stratification in the middle area of the estuary. The 2DV salinity and water temperature pattern along the longitudinal section of the estary was characterized to three specific times: March 24 (before the extreme event); April 2 (the event) and April 11 (after the event). Thus, the vertical longitudinal section was analyzed in terms of salinity (Figure 5.2), water temperature (Figure 5.3) and Brunt-Väisälä frequency (Figure 5.4), to evaluate the stratification changes in the estuary. Before the extreme event (Figure 5.2a) the salinity structure reveals a well-mixed water column (filled with freshwater) until almost 20 km from the Tagus river mouth. The mixing region, where stratification is visible, covers a region until nearly 50 km from the beginning of the longitudinal section. Here a stratified water column is visible, presenting surface values ranging from 4 to 24 between 20 and 30 km from the upstream region. Between 30 and 45 km, the isohalines became steeper, with the maximum vertical difference of 8 salinity units. This vertical gradient is relatively constant until 45 km from river mouth, near the marine region of the estuary, where the water column became mixed again. During the extreme freshwater event (Figure 5.2b), the water column is filled with freshwater, becoming well-mixed until 30 km from Tagus river mouth, with values close to 0. The mixing region is advected toward the coast, presenting a strongly stratified water column, with surface values ranging from 6 to 24 and bottom values from 18 to 36. 5.1 2013 Event Characterization In Figure 5.2: Daily average salinity along the longitudinal section to the Reference Scenario. a) March 24; b) April 2; and c) April 11, before, during and after the extreme freshwater discharge event, respectively. Figure 5.2: Daily average salinity along the longitudinal section to the Reference Scenario. a) March 24; b) April 2; and c) April 11, before, during and after the extreme freshwater discharge event, respectively. 33 the middle of the section (between 30 and 50 km), maximum stratification is reach, with top-to-bottom differences of about 24 to 28 salinity units. In the marine region of the estuary, strong stratification is also visible with vertical salinity differences of about 18 salinity units. In Figure 5.2c, the salinity structure is similar to the one previous to the extreme freshwater event, with a visible decrease of stratification process in the mixing region. It reveals a well- mixed water column, with salinity values of 0, until nearly 20 km from the Tagus river mouth. The mixing region covers a region from 20 km until the end of the section. Here a stratified water column presents values ranging from 6 to 18 and between 20 km and 30 km from the upstream region. After that, the isohalines became steeper, with the maximum vertical difference of 8 salinity units. This difference is relatively constant until the marine region of the estuary, where the water column became mixed again. Figure 5.3 depicts the longitudinal and vertical structure of water temperature in the estuary. Before the extreme freshwater event (Figure 5.3a), the upstream region of the estuary (between 0 and 20 km from river mouth) presents a well-mixed water column and a downstream thermal gradient of 16°C/20km. It is visible a warmer water column in the mixing region, with values close to 16°C, decreasing to 15°C in the marine region of the estuary (since 40 km from the beginning of the section). During the extreme freshwater event (Figure 5.3b), a well-mixed water column is visible at the upstream region of the estuary (until 25 km from Tagus river mouth). In the mixing region, the isothermic lines tilted and thermal stratification occurs. In this region, vertical water temperature differences of 4°C to 6°C are visible. In the marine region, the water column is well-mixed in terms of temperature. 5.1 2013 Event Characterization During the event, a longitudinal gradient of Figure 5.3: Daily average water temperature along the longitudinal section to the Reference Scenario. a) March 24; b) April 2; and c) April 11, before, during and after the extreme freshwater discharge event, respectively. Figure 5.3: Daily average water temperature along the longitudinal section to the Reference Scenario. a) March 24; b) April 2; and c) April 11, before, during and after the extreme freshwater discharge event, respectively. 34 6°C/50km is visible along the estuary. 6°C/50km is visible along the estuary. 6°C/50km is visible along the estuary. After the extreme freshwater event, the water column became progressively well-mixed along the longitudinal section and the horizontal thermal gradient decrease to 3.5°C/50km, following the decrease of stratification of the water column along the estuary.i In order to evaluate the vertical stratification in the water column is computed the Brunt-Väisälä frequency (N) (e.g. Prinsenberg and Rattray Jr (1975)). The Brunt-Väisälä frequency indicates the maximum frequency (ω) for internal waves that can propagate in the respective stratification, i.e., how much energy is required to exchange water parcels in the vertical (Boehrer and Schultze, 2009). Smaller N values indicate lower stratification, whereas higher values shows higher stratification. N = s −g ρ0 ∂ρ(z) ∂z (5.1) (5.1) where g is gravity acceleration (m/s2), ρ0 is the the density, ∂ρ(z) is the density at atmospheric pressure (1000 kg/m3) and z is the depth measured from the surface.i In the first 15 km from Tagus river mouth (Figure 5.4a), the Brunt-Väisälä frequency is low, with values between 0 and 20 cycles/h in the bottom, and values between 20 and 40 cycles/h at the surface. This area is well-mixed, according with Figure 5.2 and Figure 5.3. Since 15 km until 45 km from Tagus river mouth, the values of Brunt-Väisälä frequency increase substantially, mainly at the surface, reaching 180 cycles/h between 30 km and 35 km. From the middle of the section beyond, the surface has values about 2 times higher than the bottom, which means the mixing increase in depth. At 50 km, the water column de-stratify again with values of about 100 cycles/h at the most part of the water column, and values Figure 5.4: Daily average Brunt-Väisälä frequency along the longitudinal section to the Reference Scenario. a) March 24; b) April 2; and c) April 11, before, during and after the extreme freshwater discharge event, respectively. 5.1 2013 Event Characterization Figure 5.4: Daily average Brunt-Väisälä frequency along the longitudinal section to the Reference Scenario. a) March 24; b) April 2; and c) April 11, before, during and after the extreme freshwater discharge event, respectively. 35 of 140 cycles/h at the surface. In the marine region, strong stratification is also visible with values between 140 and 170 cycles/h along the water column, and 70 cycles/h in the bottom. The Figure 5.4b presents the same upstream pattern than the previous, with values lower than 30 cycles/h in the first 25 km. Since 25 km until 30 km from Tagus river mouth, N reach values between 80 cycles/h and 140 cycles/h in all water column. After 30 km, the Brunt-Väisälä frequency is high in all water column, with surface values higher than 140 cycles/h, reaching 180 cycles/h near the marine region. At the bottom, the values are about 100 cycles/h. In addition to the effects of high freshwater discharge, the water column may be more stratified, due to the increase of vertical mixing enhanced by the south-southwesterly winds during the event, according with Vaz et al. (2018). In the last panel (Figure 5.4 c), the stratification structure is similar to the one previous to the extreme freshwater event, with a visible decrease of stratification process in the surface of the mixing region. In the first 20 km, N is lower than 50 cycles/h. After that, since 20 km until the end of the section, the Brunt-Väisälä frequency is between 70 cycles/h and 120 cycles/h over the water column. At the bottom, N presents values ranging from 40 cycles/h and 80 cycles/h. The maximum Brunt-Väisälä frequency values of 140 cycles/h are reached in the navigation channel surface (at the mixing region), decreasing in the remaining area. Considering the previous results, it can be concluded that after the event, the mixing increases considerably relative to the moment before the event, according to the water temperature patterns presented in Figure 5.3. The horizontal patterns of salinity and water temperature were also evaluated (Figures 5.5 and 5.6) for the three distinct periods previously characterized in Figures 5.2, 5.3 and 5.4. The horizontal patterns are characterized on the surface and mid-water layers, in order to verify lateral and longitudinal patterns of thermohaline estuarine structure. 5.1 2013 Event Characterization In Figure 5.5, the salinity pattern for the three selected moments is represented for the surface layer (left panel), and the mid layer (right panel). Generally, the estuarine horizontal gradient is similar near the surface and at mid-depth. During the extreme freshwater event, a strong gradient is visible on the surface layer (Figure 5.5c), where a distinct salinity variation, from 0 to 36 salinity, units is visible. Before and after the event, the horizontal salinity structure is similar both on the surface and at mid-depth. Before the event (March 24 - Figures 5.5a and 5.5b), the horizontal gradient of salinity inside the estuary is visible, with values of 0 near the rivers and higher than 30 near the estuary mouth. Since nearly 20 km from Vila Franca de Xira point, the freshwater from the Tagus and Sorraia rivers starts gradually mixing with the saltwater and the estuary becomes more saline towards the marine region of the estuary, where the salinity is about 36. During the event (Figures 5.5c and 5.5d), the estuary is almost full of freshwater (with salinity values of 0). The mixing region is only visible between the navigation channel and the outside region of the estuary. The estuarine plume is intense and disperses along the adjacent coastal zone for the surface layer, according to the results obtained by Vaz et al. (2018). At the mid layer, the estuary is also filled with freshwater, but the estuarine plume is considerable smaller, covering a lower one on the coast (with values between 16 and 32). In 36 Figure 5.5: Daily mean salinity for the Tagus Estuary (Surface, on the left, and Mid Water layer, o the right) to the Reference Scenario: a), b) before - March 24; c), d) during - April and e), f) after the event - April 11. Figure 5.5: Daily mean salinity for the Tagus Estuary (Surface, on the left, and Mid Water layer, on the right) to the Reference Scenario: a), b) before - March 24; c), d) during - April 2; and e), f) after the event - April 11. 37 Figure 5.6: Daily mean water temperature for the Tagus Estuary (Surface, on the left, and Mid Water layer, on the right) to the Reference Scenario: a), b) before - March 24; c), d during - April 2; and e), f) after the event - April 11. 5.1 2013 Event Characterization Figure 5.6: Daily mean water temperature for the Tagus Estuary (Surface, on the left, and Mid Water layer, on the right) to the Reference Scenario: a), b) before - March 24; c), d) during - April 2; and e), f) after the event - April 11. 38 this layer, the mixing zone is restricted to the navigation channel and the surrounding area. The pattern observed in the Figures 5.5e and 5.5f is identical to the ones depicted on Figures 5.5a and 5.5b. However, the salinity values are lower after the event, reflecting the effect of the high discharge of freshwater. The mixing area is advected back to the the central region of the estuary and the intensity of the plume decreases again to values between 30 and 34. Figure 5.6 is the representation of the water temperature patterns, also for the three selected moments, near the surface on the left, and at mid layer of the water column on the right. Similarities between the surface layer and the mid-depth layer are visible inside the estuary for all the selected periods. In the marine region, this similarity can not be observed. In this figure, the mid layer is colder than the surface about 3°C. Before the event, Figures 5.6a and 5.6b, the freshwater is more than 2°C colder than the brackish water present in the central region of the estuary (mixing area). The southern region of the estuary is about 1.5°C warmer than the northern zone, reflecting the lower depth of this region. Moreover, as the river water is colder, the area where this water directly runs off is colder than the surrounding region. Due to this, the horizontal gradient is more intense in the southern region and in the river mouth.l During the event (Figures 5.6c and 5.6d), the influence of the colder freshwater is visible along the estuary. The difference between the northern and southern zones is of almost 8°C, and the mixing region is filled with freshwater. This influence is visible also at 60 km from Tagus river mouth, in the region outside of the estuary, where water temperatures of 14°C are reached. Vaz et al. (2018) observed the same results under these high freshwater discharge conditions. Figures 5.6e and 5.6f show a similar pattern of the water temperature patterns observed on March 24. 5.1 2013 Event Characterization The cold waters from the event still have an effects on the water temperature on April 11. The laterally variation between the north and south shores is about 1°C to 2°C. Near the river mouth, the water temperature is about 15°C and on the marine region is about 15°C to 16°C. 5.2 Predicted changes for the Tagus Estuary In this section, the influence of the predicted future change effects on extreme flow events as occurred in the 2013 winter will be studied using the idealized scenarios previously referred in section 4.4. Thus, the impact of air temperature increase (#Sc1) and SLR (#Sc2) in water properties and these two variables combined (low GHG emissions scenario - #Sc3 - and high GHG emissions scenario - #Sc4) will be evaluated. The analysis is performed for April 2 (extreme freshwater event) and all scenarios will be evaluated against the reference scenario (scenario minus the reference), which means that positive values represent an increase in the study variable value and negative values a decrease of the variable value. 39 5.2.1 #Sc1 5.2.1 #Sc1 5.2.1 #Sc1 Figure 5.7 considers the variations of vertical patterns of salinity, water temperature, and Brunt-Väisälä frequency, under a scenario of increasing air temperature. As expected, the air temperature has almost no influence in the salinity values (Figure 5.7a). For the first 30 km from Tagus river mouth, the difference between #Sc1 and #ScRef is null. After that, the salinity increases reaching the maximum in the outside of the estuary, where a residual value of 0.07 is observed in the bottom.f The longitudinal structure of water temperature presents a very low difference for #Sc1. Values lower than 0.2°C are depicted in Figure 5.7b, and well-mixed conditions are visible. The values of Brunt-Väisälä frequency (Figure 5.7c) are residual, always null along the estuary, with some regions (upstream and in the estuary mouth) with maximum values of 2 Figure 5.7: Vertical patterns under an increase of the air temperature, in the longitudinal section. Difference between #Sc1 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. Figure 5.7: Vertical patterns under an increase of the air temperature, in the longitudinal section. Difference between #Sc1 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. 40 cycles/h. For this reason, the air temperature increase of 1°C do not have a relevant influence on the section stability. Globally, the air temperature increase, per se, does not have any impact on the vertical patterns of salinity and N. An increase of the air temperature of 1°C increases the water temperature by about 0.2°C, i.e., about 25% of the air temperature increase.f For this scenario, differences in the horizontal spatial distribution of salinity and water temperature are depicted in Figure 5.8. Figure 5.8: Horizontal patterns under an increase of the air temperature, at the surface and in the middle of the water column. Difference between #Sc1 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. 41 Figure 5.8: Horizontal patterns under an increase of the air temperature, at the surface and in the middle of the water column. Difference between #Sc1 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. 41 The salinity difference between this scenario and the reference scenario at the surface layer and in the middle of the water column was represented in the Figures 5.8a and 5.8b, respectively. 5.2.1 #Sc1 The similarities between surface and middle layers are evident, inside of the estuary, where values are null. On the surface, outside the estuary, some regions have a salinity anomaly of 0.3 higher and lower than the reference scenario.f The Figures 5.8c and 5.8d show the water temperature differences, relatively to the reference scenario, also at surface and middle layers. These figures have a pattern similar to the Figures 5.6c and 5.6d. Values of variation are about 0 in the first kilometers from the river mouth. These water temperature values increase in the surrounding region, with the maximum above of 0.5°C in the southern region. The path of freshwater is evident, inducing lower differences relative to the reference scenario. Moreover, outside of the estuary, the water surface will become hotter than the middle layer, where the increase is almost null. The main difference between the north and south margins of the estuary is due to the shallowness of the south region, when compared to the north margin.l The increase air temperature will influence the horizontal patterns. The salinity remains mainly unaffected inside of the estuary. The water temperature will increase all over the estuary, except where the freshwater has the main influence. This influence tends to be larger in the shallow areas, according with a similar study developed by Fortunato et al. (2021). 5.2.2 #Sc2 The response of SLR of 0.40 m in the vertical structure of salinity, water temperature and Brunt-Väisälä frequency is depicted in Figure 5.9.f For this scenario, the maximum salinity differences of 1 are reached on the surface near the estuary mouth. This region will have more intense changes than the bottom (with salinity differences near 0). At the middle of the longitudinal section, salinity differences are about 0.8 near the bottom values that are higher than the surface differences (about 0.5). In the first 25 km near the river mouth, differences in salinity are null.l Comparatively to the previous scenario, the influence of SLR in the water temperature (Figure 5.9b) is weaker. Maximum values of 0.15°C are reached in the central region of the section. In addition, SLR does not seem to have influence in the upstream area.i In Figure 5.9c, the Brunt-Väisälä frequency values are higher than in #Sc1. Stratification decreases in all section, except between 20 km and 30 km from Tagus river mouth, where values exceeding 20 cycles/h are visible, which means a considerable decrease of mixing.f According to the results, the salinity is affected by 40 cm of SLR, except at the upstream zone of the estuary. This increase of salinity, when compared to the reference scenario, may also contribute to stratification changes in specific regions of the section. Water temperature has no contribution from the SLR. Figure 5.10 shows the horizontal patterns of the difference between this scenario and the reference of the surface and mid-layer salinity and water temperature. Salinity discrepancy between 40 cm of SLR and reference scenarios at the water surface and in the middle of the water column is represented in the Figures 5.10a and 5.10b, respectively. 42 Figure 5.9: Vertical patterns under a SLR, in the longitudinal section. Difference between #Sc2 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. Figure 5.9: Vertical patterns under a SLR, in the longitudinal section. Difference between #Sc2 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. Figure 5.9: Vertical patterns under a SLR, in the longitudinal section. Difference between #Sc2 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. The similarities between surface and middle layers patterns are evident, inside of the estuary. In the upstream region, the disparity of salinity is irrelevant in both cases. 5.2.2 #Sc2 In the mid-layer, the main differences present positive values of about 1, corresponding to an increase in the horizontal salinity, near the estuary mouth (marine region of the estuary). In the shallow areas of the south region, the differences are negative, corresponding to a decrease also about 1 units of salinity. According with Mills et al. (2020), the spatial distribution of salinity is dependent on the bathymetry. In the surface of the mixing zone, the salinity will not increase as much as the mid layer (0.5 less). At surface of the estuarine marine zone, the salinity increase is 0.5 higher than the mid-layer. In addition, the plume intensity reduction is expected, once the salinity values in this region tend to increase. Figures 5.10c and 5.10d show the water temperature differences, relatively to the reference scenario, also at surface and middle layers. Values of variation are about 0 in the first 43 kilometers from the river mouth and outside of the estuary. The water temperature values increase 0.1°C near the marine region of the estuary (in the navigation channel). The main differences are presented in the regions near the edge, which register a decrease of 0.5°C. In the central region of the estuary (the mixing region), the increase of the water temperature is about 0.3°C, comparatively to the reference scenario.i In general, the SLR have a significant impact on the vertical and horizontal patterns of In general, the SLR have a significant impact on the vertical and horizontal patterns of Figure 5.10: Horizontal patterns under a SLR scenario, at the surface and in the middle of the water column. Difference between #Sc2 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. Figure 5.10: Horizontal patterns under a SLR scenario, at the surface and in the middle of the water column. Difference between #Sc2 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. 44 salinity. This was also concluded by Mills et al. (2020), in a similar study. In the water temperatures, the SLR promotes a slight increase, according with Fortunato et al. (2021). Comparing both scenarios, SLR has a much more intense impact than the air temperature increase, in both vertical and horizontal patterns, of salinity and Brunt-Väisälä frequency. The response of water temperature is more sensible to the increase of the air temperature. 5.2.3 #Sc3 Figure 5.11 considers the variations of vertical patterns of salinity, water temperature, and N, under a scenario of increasing air temperature (+1°C) and SLR (+0.4 m). Figure 5.11 considers the variations of vertical patterns of salinity, water temperature, and N, under a scenario of increasing air temperature (+1°C) and SLR (+0.4 m).f The similarities between Figure 5.11a and Figure 5.9a are evident. The salinity difference Figure 5.11: Vertical patterns under an increase of the air temperature, in the longitudinal section. Difference between #Sc3 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. Figure 5.11: Vertical patterns under an increase of the air temperature, in the longitudinal section. Difference between #Sc3 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. 45 45 is more intense in scenario #Sc3, with values reaching 1. In the first 25 km from Tagus river mouth, differences in salinity are null. Between 25 and 35 km from the beginning of the section (mixing region), the salinity differences near the bottom (about 0.8) are higher than the surface differences (about 0.6). From 35 km to the end of the section (marine region), the differences values are higher at the surface (ranging between 0.5 and 1) than the bottom (with salinity differences between 0 and 0.5). Maximum values of about 1 unit of salinity are reached at surface of the 50 km and in the mid-layer of 45 km. In the water temperature, Figure 5.11b, the #Sc2 pattern (Figure 5.9b) is distinguished, but with values 2 times higher. In the first 20 km from Tagus river mouth, the differences in water temperature are null. From 20 km until 35 km , differences of 0.25°C are reached at the bottom and, between 0.1°C and 0.2°C, at the surface. Since 35 km until the end of the longitudinal section (near the marine region), the water temperature differences reaches maximum values of 0.3°C, at the surface, and about 0.15°C in the bottom, presenting a vertical gradient of about 1.25°C/40m. In Figure 5.11c, the oscillation of the values to Brunt-Väisälä frequency is about 4 cycles/h lower than the Figure 5.9b, but the pattern is the same. Stratification decreases in all section, except between 20 km and 30 km from Tagus river mouth, where values exceeding 15 cycles/h are visible. 5.2.3 #Sc3 For positive values of Brunt-Väisälä frequency differences, the stratification decrease is expected, once for negative values the stratification must increase substantially.f As previously concluded, the SLR scenario affects essentially the salinity and Brunt-Väisälä frequency, and the air temperature acts mainly on the water temperature. The balance in both forcing conditions intensify the three study variables. Figure 5.12 show the salinity and water temperature differences induced by #Sc3 scenario, relatively to the reference scenario, also at surface and middle layers.fi Figure 5.12a and 5.12b are a representation of the salinity differences. In these figures, the values and horizontal patterns are identical to the previous scenario, the main difference is the plume propagation at the surface. At #Sc2 scenario, the plume was confined in the estuary mouth and the adjacent coastal zone. Differently, in scenario #Sc3, the plume is extended to outside of the estuary. The reduction in the plume intensity is expected, once the salinity values in this region tend to increase between 0.25 and 1 units of salinity. The similarity between surface and middle layers patterns are evident, inside of the estuary. However, the layer in the middle of the water column will have a more intense increase along the navigation channel (about 1) than the surface layer (about 0.5). In the upstream region, the difference of salinity is irrelevant in both cases. In the south region (shallow areas), the differences are negative, corresponding to a decrease about 1 units of salinity. Regarding to the water temperature inside of the estuary, the patterns are also similar in both layers. The southern region demonstrate, in general, an increase between 0.25°C and 0.75°C, with intensification near the margins. The main difference in negative values represents a cooling higher than 0.5°C near the east land boundary. In the upstream region (freshwater region), the difference of water temperature is null in both cases. At surface after the estuary mouth, the water temperature increase of about 0.2°C is predicted. 46 Figure 5.12: Horizontal patterns under an increase of water temperature and SLR, at the surface and in the middle of the water column. Difference between #Sc3 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. Figure 5.12: Horizontal patterns under an increase of water temperature and SLR, at the surface and in the middle of the water column. 5.2.3 #Sc3 Difference between #Sc3 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. The results show that the southern regions have more changes that the upstream region. In general, as would be expected, the water temperature increase in estuary and the salinity increase only near of the navigation channel and estuary mouth. The results show that the southern regions have more changes that the upstream region. In general, as would be expected, the water temperature increase in estuary and the salinity increase only near of the navigation channel and estuary mouth. 5.2.4 #Sc4 The SLR of 0.7 m and the increase of 4.0°C was considered to represent the high GHG emissions scenario - #Sc4. In the Figure 5.13 the difference between this scenario and the reference scenario in the vertical section, in terms of salinity, water temperature, and Brunt-Väisälä frequency is presented. 47 The #Sc3 salinity vertical patterns are also visible in this scenario, although they are more intense (about 0.5). In the first 25 km from Tagus river mouth, the differences in salinity are null. From 25 km until 35 km (which corresponds to the mixing region), salinity differences near the bottom (about 1.2) are more intense than the surface differences (about 0.8). Since 35 km to the marine region in the end of the section, the differences values are higher at the surface (ranging between 0.5 and 2) than the bottom (with salinity differences between 0 and 1). Maximum values of about 2 unit of salinity are reached at surface of the 50 km and in the mid-layer of 45 km. In the Figure 5.13b, the water temperature has also the same pattern of the previous scenario, but with an increase of about 0.4°C, excluding the upstream region (the first 20 km from the river mouth) where the difference is zero. Between 20 km and 30 km from the Figure 5.13: Vertical patterns under an increase of the air temperature, in the longitudinal section. Difference between #Sc4 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. Figure 5.13: Vertical patterns under an increase of the air temperature, in the longitudinal section. Difference between #Sc4 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. Figure 5.13: Vertical patterns under an increase of the air temperature, in the longitudinal section. Difference between #Sc4 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. 48 48 river mouth, the horizontal gradient is about 0.5°C/10km. Since 30 km from the upstream region until the end of the longitudinal section (near the marine region), differences between 0.4°C and 0.7°C are reached at the bottom and, between 0.7°C and 0.9°C, at the surface, presenting a vertical gradient of about 0.4°C/40m. In relation to Brunt-Väisälä frequency (Figure 5.13c), at the regions where the stratification increase was predicted in the scenario #Sc3, this increase will intensify about 5 cycles/h. 5.2.4 #Sc4 With the same action on the negative values where the mixing increase, in #Sc4 this mixing was also intensified about 5 cycles/h. Comparatively with reference scenario, stratification decreases in all section, except between 20 km and 30 km from Tagus river mouth, where values exceeding 15 cycles/h are visible. In general, it is evident the knowledge about vertical patterns resulting from the SLR and the increase of air temperature. Although the values expected for the salinity, water temperature, and stratification will change with the amount of increase. The Figure 5.14 represent the horizontal patterns of the salinity and water temperature differences induced by #Sc3 scenario, relatively to the reference scenario, also at surface and middle layers. Analogous to the Figure 5.13 analysis, the horizontal patterns look similar to the #Sc3 scenario. Figure 5.14a and 5.14b are a representation of the salinity differences, at surface and in the middle of the water column. The salinity represented in this figure presents the same pattern that the previous scenario but with an increase of 1, representing the reduction in the estuarine plume intensity. In the upstream region, the difference in salinity is null. In the shallow areas, the differences are negative, corresponding to a decrease of about 2 units of salinity. The similarity between surface and mid layers patterns is also evident inside of the estuary. However, the mid-layer will have a higher increase along the navigation channel (about 1.5) than the surface layer (about 1).f In the Figures 5.14c and 5.14d, water temperature differences in the #Sc4 scenario, is also represented in two horizontal layers. The water temperature has a several increase (about 1.5°C), despite the patterns are also similar to the previous scenarios (in both layers). In the upstream region (freshwater region), the difference of water temperature is irrelevant. In the southern zone, an increase of 2°C relative to the reference scenario is expected. At the surface outside of the estuary, the increase between 0.5°C and 1.0°C in the water temperature is predicted. The horizontal patterns of high GHG emissions scenario show an increase of the salinity, water temperature and stratification. For the implementation applied in this dissertation, the SLR have a more significant role than the air temperature increase, in the changes of salinity, water temperature, and N patterns. 5.2.4 #Sc4 In the longitudinal section of the estuary, the first 20 km from Tagus river mouth), the three study variables will not change its values. From 20 km to the estuary mouth, the increase of air temperature and the SLR will increase the salinity, water temperature and mixing. In the freshwater region (near the Tagus river mouth), the extremely high freshwat 49 Figure 5.14: Horizontal patterns under an increase of water temperature and SLR, at the surface and in the middle of the water column Difference between #Sc4 and #ScRef in terms Figure 5.14: Horizontal patterns under an increase of water temperature and SLR, at the surface and in the middle of the water column. Difference between #Sc4 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. Figure 5.14: Horizontal patterns under an increase of water temperature and SLR, at the surface and in the middle of the water column. Difference between #Sc4 and #ScRef, in terms of: a) Salinity; b) Water Temperature; and c) Brunt-Väisälä frequency. discharge inhibit the air temperature and SLR effects. In the shallow regions of the estuary, the forcings used promote an increase of the water temperature and a decrease of salinity. At the estuary mouth (near the marine region) and in the mixing region, the increase of air temperature and sea level improves the increase of water temperature and salinity. 50 50 6 Conclusions Predicted changes and energy production choices in the next years will determine how high sea levels rise and air temperature increase will turn. Extreme freshwater events identical to the occurred in 2013 at Tagus estuary will have direct impacts in the population. The scientific evidence exposed in the introduction chapter shows a tendency to the increase of the number of extreme events and their severity, highlighting the importance of studies like this. Numerical models developed along the past years will have a key role to analyze multidisciplinary estuarine aspects. The impact of future changes in extreme events like the occurred in winter of 2013, at Tagus estuary, was studied. For that, the hydrodynamics of the Tagus estuary and coastal adjacent region were characterized. Then, a numerical model application (Delft3D-Flow) was developed to reproduce the propagation in the Tagus estuary. The changes improved by different scenarios in conditions of high freshwater discharges were characterized and the impact of changes on salinity, water temperature and stratification patterns was evaluated. Thus, the goals proposed where achieved. In this work, the Delft3D model brings valuable knowledge about the vertical and horizontal patterns of salinity, water temperature, and Brunt-Väisälä frequency of Tagus Estuary, considering changes of its natural balance. Moreover, the methodology developed in this study can be replicated to other coastal systems contributing to the increase of the knowledge about predicted climate change effects in similar occurrences. The reference scenario, which represent the actual conditions of the Tagus Estuary, was studied. Along the vertical section and during the event, the salinity values were lower than usual, with the maximum (near the estuary mouth) of about 24. Nine days later, still remaining some event effects as low salinity. In relation to the water temperature, March 21 and April 11 results showed the usual pattern, with the water being warmer at the middle of the longitudinal section, while during the event maximum values were located in the estuary mouth, revealing an influence of water with lower temperature values from the river.i The stratification in the estuary was also evaluated. In the region near Tagus river mouth, the Brunt-Väisälä frequency was low, i.e., this area is well-mixed. This frequency increases substantially, on the surface, since the middle of the section until the estuary mouth. During 51 the event, the stratification was high in all water column. Conclusions After that, mixing increase relative to before the event, according to the water temperature patterns. Before the extreme freshwater event, the horizontal mixing of salinity inside the estuary was visible. While during the event, most of the estuary were almost full of fresh water, which makes the gradient only visible since the navigation channel and outside of the estuary. In addition, the difference between the surface and the middle of the water column patterns inside of the estuary was very small. In the outside region of the estuary, these differences were considerable. The horizontal patterns revealed that the water from the river was considerably colder than the water of the central region of the estuary and the southern region of the estuary (shallow area) was warmer than the northern zone (deeper area). As during the event there was more fresh water, the estuary water temperature is lower than the normal conditions. The air temperature increase (scenario #Sc1) improve the water temperature increase but do not have an important impact on the vertical patterns of salinity and Brunt-Väisälä frequency.if The salinity and stratification were affected by 40 cm SLR, except in the upstream region of the estuary. However, the SLR do not have a significant influence in water temperature. Comparing both scenarios, SLR has a much more intense impact than the air temperature increase in both vertical and horizontal patterns, for salinity and Brunt-Väisälä frequency. In opposition, the response of water temperature was promoted by the increase of the air temperature. In the analysis of #Sc3 and #Sc4 was possible to conclude that the SLR scenario acts essentially on salinity and Brunt-Väisälä frequency, and the air temperature in the water temperature, so there was a balance in both forcing conditions, intensifying the three study variables. Although the impact of SLR was higher than the impact of air temperature increase, both are related, which means that if the air temperature values increase, it is possible that the SLR increases too. Moreover, the estuary mouth and the southern regions will have more changes in the water temperature and salinity values than the upstream region of the estuary. The air temperature and SLR do not have considerable effects in this region, possibly because the flow is so intense. The projected changes are greater for the scenario SSP5-8.5 and considerably smaller for the SSP1-2.6. Conclusions As referred by (Ferrero et al., 2021), this enhance the importance of public policies towards lower emission scenarios. Thus, it is crucial to extend the studies developed so far. In the future, the influence of another changes, as freshwater discharge, must be studied. Additionally, the analysis of velocity patterns must be done as well. Lagrangian models also provide an alternative tool for applying the implemented simulations. It is also interesting if risk maps were developed for similar events. The methodology applied in this work must be used to water quality and sediments studies, as it was done by Rodrigues et al. (2021). Despite all the knowledge given by 3D modelling, it is only useful when applied. Thereby, 52 all efforts must be focused to reverse the cycle of decline in ocean health, and consequently, estuarine systems decadence. The goal should be to ensure that ocean science supports countries in creating improved conditions for sustainable development of the ocean, together with stakeholders worldwide. all efforts must be focused to reverse the cycle of decline in ocean health, and consequently, estuarine systems decadence. The goal should be to ensure that ocean science supports countries in creating improved conditions for sustainable development of the ocean, together with stakeholders worldwide. 53 References Alvera-Azcárate A., Ferreira J. and Nunes J. (2003). Modelling eutrophication in mesotidal and macrotidal estuaries. the role of intertidal seaweeds. Estuarine, Coastal and Shelf Science, 57(4):715–724. Barros A.P. (1996). An evaluation of model parameterizations of sediment pathways: a case study for the tejo estuary. Continental Shelf Research, 16(13):1725–1749.i Boehrer B. and Schultze M. (2009). Density stratification and stability. Encyclopedia of Inland Waters, 583–593. 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https://openalex.org/W4231836142	https://www.researchsquare.com/article/rs-10623/latest.pdf	English	null	Features of alternative splicing in stomach adenocarcinoma and their clinical implication: A research based on massive sequencing data	Research Square (Research Square)	2,020	cc-by	9,059	Features of alternative splicing in stomach adenocarcinoma and their clinical implication: A research based on massive sequencing data Yuanyuan Zhang (  u201310506@hust.edu.cn ) Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology https://orcid.org/0000-0001-9778-745X Yuanyuan Zhang (  u201310506@hust.edu.cn ) Yuanyuan Zhang (  u201310506@hust.edu.cn ) Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology https://orcid.org/0000-0001-9778-745X Shengling Ma Wuhan Union Hospital Qian Niu Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Yun Han Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Xingyu Liu Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Jie Jiang Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Simiao Chen Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology Haolong Lin Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology DOI: https://doi.org/10.21203/rs.2.20011/v6 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 on August 24th, 2020. See the published version at https://doi.org/10.1186/s12864-020-06997-x. Research article Keywords: bioinformatic analysis, alternative splicing, stomach adenocarcinoma, survival, splicing factor, prognosis Posted Date: August 18th, 2020 DOI: https://doi.org/10.21203/rs.2.20011/v6 Version of Record: A version of this preprint was published on August 24th, 2020. See the published version at https://doi.org/10.1186/s12864-020-06997-x. Page 1/26 Page 1/26 Page 1/26 Abstract Background: Alternative splicing (AS) offers a main mechanism to form protein polymorphism. A growing body of evidence indicates the correlation between splicing disorders and carcinoma. Nevertheless, an overall analysis of AS signatures in stomach adenocarcinoma (STAD) is absent and urgently needed. Results: 2042 splicing events were confirmed as prognostic molecular events. Furthermore, the final prognostic signature constructed by 10 AS events gave good result with an area under the curve (AUC) of receiver operating characteristic (ROC) curve up to 0.902 for 5 years, showing high potency in predicting patient outcome. We built the splicing regulatory network to show the internal regulation mechanism of splicing events in STAD. QKI may play a significant part in the prognosis induced by splicing events. Conclusions: In our study, a high-efficiency prognostic prediction model was built for STAD patients, and the results showed that AS events could become potential prognostic biomarkers for STAD. Meanwhile, QKI may become an important target for drug design in the future. Background Gastric cancer (GC) is the fourth major cancer threat to human health in the world whose etiology remains unclear, with 989,000 new cases and 738,000 deaths every year [1]. Most (about 90%) of gastric cancers are adenocarcinomas, which originate from the epithelial cells in the most superficial layer of the gastric wall and are caused by malignant changes in gastric gland cells. Although significant progress has been made in the study of epidemiology, pathological mechanisms and treatment options, the medical burden still exceeds expectations [2]. It is not optimistic that complete surgical resection is still the only solution for doctors to treat gastric cancer [3, 4]. The widespread implementation and application of adjuvant and neoadjuvant therapy have increased the 5-year overall survival rate by 10-15%, but it is worth thinking that there is no global consensus on the best treatment scheme [2]. Therefore, it is urgent to explore new and accurate biomarkers to evaluate the diagnosis and prognosis of STAD patients. Eukaryotic cells produce various regulatory changes and perform complex functions to adapt to changes in the environment, largely due to the diversity of proteins. A common mechanism is that a limited number of gene sets produce a large number of mRNA isoforms through alternative splicing of pre-mRNA [5]. Alternative splicing actually regulates gene expression at the intron/exon level [6-8]. In addition, alternative splicing causes the premature occurrence of termination codon in mRNA, which degrades immediately upon discovery to prevent its translation [9]. Therefore, alternative splicing is a key biological process in cells, and different mRNA splicing isoforms make the final protein products perform different functions. More and more studies have found that splicing disorder can be used as a marker of tumor development [10] and as a key mechanism involved in the broad biological process of cancer [11, 12]. It is noteworthy that some important splicing factors can change the alternative splicing mode of target genes, thus forming a favorable environment for promoting the occurrence and development of cancer [13]. In Page 2/26 Page 2/26 general, comprehensive and in-depth analysis of alternative splicing can dig out potential biomarkers of malignant tumors, so as to assist physicians in clinical diagnosis and prognosis judgment [12, 14]. general, comprehensive and in-depth analysis of alternative splicing can dig out potential biomarkers of malignant tumors, so as to assist physicians in clinical diagnosis and prognosis judgment [12, 14]. Background We used a variety of bioinformatics analysis methods to explore prognostic factors in STAD. COX regression analysis helped us screen out significant prognostic markers for further study. According to the regulatory relationship between AS events and splicing factors in STAD, a clear network diagram was drawn to find out the potential mechanism. These results provide a basic direction for further exploration of the molecular mechanism and diagnostic markers of STAD. 2.2. Molecular characteristics of survival related AS events The distributions of AS events significantly correlated with patient survival are displayed in Figure 2A. The 20 most significant prognosis-related AS events are also shown (Figure 2B-H). To reveal the molecular characteristics of genes with survival-associated AS events, several bioinformatics analyses were conducted. First, a PPI network was constructed to demonstrate the relationships among these genes. UBA52, STAT3 and PLK4 ranked at the core in the network (Figure 3). According to the functional annotations, "organelle organization", "positive regulation of cellular process" and "protein localization" were the three most significant biological process terms. “intracellular organelle”, “membrane-bounded organelle” and “intracellular membrane-bounded organelle” were the three most significant cellular component terms. For molecular function, “enzyme binding” and “GTPase binding” were two most enriched categories (Figure 4). 2.1. Survival associated AS events As a whole, there are 4006 AA events in 2799 genes, 3450 AD events in 2401 genes, 10004 AP events in 4025 genes, 8390 AT events in 3666 genes, 19121 ES events in 6973 genes, 226 ME events in 219 genes, and 2944 RI events in 1956 genes for evaluation of prognostic value (Figure 1A). The initial clinical data downloaded from the TCGA website is in the supplementary files (Additional file 1). A total of 157 AA events in 153 genes, 174 AD events in 164 genes, 461 AP events in 304 genes, 297 AT events in 203 genes, 805 ES events in 660 genes, 18 ME events in 18 genes, and 130 RI events in 113 genes were identified as prognostic AS events (P<0.05) (Figure 1B, Additional file 2). Thus, one gene might have two or more AS events that were markedly related to the survival of STAD patients. The ES which was vividly revealed by the UpSet plot was the most common prognosis-related event, and a gene could have up to seven prognosis-related events (Figure 1C). Results 2.1. Survival associated AS events 2.4. Survival-associated SF-AS network Because events are primarily orchestrated by SFs that often bind with pre-mRNAs and regulate RNA splicing via influencing exon selection and splicing site. Therefore, exploration of the SF-AS regulatory network is imperative in STAD. Next, correlation analyses between the SFs’ expression and the most significant AS events’ PSI value (P<0.001) were conducted (Figure 11A). We observed that QKI was most significantly connected in the network, so we compared the influence of QKI expression on STAD’s survival rate. The consequence showed that low QKI expression significantly improved the survival rate of patients with STAD, and the five-year survival rate of the patients with low QKI expression was almost twice that of the patients with high QKI expression (Figure 11B, Figure 11C). 2.3. Prognostic signatures for STAD patients By applying the LASSO Cox analysis following univariate Cox, which aims to filter out redundant genes and prevent overfitting of the model, we developed seven types of optimal prognostic signatures based on AA, AD, AP, AT, ES, ME and RI (Figure 5, Table 2). According to Figure 6, the eight models we Page 3/26 Page 3/26 Page 3/26 constructed were able to separate the high and low risk groups well, because the differences between the high and low risk groups were very significant (although there were some overlaps of confidence intervals in the several subfigures, the overlaps were less, and the p values were very small, and the differences were significant). Therefore, the eight models could be used to predict the clinical results of STAD patients in clinical practice (Figure 6). Eight ROC curves validated the performance of prognostic signatures in prognosis prediction, and their AUC values were all greater than 0.7, indicating that these eight models had certain accuracy (Figure 7). Figure 8 shows the patient's survival status and risk score, as well as the splicing pattern of AS signatures for each AS type or a combination of seven AS types. The upper risk score curve classified patients with low and high risk. The middle survival status figure indicated that the risk value was related to survival; although the decline of survival time was not obvious, the survival status was different. The bottom heat map shows the PSI value change of AS events with the increase of the risk value, in which if the PSI value of an AS event increases with the risk value, it indicated that the AS event was a high-risk AS event (Figure 8). In univariate Cox analysis, the eight riskScores we constructed were all correlated with prognosis and high-risk factors (Figure 9). According to multivariate independent prognostic analysis, riskScores obtained by the eight models could be used as independent prognostic factors, and all of them were high-risk factors (Figure 10). Discussion Currently, scientific research on the role of AS events in STAD still has many unanswered questions owing to the shortage of available large-sample public AS profiles and the paucity of systematic analysis referring to their clinical significance and deep molecular function. These bottlenecks have prevented cancer researchers from effectively recognizing the widespread applicability of AS events in STAD. Exploration of AS patterns broadens our vision and our understanding of traditional transcriptome molecular biomarkers. In this project, we adopted several biomedical computational approaches, which integrate the AS event profiles and clinical information of STAD patients to mine prognosis-related AS and construct splicing prognostic signatures that could stratify STAD patients into subgroups with distinct survival outcomes. Moreover, the SF-AS network could provide further insights into regulatory mechanisms in patients with STAD from the perspective of splicing. Page 4/26 Page 4/26 Gastric cancer is a highly heterogeneous malignant tumor. Therefore, a single drug is not significantly useful for various types of gastric cancer. Classical cytotoxic therapy cannot be fully effective because of the presence of patients resistant to specific drugs. At present, the diagnosis and treatment of gastric cancer rely on histopathological diagnosis and definite classification. Therefore, in addition to targeted treatment with trastuzumab, we need to develop new targeted drugs to provide better treatment for patients. Potential biomarkers can be mined and used to predict patient outcomes, and treatment strategies can be developed for specific tumor parameters. The next-generation sequencing technology developed in recent years adopts the whole- genome sequencing method, which has great advantages in exploring alternative splicing. Previously, several studies conducted SpliceSeq analyses to generate alterative splicing profiles for some types of cancer, as well as to construct prognostic signatures for cancer prognosis monitoring, including non-small cell lung cancer [15], colorectal cancer [16], and esophageal cancer [17]. This computational bioinformatics analysis could open up different perspectives on the clinical application and potential pathological mechanism of AS on a macro level. Previously, several studies have proposed transcriptomic signatures related to epithelial-to-mesenchymal transition and diagnosis of gastric cancer [18, 19]. The present in- depth study further explored alterations of transcriptomes used as prognostic predictors and could broaden our horizons in the clinical significance of transcriptomic signatures. Given the multitude of AS events impacted by their own pre-mRNAs, the downstream functional impact is partly used to describe the molecular function of AS alteration events. Discussion RhoA-mediated Fbxw7 regulates the apoptosis of tumor cells and other phenotypes in gastric cancer [33]. Similarly, Gastrokine 1's inhibition of gastric cancer progression may also be dependent on RhoA [34]. Our findings suggest that a group of AS events play a biological role in the alteration of GTPase in STAD. The highlight of the current study was that we proposed prognostic signatures based on AS events for monitoring the prognosis of STAD patients. Recently, some prognostic signatures in STAD have been proposed. Zhang H et al. found that the efficacy of postoperative adjuvant chemotherapy for gastric cancer was affected by the degree of neutrophil infiltration of the tumor [35]. Jiang Y et al. developed an immune score GC classifier that can effectively predict the recurrence and survival of patients with gastric cancer, which plays a good role in complementation of the prognosis judgment for the TNM staging system [36]. The clinical management of STAD patients still needs to be improved, and the above mentioned molecular biomarkers have broad prospects. In order to facilitate clinical practice, we selected a group of AS events using the LASSO Cox regression model, and the prognostic model proposed on which showed satisfactory results. However, our prognostic model has limitations because our work completely based on the bioinformatic analysis and lacked an independent validation cohort. In addition, in order to provide more explanation details, our study also needed a wet lab validation. Due to the limited public alternative splicing data currently, the sample size used to construct a prognostic model is small. If these samples are forcibly divided into a training group and a validation group, the sample size used to construct the prognostic model will be less, leading to poor accuracy of the prognostic model. Besides, on account of the Corona virus disease 2019 pandemic, our Wuhan laboratory has been closed for a long time, and there is no objective condition to collect clinical samples for genetic testing. We believe that the TCGA data we used were appropriately standardized. However, the Lasso model adopts the square loss function and applies the same tuning parameter to all variables. Once outliers exist in the data, the estimator obtained is biased, resulting in poor robustness. Therefore, it may be a potential problem that leads to the imperfect accuracy of the prognostic model. Discussion In the PPI network analysis, UBA52, STAT3 and PLK4 were the hub genes. Previous studies have shown that UBA52 and STAT3 are all considered to be related molecules involved in the biological process of STAD. For example, bioinformatics analysis has verified the correlation between UBA52 and GC progress and metastasis [20]. STAT3 is a crucial transcription factor that regulates the transcription of many genes. It plays an extremely important role in promoting the occurrence and development of gastric cancer, and chronic STAT3 activation is a key event to induce the occurrence and development of gastric cancer [21]. STAT3 can directly up-regulate the epithelial expression of TLR2 in gastric tumors, which is related to the low survival rate of GC patients [22]. STAT3 signaling drives transcription activation of EZH2 and mediates poor prognosis in gastric cancer [23]. STAT3 promotes the increased expression of lncRNA HAGLROS, which leads to further progress of gastric cancer [24]. PLK4 is a serine/threonine protein kinase that regulates centriole duplication. Its maladjustment can lead to abnormal centrosomal numbers, mitotic defects, chromosomal instability, and ultimately tumorigenesis [25]. The relevant study has also shown that PLK4 overexpression in gastric cancer induces centrosome amplification and chromosomal instability, and leads to inhibition of primary cilia formation [26]. These findings also pave the way for future clinical applications, and related target inhibitors are being widely studied and clinically tested as new anticancer drugs. Functional enrichment analysis showed that in STAD, the main molecular function of AS event gene related to prognosis is to bind to GTPase, so it may provide selective advantages for cancer cells by regulating GTPase. Increased RhoA activity leads to poorer survival outcomes for the Lauren diffuse type of gastric adenocarcinoma (DGA), and inhibition of RhoA can correct the drug resistance of DGA [27]. RacGAP1 is closely associated with malignant progression and poor survival [28]. Page 5/26 Page 5/26 Leptin promotes GC migration through the Rho/ROCK mechanism [29]. RASSF6 partially regulates the effect of mir-181a-5p on GC progression through the MAKP pathway [30]. It is worth considering that, in gastric cancer cells, RhoA promotes cell proliferation and RhoC stimulates cell migration and invasion, while RhoB functions contrary to RhoA and/or RhoC [31]. Therefore, targeted GTPase therapy is also being explored. For example, ALEX1 functions in gastric cancer through the PAR-1/Rho GTPase signaling pathway, becoming a new target for tumor inhibition [32]. 5.1. Data acquisition TCGA SpliceSeq [48] is a data portal that provides AS profiles across 33 tumors based on TCGA RNA-seq data. SpliceSeq evaluates seven types of splice events, including alternate acceptor (AA), alternate donor (AD), alternate promoter (AP), alternate terminator (AT), exon skip (ES), mutually exclusive exon (ME) and retained intron (RI). TCGA SpliceSeq processed the percent spliced in (PSI) value for cancer research analysis, which indicates the inclusion of a transcript element divided by the total number of reads for that AS event. Alterations in PSI values range from 0 to 100 (%), which suggests a shift in splicing events. The filtering condition for downloading data from the TCGA SpliceSeq website was the percentage of samples with PSI value ≥ 75. The AS events with standard diversion < 1 were removed. Clinical information of STAD patients was also obtained from the TCGA database. Only pathologically confirmed STAD patients with both follow-up and AS event data were included for our analysis. For clinical information downloaded from TCGA, we deleted patients with survival time <90 days or null data, and included a total of 338 patients (Table 1) for subsequent analysis. For advanced cancer patients with survival time <90 days (usually along with severe metabolic disorders such as cachexia and endotoxin), the effect of alternative splicing events in promoting cancer development is no longer accurate, so patients with survival time <90 days were removed. The same TCGA ID was used to integrate clinical information and AS events data. Discussion Robust analysis methods using outliers to process data for high-dimensional genetic data analysis have been developed and are rapidly gaining popularity. Among them, LAD (least absolute deviation) –LASSO is a method to combine the regression shrinkage and selection of LASSO and robustness of LAD for outliers and heavy-tailed errors [37, 38]. In theory, the test results may be more meaningful through robust variable selection. A large number of AS events are programmed by finite SFs in cells [39]. The altered profile of AS events in multiple tumor types emphasizes the important mechanism of splicing factors in cancer, which is disordered splicing [40]. It is increasingly believed that changes of SFs in STAD can be involved in tumorigenesis and progression through various mechanisms [41-43]. The splicing correlation network Page 6/26 Page 6/26 analysis has also found out the larger regulated nodes, indicating that they occupy a significant position in the SF-AS network. QKI, which is recognized as a tumor suppressor in a wide range of cancers, is highly connected in the network, which can play a significant part in the prognosis induced by splicing events [44-46]. Multiple-factor analysis of a related study shows that QKI expression is an independent prognostic factor for the survival of GC patients [47]. But the role of QKI in STAD has not been fully discussed yet. Our study indicates that the level of QKI expression is significantly correlated with the survival rate of patients with STAD, and it can become an important target for drug design in the future. Nevertheless, our algorithm suggested deregulated AS events as a hallmark of STAD. However, there are some limitations inevitably affecting the reliability of the study. Firstly, we didn’t use a separate cohort for more validation. Secondly, more functional experiments are needed to further investigate the impact of dysregulated AS events and SFs on carcinogenesis. 5.4. SF-AS regulatory network A compendium of 404 splicing factors was obtained from a previous study [53]. The expression profiles of SF genes were curated from the TCGA dataset. We selected axes between the expression value of SFs and PSI values of prognosis-related AS events to construct the SF-AS regulatory network according to the following conditions: P value less than 0.001 and the absolute value of Pearson's correlation coefficient more than 0.6. Then, we built the correlation plots via Cytoscape version 3.7.1. All R code and annotations have also been submitted (Additional file 3). 5.2. Survival analysis Page 7/26 In the survival analysis, the follow-up periods ranged from 90 days to 3720 days after removal of patients with survival less than 90 days. Univariate Cox analysis was conducted to assess the correlations between the PSI value (from 0 to 100) of each AS event and the survival data of STAD patients (P < 0.05). We input the corresponding genes into the Search Tool for the Retrieval of Interacting Genes (STRING) database [49], and the constructed protein-protein interaction (PPI) network was adjusted by Cytoscape software [50]. Meanwhile, we applied the ClueGO plug-in [51] in Cytoscape software for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and drew the enrichment network. The least absolute shrinkage and selection operator (LASSO) method is a widely used regression analysis method of high-dimensional predictors [52]. LASSO has been extended for use in Cox regression survival analysis and is ideal for high- dimensional data. We selected the LASSO Cox regression model to determine the accurate coefficient for each prognostic feature and to estimate the deviance likelihood via 1-standard error (SE) criteria. The coefficients and partial likelihood deviance were calculated with the “glmnet” package in R. 5.3. Prognostic signature construction The significant AS events in univariate Cox analysis were submitted to LASSO regression analysis to develop prognostic signatures based on seven types of AS events. Finally, prognostic signatures for survival prediction were calculated by multiplying the PSI values of prognostic indictors and the coefficient assigned by LASSO regression analysis. The riskScore of each patient was calculated according to the constructed prognostic signatures. Based on the median value of all patients' riskScores, all patients were divided into high and low risk groups. Then, survival analysis was carried out for the high and low risk groups to obtain the P-values of survival difference and survival curves. The ROC curve was plotted using the survivalROC package, primarily to determine the accuracy of the prognostic model. By incorporating the following parameters into multivariate Cox regression analysis, splicing-based prognostic signature was evaluated as independent predictors: age, gender, grade, stage, TMN stage. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials Not applicable Acknowledgements The authors would like to thank the TCGA Spliceseq and TCGA databases for the availability of the data. The authors would like to thank the TCGA Spliceseq and TCGA databases Not applicable Availability of data and materials Page 8/26 Page 8/26 Gene expression data and clinical information of STAD were publicly available in The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov/repository?facetTab=cases). In the left box, select TCGA-STAD and HTSeq - FPKM, add all files to Cart, and then Gene expression data of STAD can be downloaded; select TCGA-STAD, clinical and bcr xml in turn, add all files to Cart, and then clinical information of STAD can be downloaded. Alternative splicing data of STAD were downloaded from TCGA SpliceSeq website (http://projects.insilico.us.com/TCGASpliceSeq/PSIdownload.jsp). Select the Stomach Adenocarcinoma [STAD], all Splice Event Types in turn, and then the Alternative splicing data of STAD can be downloaded. Competing interests Competing interests The authors declare that they have no competing interests. No funding was received. No funding was received. Authors' contributions Authors' contributions YZ conceived of and designed the study. YZ, QN, YH and XL performed the literature search, generated the figures and tables, and wrote the manuscript. SM, QN, YH and JJ collected and analyzed the data, and critically reviewed the manuscript. SM, XL, SC and HL supervised the study and reviewed the manuscript. All authors read and approved the final manuscript. Acknowledgements Abbreviations Alternative splicing (AS) Stomach adenocarcinoma (STAD) The Cancer Genome Atlas (TCGA) Gene Ontology (GO) The least absolute shrinkage and selection operator (LASSO) The Lauren diffuse type of gastric adenocarcinoma (DGA) Least absolute deviation (LAD) Alternate donor (AD) Alternate donor (AD) Alternate promoter (AP) Alternate terminator (AT) Mutually exclusive exon (ME) Mutually exclusive exon (ME) Retained intron (RI) Percent spliced in (PSI) Search Tool for the Retrieval of Interacting Genes (STRING) Protein-protein interaction (PPI) Gene Ontology (GO) Area under the curve (AUC) Page 9/26 Page 9/26 References 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D: Global cancer statistics. CA Cancer J Clin 2011, 61(2):69-90. 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D: Global cancer statistics. CA Cancer J Clin 2011, 61(2):69-90. 2. Cutsem EV, Sagaert X, Topal B, Haustermans K, Prenen H: Gastric cancer. 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Zong FY, Fu X, Wei WJ, Luo YG, Heiner M, Cao LJ, Fang Z, Fang R, Lu D, Ji H et al: The RNA-binding protein QKI suppresses cancer-associated aberrant splicing. PLoS Genet 2014, 10(4):e1004289. 46. Zong FY, Fu X, Wei WJ, Luo YG, Heiner M, Cao LJ, Fang Z, Fang R, Lu D, Ji H et al: The RNA-binding protein QKI suppresses cancer-associated aberrant splicing. PLoS Genet 2014, 10(4):e1004289. 47. Bian Y, Wang L, Lu H, Yang G, Zhang Z, Fu H, Lu X, Wei M, Sun J, Zhao Q et al: Downregulation of tumor suppressor QKI in gastric cancer and its implication in cancer prognosis. Biochem Biophys Res Commun 2012, 422(1):187-193. 47. Bian Y, Wang L, Lu H, Yang G, Zhang Z, Fu H, Lu X, Wei M, Sun J, Zhao Q et al: Downregulation of tumor suppressor QKI in gastric cancer and its implication in cancer prognosis. Biochem Biophys Res Commun 2012, 422(1):187-193. 48. Ryan M, Wong WC, Brown R, Akbani R, Su X, Broom B, Melott J, Weinstein J: TCGASpliceSeq a compendium of alternative mRNA splicing in cancer. Nucleic Acids Res 2016, 44(D1):D1018-1022. 49. Szklarczyk D, Morris JH, Cook H, Kuhn M, Wyder S, Simonovic M, Santos A, Doncheva NT, Roth A, Bork P et al: The STRING database in 2017: quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res 2017, 45(D1):D362-D368. Page 13/26 Page 13/26 50. Su G, Morris JH, Demchak B, Bader GD: Biological network exploration with Cytoscape 3. Curr Protoc Bioinformatics 2014, 47:8 13 11-24. 51. Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A, Fridman WH, Pages F, Trajanoski Z, Galon J: ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics 2009, 25(8):1091-1093. 52. Tibshirani R: The lasso method for variable selection in the cox model. Statistics In Medicine 1997, 16:385-395. 53. Seiler M, Peng S, Agrawal AA, Palacino J, Teng T, Zhu P, Smith PG, Cancer Genome Atlas Research N, Buonamici S, Yu L: Somatic Mutational Landscape of Splicing Factor Genes and Their Functional Consequences across 33 Cancer Types. Cell Rep 2018, 23(1):282-296 e284. Tables 50. Table 2. Prognostic signatures for STAD. Tables Table 1. Clinical characteristics of STAD patients in the TCGA database. Table 1. Clinical characteristics of STAD patients in the TCGA database. Table 1. Clinical characteristics of STAD patients in the TCGA database. Table 1. Clinical characteristics of STAD patients in the TCGA database. Characteristics Total % All 338 100.00 Age (y) ≥65 189 55.92 <65 149 44.08 Gender Male 216 63.91 Female 122 36.09 Grade G1 7 2.07 G2 116 34.32 G3 215 63.61 G4 0 0.00 Stage I 42 12.43 II 112 33.14 III 153 45.27 IV 31 9.17 T stage T1 15 4.44 T2 72 21.30 T3 167 49.41 T4 84 24.85 M stage M0 319 94.38 M1 19 5.62 N stage N0 104 30.77 N1 95 28.11 N2 71 21.01 N3 68 20.12 Page 14/26 Type Formula AA AD AP KIAA1147-82046-AP(-26.932098)+CDKN3-27569- AP(-16.593603)+WEE1-14328-AP* (-4.589803)+RCAN1-60494-AP0.979582+MID1- 88465-AP3.529023+TUBA1A-21538- AP7.031309+PDZK1IP1-2893-AP(-77.071138) 1.072(1.047−1.099) 0.71 AP 7.031309+PDZK1IP1 2893 AP ( 77.071138) AT PPHLN1-21214-AT4.712066+ABCB5-78909- AT1.406777+TLN2-30978-AT(-3.649182)+TET2- 70188-AT2.736038+MFSD2B-52798-AT* (-4.976167)+BRSK1-52060-AT* (-3.402968)+ZC3H12D-78076-AT1.091190+IL7R- 71774-AT(-4.704211)+ZNF846-47399- AT1.093000+ZFYVE28-68559-AT(-1.498791) 1.183(1.130−1.239) 0.77 1.636(1.436−1.863) 0.781 ME Page 15/26 ME2.671001+GRB10-79717- ME1.261053+ITGB1-126615-ME1.273771 RI SRSF7-53276-RI(-1.721712)+RPS15-46490-RI* (-2.387697)+DTD2-27118-RI(-6.362278)+DUSP18- 61796-RI(-5.322993)+ADRA2C-68651-RI* (-5.026148)+LGALS3BP-43940-RI* (-2.549496)+BICD2-86883-RI* (-1.137099)+ALS2CL-64462-RI1.664472+THAP7- 61211-RI(-1.872850)+KAT5-16914-RI(-1.868716) 1.138(1.097−1.180) 0.902 All CD44-14986-ES(-6.883160)+PPHLN1-21214- AT5.396354+RASSF4-11351-ES (-14.293402)+KIAA1147-82046-AP* (-22.278949)+PPP2R5D-76200-ES* (-6.035515)+LOH12CR1-20507-ES* (-10.075293)+CDKN3-27569-AP* (-18.653016)+UBA52-48486-AD* (-3.222755)+CADPS-65499-AT(-1.957771)+SRSF7- 53276-RI(-2.217781)+WEE1-14328-AP* (-5.279043) 1.043(1.030−1.057) 0.882 All Figures Page 16/26 Figure 1 P i l t d lt ti li i (AS) t (A) Th b f AS t d di Figure 1 Prognosis-related alternative splicing (AS) events. (A) The number of AS events and corresponding gen included in the present study; (B) The number of prognosis-related AS events and corresponding genes obtained by using univariate COX analysis; (C) UpSet plot of interactions between the seven types of survival associated AS events in STAD. One gene may have up to seven types of alternative splicing to associated with patient survival. Figure 1 Figure 1 Prognosis-related alternative splicing (AS) events. (A) The number of AS events and corresponding genes included in the present study; (B) The number of prognosis-related AS events and corresponding genes obtained by using univariate COX analysis; (C) UpSet plot of interactions between the seven types of survival associated AS events in STAD. One gene may have up to seven types of alternative splicing to be associated with patient survival. Page 17/26 Figure 2 Figure 2 Top 20 most significant alternative splicing (AS) events in STAD. (A) Volcano plot of AS events. Each dot represents an AS event that occurs in a gene. The red dots represent AS events that are significantly correlated with patient survival. The blue dots represent AS events without correlation. Both z-score and p- value are statistical values generated by the previous univariate COX analysis, and they have a corresponding relationship (that is, the p-value can be obtained by searching the table according to z- score). Z-score represents Wald statistic, z-score > 0 corresponds to high risk AS events, z-score < 0 corresponds to low risk AS events. So all dots form a parabola finally. The top 20 AS events correlated Figure 2 Top 20 most significant alternative splicing (AS) events in STAD. (A) Volcano plot of AS events. Each dot represents an AS event that occurs in a gene. The red dots represent AS events that are significantly correlated with patient survival. The blue dots represent AS events without correlation. Both z-score and p value are statistical values generated by the previous univariate COX analysis, and they have a corresponding relationship (that is, the p-value can be obtained by searching the table according to z- score). Z-score represents Wald statistic, z-score > 0 corresponds to high risk AS events, z-score < 0 corresponds to low risk AS events. So all dots form a parabola finally. The top 20 AS events correlated Figure 2 Top 20 most significant alternative splicing (AS) events in STAD. (A) Volcano plot of AS events. Each dot represents an AS event that occurs in a gene. The red dots represent AS events that are significantly correlated with patient survival. The blue dots represent AS events without correlation. Both z-score and p- value are statistical values generated by the previous univariate COX analysis, and they have a corresponding relationship (that is, the p-value can be obtained by searching the table according to z- score). Z-score represents Wald statistic, z-score > 0 corresponds to high risk AS events, z-score < 0 corresponds to low risk AS events. So all dots form a parabola finally. The top 20 AS events correlated Top 20 most significant alternative splicing (AS) events in STAD. (A) Volcano plot of AS events. Each dot represents an AS event that occurs in a gene. The red dots represent AS events that are significantly correlated with patient survival. The blue dots represent AS events without correlation. Both z-score and p value are statistical values generated by the previous univariate COX analysis, and they have a corresponding relationship (that is, the p-value can be obtained by searching the table according to z- score). Z-score represents Wald statistic, z-score > 0 corresponds to high risk AS events, z-score < 0 corresponds to low risk AS events. So all dots form a parabola finally. The top 20 AS events correlated Page 18/26 Page 18/26 Page 18/26 with clinical outcome based on acceptor sites (B), alternate donor sites (C), alternate promoters (D), alternate terminators (E), exon skips (F), mutually exclusive exons (G), and retained introns (H). Figure 3 Protein-protein interaction network of genes with survival-associated alternative splicing events in STAD. For nodes, low degrees correspond to small sizes and bright colors, and high degrees correspond to large sizes and dark colors; For edges, low combined_scores correspond to small sizes and bright colors, and high combined_scores correspond to large sizes and dark colors. Figure 3 Protein-protein interaction network of genes with survival-associated alternative splicing events in STAD. For nodes, low degrees correspond to small sizes and bright colors, and high degrees correspond to large sizes and dark colors; For edges, low combined_scores correspond to small sizes and bright colors, and high combined_scores correspond to large sizes and dark colors. Protein-protein interaction network of genes with survival-associated alternative splicing events in STAD. Figure 2 For nodes, low degrees correspond to small sizes and bright colors, and high degrees correspond to large sizes and dark colors; For edges, low combined_scores correspond to small sizes and bright colors, and high combined_scores correspond to large sizes and dark colors. Page 19/26 Page 19/26 Figure 4 Cluego analysis for GO terminology (show only pathways with P-value ≤ 0.001). And each node represents a GO term, each line reflects the correlation between the terms, and the color embodies the function enrichment classification of the nodes, with same function aggregating together in same color. Figure 4 Cluego analysis for GO terminology (show only pathways with P-value ≤ 0.001). And each node represents a GO term, each line reflects the correlation between the terms, and the color embodies the function enrichment classification of the nodes, with same function aggregating together in same color. Cluego analysis for GO terminology (show only pathways with P-value ≤ 0.001). And each node represents a GO term, each line reflects the correlation between the terms, and the color embodies the function enrichment classification of the nodes, with same function aggregating together in same color. Cluego analysis for GO terminology (show only pathways with P-value ≤ 0.001). And each node represents a GO term, each line reflects the correlation between the terms, and the color embodies the function enrichment classification of the nodes, with same function aggregating together in same color. Page 20/26 Page 20/26 Figure 5 Construction of prognostic signatures based on LASSO COX analysis. Each right subfigure shows the Log Lambda value corresponding to the minimum cross-validation error point. And the AS events with non-zero coefficient corresponding to the same Log Lambda value were selected in the left figure for subsequent model construction. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons Figure 5 Figure 5 Construction of prognostic signatures based on LASSO COX analysis. Each right subfigure shows the Log Lambda value corresponding to the minimum cross-validation error point. And the AS events with non-zero coefficient corresponding to the same Log Lambda value were selected in the left figure for subsequent model construction. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. Page 21/26 Page 21/26 Page 21/26 Figure 6 Kaplan-Meier curves of prognostic predictors for STAD. The lighter blue and red regions of each subfigure are the range of fluctuations, meaning 95%CI of the curve. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. Figure 6 Figure 6 Kaplan-Meier curves of prognostic predictors for STAD. The lighter blue and red regions of each subfigure are the range of fluctuations, meaning 95%CI of the curve. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. Page 22/26 Figure 7 Figure 7 Page 22/26 ROC curves of prognostic predictors for STAD. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. ROC curves of prognostic predictors for STAD. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. Figure 8 Determination and analysis of the prognostic AS signatures in the STAD cohort. STAD patients were divided into high- and low-risk subgroups based on the median cut of the risk score calculated separate The upper part of each assembly represents the risk score curve (low risk patients are represented by green dots, high risk patients by red dots, and dash lines correspond to the median of all samples riskScore). The middle section represents the distribution of survival status and survival time of patient ranked by risk score (more green dots on the left for low-risk patients, and more red dots on the right for high-risk patients. From left to right, with the increase of the risk value, more and more patients died, indicating that the risk value is related to survival). The bottom heatmap displays the splicing pattern o the AS signature from each AS type or all seven AS types (the color transition from green to red indicate that the PSI value of the corresponding AS event increases from low to high). (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Figure 9 Univariate Cox regression analysis of clinical parameters and riskScore in STAD. For a clinical parameter or riskScore, if the P-value is less than 0.05, it is related to survival; if Hazard ratio is greater than 1, it is a high-risk factor. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. Univariate Cox regression analysis of clinical parameters and riskScore in STAD. For a clinical parameter or riskScore, if the P-value is less than 0.05, it is related to survival; if Hazard ratio is greater than 1, it is a high-risk factor. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. Univariate Cox regression analysis of clinical parameters and riskScore in STAD. For a clinical parameter or riskScore, if the P-value is less than 0.05, it is related to survival; if Hazard ratio is greater than 1, it is a high-risk factor. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. Figure 10 Multivariate Cox regression analysis of clinical parameters and riskScore in STAD. For the results of univariate and multivariate independent prognostic analysis, if the riskScore P-value of both is less than 0.05, it indicates that riskScore is independent of other clinical parameters and can be used as an independent prognostic factor in clinical practice. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. Fi 10 Figure 8 Determination and analysis of the prognostic AS signatures in the STAD cohort. STAD patients were divided into high- and low-risk subgroups based on the median cut of the risk score calculated separately. The upper part of each assembly represents the risk score curve (low risk patients are represented by green dots, high risk patients by red dots, and dash lines correspond to the median of all samples riskScore). The middle section represents the distribution of survival status and survival time of patients ranked by risk score (more green dots on the left for low-risk patients, and more red dots on the right for high-risk patients. From left to right, with the increase of the risk value, more and more patients died, indicating that the risk value is related to survival). The bottom heatmap displays the splicing pattern of the AS signature from each AS type or all seven AS types (the color transition from green to red indicates that the PSI value of the corresponding AS event increases from low to high). (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. Page 23/26 Figure 9 Univariate Cox regression analysis of clinical parameters and riskScore in STAD. For a clinical parameter or riskScore, if the P-value is less than 0.05, it is related to survival; if Hazard ratio is greater than 1, it is a high-risk factor. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. Figure 9 Figure 11 SF-AS network and survival analysis. (A) Survival-associated SF-AS network in STAD. AS is represented by circles (red for high-risk AS, green for low-risk AS), and SF is represented by triangles. A line between AS and SF indicates a regulatory relationship between them (the red line represents positive regulation and the green line represents negative regulation); (B) Overall survival of STAD patients; (C) Correlation analysis between the expression of QKI and the survival rate of STAD patients. Figure 10 Multivariate Cox regression analysis of clinical parameters and riskScore in STAD. For the results of univariate and multivariate independent prognostic analysis, if the riskScore P-value of both is less than 0.05, it indicates that riskScore is independent of other clinical parameters and can be used as an independent prognostic factor in clinical practice. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. Multivariate Cox regression analysis of clinical parameters and riskScore in STAD. For the results of univariate and multivariate independent prognostic analysis, if the riskScore P-value of both is less than 0.05, it indicates that riskScore is independent of other clinical parameters and can be used as an independent prognostic factor in clinical practice. (A) Alternate Acceptor site (AA); (B) Alternate Donor site (AD); (C) Alternate Promoter (AP); (D) Alternate Terminator (AT); (E) Exon Skip (ES); (F) Mutually Exclusive Exons (ME); (G) Retained Intron (RI); and (H) All types of AS. Page 24/26 Page 24/26 Additionalfile3.txt Additionalfile1.xls Additionalfile2.txt Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 25/26 Page 25/26 Page 25/26 Additionalfile3.txt Additionalfile1.xls Additionalfile2.txt Additionalfile3.txt Additionalfile1.xls Additionalfile2.txt Page 26/26
https://openalex.org/W3033644579	https://www.mdpi.com/1424-8220/20/11/3188/pdf?version=1591961839	English	null	Implementation of a Phase Synchronization Scheme Based on Pulsed Signal at Carrier Frequency for Bistatic SAR	Sensors	2,020	cc-by	10,194	Received: 20 April 2020; Accepted: 1 June 2020; Published: 4 June 2020 Abstract: Phase synchronization is one of the key technical challenges and prerequisites for the bistatic synthetic aperture radar (SAR) system, which can form a single-pass interferometry system to perform topographic mapping. In this paper, an advanced phase synchronization scheme based on a pulsed signal at carrier frequency is proposed for a bistatic SAR system and it is veriﬁed by a ground validation system. In the proposed phase synchronization scheme, the pulsed signal at carrier frequency is used for phase synchronization link, and it is exchanged by virtue of a time slot between radar signals. The feasibility of the scheme is proven by theoretical analysis of various factors aﬀecting the performance of phase synchronization, and the reliability of the scheme is veriﬁed by the test results of the ground validation system. Keywords: phase synchronization; pulsed signal; carrier frequency; bistatic SAR sensors sensors sensors Article Implementation of a Phase Synchronization Scheme Based on Pulsed Signal at Carrier Frequency for Bistatic SAR Yafeng Chen 1, Da Liang 1,2 , Haixia Yue 1, Dacheng Liu 1, Xiayi Wu 1, Heng Zhang 1, Yuanbo Jiao 1 , Kaiyu Liu 1,* and Robert Wang 1 Yafeng Chen 1, Da Liang 1,2 , Haixia Yue 1, Dacheng Liu 1, Xiayi Wu 1, Heng Zhang 1, Yuanbo Jiao 1 , Kaiyu Liu 1,* and Robert Wang 1 Yafeng Chen 1, Da Liang 1,2 , Haixia Yue 1, Dacheng Liu 1, Xiayi Wu 1, Heng Zhang 1, Yuanbo Jiao 1 , Kaiyu Liu 1,* and Robert Wang 1 1 Space Microwave Remote Sensing System Department, Aerospace Information Research Institute, Chinese Academy of Sciences, Beijing 100094, China; chenyf@aircas.ac.cn (Y.C.); liangda16@mails.ucas.edu.cn (D.L.); yuehx@aircas.ac.cn (H.Y.); dcliu@mail.ie.ac.cn (D.L.); wuxy@aircas.ac.cn (X.W.); zhangheng@aircas.ac.cn (H.Z.); jiaoyb@aircas.ac.cn (Y.J.); yuwang@mail.ie.ac.cn (R.W.) 1 Space Microwave Remote Sensing System Department, Aerospace Information Research Institute, Chinese Academy of Sciences, Beijing 100094, China; chenyf@aircas.ac.cn (Y.C.); liangda16@mails.ucas.edu.cn (D.L.); yuehx@aircas.ac.cn (H.Y.); dcliu@mail.ie.ac.cn (D.L.); wuxy@aircas.ac.cn (X.W.); zhangheng@aircas.ac.cn (H.Z.); jiaoyb@aircas.ac.cn (Y.J.); yuwang@mail.ie.ac.cn (R.W.) 1 Space Microwave Remote Sensing System Department, Aerospace Information Research Institute, Chinese Academy of Sciences, Beijing 100094, China; chenyf@aircas.ac.cn (Y.C.); liangda16@mails.ucas.edu.cn (D.L.); yuehx@aircas.ac.cn (H.Y.); dcliu@mail.ie.ac.cn (D.L.); wuxy@aircas.ac.cn (X.W.); zhangheng@aircas.ac.cn (H.Z.); jiaoyb@aircas.ac.cn (Y.J.); yuwang@mail.ie.ac.cn (R.W.) y g ( ) 2 School of Electronic, Electrical and Communication Engineering, University of Chinese Academy of Sciences, Beijing 100039, China y g 2 School of Electronic, Electrical and Communication Engineering, University of Chinese Academy of Sciences, Beijing 100039, China * Correspondence: liuky@aircas.ac.cn; Tel.: +86-10-5888-7368 Sensors 2020, 20, 3188; doi:10.3390/s20113188 1. Introduction Synthetic aperture radar (SAR) is a fast-developing tool for Earth observation in use for the last 40 years [1,2]. Because of its outstanding advantages of all-weather and all-day observation of the Earth, it has been widely used in military reconnaissance, land resources surveys, disaster monitoring, ocean monitoring and yield estimation of crops, surveying and mapping and so on [3–5]. In recent years, bistatic SAR (BiSAR) systems have attracted more and more interest as they have various unique features, which are characterized by the diﬀerent locations of the transmitter and receiver, ﬂexible baseline conﬁgurations, costs saving using existing systems as a source of opportunity with several receive-only systems and the possibility to generate highly accurate digital elevation models (DEM) using bistatic interferometry [6–9]. The BiSAR stripmap mode is shown in Figure 1, which can be used for generating a DEM of the Earth’s surface. In this mode, the primary satellite transmits the radar signal to the ground, and then the echoes are received by the primary satellite and the slave satellite. Sensors 2020, 20, 3188; doi:10.3390/s20113188 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors 2 of 14 2 of 16 Sensors 2020, 20, 3188 Sensors 2020, 20, x FOR Figure 1. Example of bistatic synthetic aperture radar BiSAR stripmap mode. Figure 1. Example of bistatic synthetic aperture radar BiSAR stripmap mode. Figure 1. Example of bistatic synthetic aperture radar BiSAR stripmap mode. Figure 1. Example of bistatic synthetic aperture radar BiSAR stripmap mode. However, the unique characteristics of BiSAR also add new challenges in technology implementation, such as measurement, control of the satellite platforms, the synchronization of the time, beam and phase, and BiSAR imaging formation algorithms and so on. Among these challenges, phase synchronization is one of the most critical factors for BiSAR interferometry [10–12]. Phase synchronization can be realized by exchanging the synchronization signal between two platforms in BiSAR systems, as has been investigated in [13–22]. The pulse alternate synchronization scheme is proposed in and has been used for the TanDEM‐X [16,18]. Each satellite in the TanDEM‐X mission is equipped with six synchronization antennas used for transmitting and receiving the synchronization signal [18–20]. The phase synchronization accuracy in the TanDEM‐X mission is less than 1° [20]. A linear frequency modulation (LFM) signal is used for exchanging. The LFM signal with the same carrier frequency as the transmitted radar signal is used for synchronization in order to simplify the system design. 1. Introduction To date, the carrier frequency signal used for synchronization in BiSAR systems has not been demonstrated in the literature. In this paper, we investigate a pulsed signal at carrier frequency used for phase synchronization. From the test results, we can see that the synchronization scheme with the pulsed signal at carrier frequency has good performance. The test results of the synchronization scheme in the paper have guiding significance for the synchronization design of bistatic and multistatic SAR systems. However, the unique characteristics of BiSAR also add new challenges in technology implementation, such as measurement, control of the satellite platforms, the synchronization of the time, beam and phase, and BiSAR imaging formation algorithms and so on. Among these challenges, phase synchronization is one of the most critical factors for BiSAR interferometry [10–12]. Phase synchronization can be realized by exchanging the synchronization signal between two platforms in BiSAR systems, as has been investigated in [13–22]. The pulse alternate synchronization scheme is proposed in and has been used for the TanDEM-X [16,18]. Each satellite in the TanDEM-X mission is equipped with six synchronization antennas used for transmitting and receiving the synchronization signal [18–20]. The phase synchronization accuracy in the TanDEM-X mission is less than 1◦[20]. A linear frequency modulation (LFM) signal is used for exchanging. The LFM signal with the same carrier frequency as the transmitted radar signal is used for synchronization in order to simplify the system design. To date, the carrier frequency signal used for synchronization in BiSAR systems has not been demonstrated in the literature. In this paper, we investigate a pulsed signal at carrier frequency used for phase synchronization. From the test results, we can see that the synchronization scheme with the pulsed signal at carrier frequency has good performance. The test results of the synchronization scheme in the paper have guiding signiﬁcance for the synchronization design of bistatic and multistatic SAR systems. y This paper is organized as follows. In Section 2, the proposed pulsed signal phase synchronization scheme is introduced, including a pulsed synchronization signal exchange strategy and performance analysis. The performance verification of the phase synchronization is analyzed in Section 3. In Section 4, the validation experiment is conducted using the LuTan‐1 ground test system. The performance of the proposed synchronization scheme is evaluated in Section 4. Finally, Section 5 concludes this paper with a short summary. This paper is organized as follows. 1. Introduction In Section 2, the proposed pulsed signal phase synchronization scheme is introduced, including a pulsed synchronization signal exchange strategy and performance analysis. The performance veriﬁcation of the phase synchronization is analyzed in Section 3. In Section 4, the validation experiment is conducted using the LuTan-1 ground test system. The performance of the proposed synchronization scheme is evaluated in Section 4. Finally, Section 5 concludes this paper with a short summary. 2.1. Synchronization Scheme Description  The phase synchronization signal is exchanged by virtue of a time slot between radar signals; thus, the normal work of the BiSAR satellites can be prevented from being interrupted. Time diagrams of the synchronization pulse exchange are shown in Figure 2 • The primary satellite and the slave satellite demodulate and collect the phase synchronization pulses received, respectively. In the data processing, the compensation phase is extracted by fast Fourier transform (FFT) operation of the pulsed signal, and the echo of the slave satellite is compensated by the compensation phase to complete the phase synchronization. diagrams of the synchronization pulse exchange are shown in Figure 2.  The primary satellite and the slave satellite demodulate and collect the phase synchronization pulses received, respectively. In the data processing, the compensation phase is extracted by fast Fourier transform (FFT) operation of the pulsed signal, and the echo of the slave satellite is compensated by the compensation phase to complete the phase synchronization • The primary satellite and the slave satellite demodulate and collect the phase synchronization pulses received, respectively. In the data processing, the compensation phase is extracted by fast Fourier transform (FFT) operation of the pulsed signal, and the echo of the slave satellite is compensated by the compensation phase to complete the phase synchronization. diagrams of the synchronization pulse exchange are shown in Figure 2.  The primary satellite and the slave satellite demodulate and collect the phase synchronization pulses received, respectively. In the data processing, the compensation phase is extracted by fast Fourier transform (FFT) operation of the pulsed signal, and the echo of the slave satellite is compensated by the compensation phase to complete the phase synchronization Figure 2. Timing diagrams of synchronization pulse exchange. Figure 2. Timing diagrams of synchronization pulse exchange. Figure 2. Timing diagrams of synchronization pulse exchange. Figure 2. Timing diagrams of synchronization pulse exchange. In the first pulse repetition time (PRT), the primary satellite transmits the synchronization signal to the slave satellite and the slave satellite receives the synchronization signal. Correspondingly, at the next PRT, the slave satellite transmits the synchronization signal to the primary satellite and the primary satellite receives the synchronization signal. The synchronization signal is at carrier frequency; therefore, the phase with a high signal‐noise ratio (SNR) can be obtained at the frequency domain by fast Fourier transform (FFT) operation. 2. Pulsed Signal at Carrier Frequency Synchronization Implementation Scheme 2. Pulsed Signal at Carrier Frequency Synchronization Implementation Scheme In this section, the phase synchronization implementation scheme based on a pulsed signal at carrier frequency is established, which is later used as a basis for deriving quantitative estimates for the performance of the synchronization link. In this section, the phase synchronization implementation scheme based on a pulsed signal at carrier frequency is established, which is later used as a basis for deriving quantitative estimates for the performance of the synchronization link. 3 of 14 Sensors 2020, 20, 3188 2.1. Synchronization Scheme Description In the BiSAR system, frequency deviation and phase noise of the radar carrier are the main factors that cause a phase synchronization error between the primary satellite and the slave satellite. In principle, phase synchronization can be achieved as long as the frequency diﬀerence and phase noise signal of the primary satellite and the slave satellite are obtained through the bidirectional transmission of the radar carrier, and the echo received by the slave satellite is compensated by it [22]. In this paper, on the basis of the phase synchronization scheme proposed by Younis et al. [15,16] and applied to the TanDEM-X system with a bidirectional pair of phase synchronization pulses, an improved phase synchronization scheme was proposed for BiSAR satellites, which was based on a pulsed signal at carrier frequency as an uninterruptable bidirectional pair of synchronization pulses. The key points of the scheme are as follows: Sensors 2020, 20, x FOR PEER REVIEW 3 of 16 principle, phase synchronization can be achieved as long as the frequency difference and phase noise signal of the primary satellite and the slave satellite are obtained through the bidirectional transmission of the radar carrier, and the echo received by the slave satellite is compensated by it [22]. In this paper, on the basis of the phase synchronization scheme proposed by Younis et al. [15,16] and applied to the TanDEM‐X system with a bidirectional pair of phase synchronization pulses, an improved phase synchronization scheme was proposed for BiSAR satellites, which was based on a pulsed signal at carrier frequency as an uninterruptable bidirectional pair of synchronization pulses. The key points of the scheme are as follows: • The phase synchronization signal is exchanged by virtue of a time slot between radar signals; thus, the normal work of the BiSAR satellites can be prevented from being interrupted. Time diagrams of the synchronization pulse exchange are shown in Figure 2.  The phase synchronization signal is exchanged by virtue of a time slot between radar signals; thus, the normal work of the BiSAR satellites can be prevented from being interrupted. Time diagrams of the synchronization pulse exchange are shown in Figure 2 • The phase synchronization signal is exchanged by virtue of a time slot between radar signals; thus, the normal work of the BiSAR satellites can be prevented from being interrupted. Time diagrams of the synchronization pulse exchange are shown in Figure 2. 2.1. Synchronization Scheme Description The processing of the synchronization signal can be summarized as follows: In the ﬁrst pulse repetition time (PRT), the primary satellite transmits the synchronization signal to the slave satellite and the slave satellite receives the synchronization signal. Correspondingly, at the next PRT, the slave satellite transmits the synchronization signal to the primary satellite and the primary satellite receives the synchronization signal. The synchronization signal is at carrier frequency; therefore, the phase with a high signal-noise ratio (SNR) can be obtained at the frequency domain by fast Fourier transform (FFT) operation. The processing of the synchronization signal can be summarized as follows: y g P l b FFT Th d f d f d • Pulse compression by FFT operation. The data are transformed into frequency domain; y g P l i b FFT ti Th d t t f d i t f d i • Pulse compression by FFT operation. The data are transformed into frequency domain;  Pulse compression by FFT operation. The data are transformed into frequency domain;  The peak phases are extracted at peak position of amplitude in the comp • The peak phases are extracted at peak position of amplitude in the compression synchronizatio synchronization data. 2.2. Synchronization Scheme Performance Analysis synchronization data. 2.2. Synchronization Scheme Performance Analysis 2.2. Synchronization Scheme Performance Analysis The frequency of oscillator at the start of data take t is f f f  where {1 2} i  which The frequency of oscillator at the start of data take t0 is fi = f0 + ∆fi, where i ∈{1, 2}, which represents the primary satellite and slave satellite. f0 represents the nominal frequency, Sensors 2020, 20, 3188 4 of 14 fi is the carrier frequency and ∆fi denotes frequency oﬀset [23–26]. The phases of two satellites at time are: Z t fi is the carrier frequency and ∆fi denotes frequency oﬀset [23–26]. The phases of two satellites at time are: Z t ϕi(t) = 2π Z t 0 ( f0 + ∆fi)dt + ϕ0i + nϕi(t) (1) (1) where ϕ0i is the initial time-independent phase and nϕi(t) is the oscillator phase noise. where ϕ0i is the initial time-independent phase and nϕi(t) is the oscillator phase noise. The demodulated phase ϕij(t) available at satellite i for a signal transmitted by satellite j. Thus, the demodulated phase ϕ21 is available at the slave satellite for a signal transmitted by the primary satellite [15,16]. synchronization data. 2.2. Synchronization Scheme Performance Analysis ϕ21(t) = ϕ1(t) −ϕ2(t + τ12) = 2π(∆f1 −∆f2)t −2π( f0 + ∆f1)τ12 +ϕ01 −ϕ02 + nϕ1(t) −nϕ1(t + τ12) (2) (2) The output phase when the slave satellite transmits a synchronization pulse at t = t + τsys while the primary satellite receives at t + τsys + τ21, and the demodulated phase ϕ12 is [15,16]: The output phase when the slave satellite transmits a synchronization pulse at t = t + τsys while the primary satellite receives at t + τsys + τ21, and the demodulated phase ϕ12 is [15,16]: ϕ12(t) = 2π(∆f2 −∆f1) t + τsys −2π( f0 + ∆f1)τ21 +ϕ02 −ϕ01 + nϕ2(t + τsys) −nϕ1(t + τsys + τ21) (3) (3) The ﬂow diagram for synchronization signal processing is shown in Figure 3 and the compensation phase can be represented as [15,16]: The ﬂow diagram for synchronization signal processing is shown in Figure 3 and the compensation phase can be represented as [15,16]: ϕc(t) = 1 2(ϕ12(t) −ϕ21(t)) = 2π(∆f2 −∆f1)t + π(∆f2 −∆f1) τ12 + τsys −πfDτsys+ 1 2 nϕ2(t + τsys) + nϕ2(t + τ) −nϕ1(t) −nϕ1(t + τ + τsys) ≈2π∆f(t)t + π∆f(t)(τ + τsys) −πfDτsys + ϕres (4) (4) where ∆f(t) = ∆f2(t) −∆f1(t) the frequency is oﬀset between two oscillators, fD is the Doppler frequency due to the relative velocity between the two satellites and ϕres is residual phase, which includes the initial phase diﬀerence between two oscillators, oscillator noise phase error and hardware system phase error. Sensors 2020, 20, x FOR PEER REVIEW 5 of 16 Primary phase synchronization signal at carrier frequency Slave phase synchronization signal at carrier frequency Extract the peak phase ( ) by FFT Extract the peak phase ( ) by FFT Calculate compensation phase    12 21 1 ( ) ( ) 2 c t t t      Compensation phase interpolation Point-by-point compensation of slave satellite data by compensation phase 12( )t  21( )t  Figure 3. The flow diagram of synchronization signal processing. Figure 3. The ﬂow diagram of synchronization signal processing. 2.3. Comparison between Pulsed Signal at Carrier Frequency and LFM Signal 2.3. Comparison between Pulsed Signal at Carrier Frequency and LFM Signal Here, a comparison between a pulsed signal at carrier frequency and an LFM signal is given. The LFM signal sLFM(τ) is written as follows: sLFM(τk) = rect(τk T )A exp(jπKrτk 2) (5) (5) where rect() is the rectangular function, A is the amplitude, Kr is the frequency modulation rate and T is the pulse width of the LFM signal. τk is fast time, where k = 1,2,···, [TFs] and Fs is the sampling frequency. Suppose the bandwidth of the LFM signal sLFM(τ) is B. In the receiving signal, suppose the average noise power is nniose and there are Nr samples. Therefore, the total noise power Pniose of Nr samples can be expressed as (6) Pnoise = Nrnnoise (6) After matching ﬁltering in the frequency domain, the LFM signal sLFM(τ) is compressed. The peak power in the peak position can be written as After matching ﬁltering in the frequency domain, the LFM signal sLFM(τ) is compressed. The peak power in the peak position can be written as PLFM = A2TB (7) (7) In addition, the total noise power after ﬁltering can be expressed as Pnoise, filter = NrnnoiseB/Fs. The average noise power after ﬁltering is nnoise, filter = nnoiseB/Fs. Therefore, the SNR of the LFM signal after matching ﬁltering is SNRLFM = PLFM nnoise, filter = A2BT nnoiseB/Fs = A2TFs nnoise (8) (8) Suppose the pulse signal at carrier frequency (after demodulation) is written as follows: Suppose the pulse signal at carrier frequency (after demodulation) is written as follows: scarrier(τk) = rect(τk T )A exp(−j2π∆f(t)τk) (9) (9) For the purpose of comparison, the signal scarrier(τ) has the same amplitude A and pulse width T as the LFM signal. After FFT operation, the peak power in the peak position can be expressed as follows: Pcarrier = NrA2TFs (10) (10) The average noise power in the frequency domain can be written as nnoise, f = Nrnnoise. Therefore, the SNR of the scarrier(τ) is given by SNRcarrier = Pcarrier nnoise,f = NrA2TFs Nrnnoise = A2TF nnoise (11) (11) It can be seen from Equations (8) and (11) that the SNRs of the LFM signal and pulse signal at carrier frequency are the same. It can be seen from Equations (8) and (11) that the SNRs of the LFM signal and pulse signal at carrier frequency are the same. synchronization data. 2.2. Synchronization Scheme Performance Analysis Primary phase synchronization signal at carrier frequency Slave phase synchronization signal at carrier frequency Extract the peak phase ( ) by FFT 12( )t  Extract the peak phase ( ) by FFT 21( )t  Calculate compensation phase    12 21 1 ( ) ( ) 2 c t t t      Compensation phase interpolation Figure 3. The flow diagram of synchronization signal processing. Figure 3. The ﬂow diagram of synchronization signal processing. Sensors 2020, 20, 3188 5 of 14 The time synchronization error can be up to 10−8 s, so in a short time, it can be considered stable [27–29]. As shown in Figure 3, phase unwrapping starts the primary and secondary synchronization phases, and after phase unwrapping, interpolation is performed to obtain a synchronization phase consistent with the phase dimension of the echo and the compensation phase can be used to compensate the satellite data point-by-point. 3.1. Synchronization Scheme Performance Analysis The phase jitter caused by synchronization antenna is i ant  and the phase jitter caused by the Doppler effect of relative motion of the primary satellite and slave satellite is 2 / d   , where 1 i  represents the primary satellite and 2 i  represents the slave satellite The purpose of extracting the compensated phase is to obtain the phase synchronization error caused by the frequency deviation of the primary and slave satellites and other phase noise. The other phases introduced in this process will become the interference phase that aﬀects the phase synchronization performance. It can be seen from Figure 4 that during the phase synchronization pulse transmission, the factors that cause the phase change include: primary satellite and slave satellite frequency oﬀset ∆f(t), the phase jitter of transmit and receive channels ϕsysTi and ϕsysRi and the phase jitter caused by receiver noise is ϕSNRi. The phase jitter caused by synchronization antenna is ϕanti and the phase jitter caused by the Doppler eﬀect of relative motion of the primary satellite and slave satellite is 2π △d/λ, where i = 1 represents the primary satellite and i = 2 represents the slave satellite. The compensation phase interpolation can be represented as [25]: represents the slave satellite. 3.1. Synchronization Scheme Performance Analysis The hardware structure diagram of the phase synchronization link is illustrated in Figure 4. The system consists of a global navigation satellite system (GNSS) disciplined oscillator, a transmit channel, a receive channel and a synchronization antenna. The GNSS disciplined oscillator employs disciplined rubidium clocks to provide a time frequency signal for the reference frequency source module. The GNSS disciplined oscillator combines the excellent short-term stability characteristics of quartz crystal oscillators with the long-term stability characteristics of GPS signals [25,30]. In the previous test of the ground validation system of LuTan-1, an oscillator with 10−7 accuracy was used and the experiments presented in the paper were conducted. However, when satellites are in orbit, a GNSS disciplined oscillator is used in the system and accuracy can reach 10−11. Sensors 2020, 20, x FOR PEER REVIEW 7 of 16 GNSS disciplined oscillator Transmit Channel ϕsysT1 Receive Channel ϕsysR1 Receiver noise ϕSNR1 Sync antenna ϕant1 Transmit Channel ϕsysT2 Receive Channel ϕsysR2 GNSS disciplined oscillator Receiver noise ϕSNR2 Sync antenna ϕant2 Primary satellite Slave satellite Figure 4. The hardware structure of the phase synchronization link. Figure 4. The hardware structure of the phase synchronization link. Transmit Channel ϕsysT2 Receive Channel ϕsysR2 GNSS disciplined oscillator Receiver noise ϕSNR2 Sync antenna ϕant2 Slave satellite of the phase synchronization link igure 4. The hardware structure of the phase synchronization link Figure 4. The hardware structure of the phase synchronization link. The purpose of extracting the compensated phase is to obtain the phase synchronization error caused by the frequency deviation of the primary and slave satellites and other phase noise. The other phases introduced in this process will become the interference phase that affects the phase synchronization performance. It can be seen from Figure 4 that during the phase synchronization pulse transmission, the factors that cause the phase change include: primary satellite and slave satellite frequency offset ( ) f t  , the phase jitter of transmit and receive channels i sysT  and i sysR  and the phase jitter caused by receiver noise is i SNR  . 3. Pulsed Signal at Carrier Frequency Phase Synchronization Scheme Veriﬁcation In order to evaluate the performance of the pulsed signal at carrier frequency synchronization scheme, the test experiment based on the ground validation system of the LuTan-1 is performed. 6 of 14 Sensors 2020, 20, 3188 As mentioned above, the LFM signal is used for synchronization in the LuTan-1 mission. However, with some modiﬁcation, the ground validation system of LuTan-1 can be used for demonstrating the pulsed signal at carrier frequency synchronization scheme. In this section, the hardware system is introduced. The test experiment is described in detail. 3.1. Synchronization Scheme Performance Analysis The compensation phase interpolation can be represented as [25]: ensation phase interpolation can be represented as [25]:         12 21 2 1 2 1 12 1 ( ) ( ) 2 2 c sys D sys t t t f f t f f f                    ϕc(t) = 1 2(ϕ12(t) −ϕ21(t)) = 2π(∆f2 −∆f1)t + π(∆f2 −∆f1) τ12 + τsys −π fDτsys + 1 2 h nϕ2(t + τsys) + nϕ2(t + τ) i −1 2 h nϕ1(t) + nϕ1(t + τ + τsys) i + 1 2∆ϕsys(t) + 1 2∆ϕant(t) + 1 2∆ϕSNR(t) (12) (12) 2 2 1 1 1 1 ( ) ( ) - ( )+ ( ) 2 2 1 1 1 ( ) ( ) ( ) sys sys n t n t n t n t                          (12) where ∆ϕsys(t) represents the diﬀerence in transmit/receive channel phase, ∆ϕant(t) the inﬂuence of the phase of the antenna pattern and ∆ϕSNR(t) denotes the diﬀerence of phase error caused by receiver noise. 1 1 1 ( ) ( ) ( ) 2 2 2 sys ant SNR t t t          where ( ) sys t   represents the difference in transmit/receive channel phase, ( ) ant t   the influence of the phase of the antenna pattern and ( ) SNR t   denotes the difference of phase error caused by According to Equation (12) of the analysis, the phase error introduced by the synchronous antenna pattern is much smaller than 0.01◦and can be ignored. The phase jitter of the BiSAR transmit and receive channels can generally be controlled within 0.01◦, and the impact on the phase synchronization performance is also small, having little eﬀect on the phase synchronization performance. The receiver 7 of 14 8 of 16 Sensors 2020, 20, 3188 Sensors 2020 20 x FOR noise phase jitter, which can be regarded as the total synchronization link error caused by the receiver noise and sampling, after azimuth compression, can be further reduced. 3.2. Hardware Implementation of Phase Synchronization Scheme transmits the radar signal. The radar signal passes through th by the primary satellite and slave satellite. After range com In accordance with the above phase synchronization scheme, the L-band radar central electronic equipment with the phase synchronization function of the primary satellite and the slave satellite is developed, and the ground veriﬁcation test of phase synchronization is carried out. The structure block diagram of the LuTan-1 ground test system is in Figure 5. y p y g p , p p echo signals, which can be referred to as the reference phases, are extracted in the peak position for primary satellite and slave satellite, respectively. The synchronization signals are exchanges between two synchronization antennas. The phase synchronization scheme performance of the system can be obtained by analyzing the experimental data with a 120 s data take. Data former Receiver Inner calibrator Synchronization transceiver GNSS disciplining module Reference frequency source LFM signal source Data former Receiver Inner calibrator Synchronization transceiver GNSS disciplining module Reference frequency source LFM signal source Optical delay line LFM signal Two-Channel Data Acquisition and Data Processing system Satellite Interface Analog system Control instruction Control instruction Telemetry values Telemetry values 1553 Bus 1553 Bus Primary satellite Slave satellite Sync anntenna Sync anntenna Figure 5. The structure block diagram of the ground test system. Figure 5. The structure block diagram of the ground test system. Two-Channel Data Acquisition and Data Processing system Figure 5. The structure block diagram of the ground test system. Figure 5. The structure block diagram of the ground test system. As shown in Figure 5, the GNSS disciplined oscillator module employs a disciplined rubidium clock to provide a time frequency signal for the reference frequency source. The LFM signal source is composed of three parts: a programmable digital chirp baseband generation module, quadrature modulation and power amplification and a multimode output module. According to the programming control signal, a baseband linear frequency modulation signal with a certain bandwidth, a certain pulse width and a specified slope that meets the needs of the system is generated. Mode combination gating filtering and power amplification to obtain RF chirp signals are used to meet the requirements in time domain frequency domain modulation domain and signal In the test experiment, two satellites are placed in a microwave anechoic chamber. An optical delay line is used to simulate the echo-receiving process for the two satellites. The primary satellite transmits the radar signal. 3.1. Synchronization Scheme Performance Analysis From Equation (12), it can be seen that the ﬁrst term is the phase diﬀerence formed by the frequency deviation of the primary satellite and the slave satellite, where the accuracy of the atomic clock frequency used on GNSS is more than 10−12 orders of magnitude [25]. It can be compensated by the frequency and time diﬀerence data in the primary and slave satellites. The ﬁfth and sixth terms are the phase diﬀerence formed by the phase noise, which can be obtained from the phase synchronization signal as an FFT algorithm. 3.2. Hardware Implementation of Phase Synchronization Scheme In accordance with the above phase synchronization scheme, the L‐band radar central electronic equipment with the phase synchronization function of the primary satellite and the slave satellite is developed, and the ground verification test of phase synchronization is carried out. The structure block diagram of the LuTan‐1 ground test system is in Figure 5. In the test experiment, two satellites are placed in a microwave anechoic chamber. An optical delay line is used to simulate the echo‐receiving process for the two satellites. The primary satellite 3.2. Hardware Implementation of Phase Synchronization Scheme transmits the radar signal. The radar signal passes through th by the primary satellite and slave satellite. After range com The radar signal passes through the optical delay line and then is received by the primary satellite and slave satellite. After range compression, the peak phases of the radar echo signals, which can be referred to as the reference phases, are extracted in the peak position for primary satellite and slave satellite, respectively. The synchronization signals are exchanges between two synchronization antennas. The phase synchronization scheme performance of the system can be obtained by analyzing the experimental data with a 120 s data take. used to meet the requirements in time domain, frequency domain, modulation domain and signal power. The former data module completes the analog‐to‐digital conversion (ADC) of the intermediate frequency. An optical delay line is used to simulate the echo‐receiving process for the two satellites. The primary satellite transmits the radar signal. The radar signal passes through the optical delay line and then is received by the primary satellite and slave satellite. The test system consists of the satellite interface analog system, the primary satellite central electronic equipment, the slave satellite central electronic equipment, the two‐channel data acquisition and data processing system and the quadrifilar helix antenna used for the LuTan‐1 phase As shown in Figure 5, the GNSS disciplined oscillator module employs a disciplined rubidium clock to provide a time frequency signal for the reference frequency source. The LFM signal source is composed of three parts: a programmable digital chirp baseband generation module, quadrature modulation and power ampliﬁcation and a multimode output module. According to the programming control signal, a baseband linear frequency modulation signal with a certain bandwidth, a certain pulse width and a speciﬁed slope that meets the needs of the system is generated. Mode combination gating ﬁltering and power ampliﬁcation to obtain RF chirp signals are used to meet the requirements Sensors 2020, 20, 3188 Sensors 2020, 20, 3188 8 of 14 in time domain, frequency domain, modulation domain and signal power. The former data module completes the analog-to-digital conversion (ADC) of the intermediate frequency. An optical delay line is used to simulate the echo-receiving process for the two satellites. The primary satellite transmits the radar signal. The radar signal passes through the optical delay line and then is received by the primary satellite and slave satellite. 3.2. Hardware Implementation of Phase Synchronization Scheme transmits the radar signal. The radar signal passes through th by the primary satellite and slave satellite. After range com ors 2020, 20, x FOR PEER REVIEW 9 o Table 1 Experimental parameters The test system consists of the satellite interface analog system, the primary satellite central electronic equipment, the slave satellite central electronic equipment, the two-channel data acquisition and data processing system and the quadriﬁlar helix antenna used for the LuTan-1 phase synchronization link. The experimental parameters are shown in Table 1. Table 1. Experimental parameters. Parameter Value Carrier frequency 1.26 GHz Table 1. Experimental parameters. Parameter Value Carrier frequency 1.26 GHz Signal bandwidth 80 MHz Sampling rate 360 Mbps SNR 30 dB PRF 3000 Hz Total recorded pulse number 360,000 Signal bandwidth 80 MHz Sampling rate 360 Mbps SNR 30 dB PRF 3000 Hz Total recorded pulse number 360,000 interface ground analog system sends control instructions to the cen Table 1. Experimental parameters. bandwidth The satellite interface ground analog system sends control instructions to the central electronic equipment of the two satellites through a 1553 data bus and obtains their important telemetry values in real time to ensure the normal operation of the two satellites. The two-channel data acquisition and data processing system collects the echo signal and synchronization signal at the same time, calculates the compensation phase and compensates the echo data of the slave satellite point-to-point. The central electronic equipment of the SAR system consists of a signal generation unit, a transmit/receive unit and an inner calibration unit. The LFM signals transmitted by the primary satellite pass through the optical delay line, and then they are received by the two satellites to simulate the echo-receiving process. The synchronization pulses are transmitted using the quadriﬁlar helix antenna between the two satellites. ipment of the two satellites through a 1553 data bus and obtains their important telemetry val eal time to ensure the normal operation of the two satellites. The two‐channel data acquisit data processing system collects the echo signal and synchronization signal at the same tim ulates the compensation phase and compensates the echo data of the slave satellite point‐to‐po central electronic equipment of the SAR system consists of a signal generation unit nsmit/receive unit and an inner calibration unit. The LFM signals transmitted by the prim llite pass through the optical delay line, and then they are received by the two satellites ulate the echo‐receiving process. The synchronization pulses are transmitted using the quadrif x antenna between the two satellites. 4.1. Theoretical Error Analysis In the test system, the GNSS disciplined oscillator module employs a disciplined rubidium clock to provide a time frequency signal. The ground GNSS disciplined oscillator has added the function of correcting phase every 10 s. For LuTan-1 synchronization, where the maximum value can be set to 1/2 PRF, the system works at a high phase synchronization frequency, so as to avoid a phase synchronization precision error and ensure phase synchronization accuracy. The receiver noise, which consists of thermal noise and the noise collected by the synchronization, will introduce both amplitude and phase ﬂuctuations to the synchronization signal and reduce the accuracy of phase synchronization [15]. Sensors 2020, 20, x FOR PEER REVIEW 10 of 16 he total phase variance in phase synchronization can be written as [15] 2 2 2 2 2 1 The total phase variance in phase synchronization can be written as [15] 2 2 2 2 2 1 σ2 link = σ2 i + σ2 a + σ2 f + 1 2σ2 SNR (13) 2 link i a f SNR σ σ σ σ σ + + + (13) rpolation variance, 2 a σ denotes the aliasing variance, 2 f σ is filter (13) where σ2 i denotes the interpolation variance, σ2 a denotes the aliasing variance, σ2 f is ﬁlter mismatch variance and 1 2σ2 SNR is the receiver noise variance. In the proposed synchronization scheme, the synchronization rate can be very high. As a result, the interpolation variance and aliasing variance are very small and can be ignored in the analysis. In addition, supposing there is no ﬁlter mismatch error, σ2 f is also ignored for the sake of simpliﬁcation. Therefore, the total error variance can be expressed as follows [15] f mismatch variance and 2 1 2 SNR σ is the receiver noise variance. In the proposed synchronization scheme, the synchronization rate can be very high. As a result, the interpolation variance and aliasing variance are very small and can be ignored in the analysis. In addition, supposing there is no filter mismatch error, 2 f σ is also ignored for the sake of simplification. 3.2. Hardware Implementation of Phase Synchronization Scheme transmits the radar signal. The radar signal passes through th by the primary satellite and slave satellite. After range com The quadriﬁlar helix antenna used for synchronization link in the test system is shown in Figure 6. This quadriﬁlar helix antenna incorporated broadband antennas, a wide beam, circular polarization, light weight and low-proﬁle design. The actual measurement results show that the antenna standing wave bandwidth can reach 61.1%, and the 3 dB beam width is greater than 120◦. The quadrifilar helix antenna used for synchronization link in the test system is shown ure 6. This quadrifilar helix antenna incorporated broadband antennas, a wide beam, circu arization, light weight and low‐profile design. The actual measurement results show that enna standing wave bandwidth can reach 61.1%, and the 3 dB beam width is greater than 120 Figure 6. The quadrifilar helix antenna used for synchronization link. Figure 6. The quadriﬁlar helix antenna used for synchronization link. gure 6. The quadrifilar helix antenna used for synchronization lin Figure 6. The quadriﬁlar helix antenna used for synchronization link. 9 of 14 Sensors 2020, 20, 3188 4.1. Theoretical Error Analysis Therefore, the total error variance can be expressed as follows [15] σ2 link = 1 2σ2 SNR = 1 4 fsynSNR Z fsyn/2 −fsyn/2 Hsyn( f) 2df (14) /2 2 2 2 /2 1 1 = ( ) 2 4 SNR syn syn f link SNR syn f syn H f df f σ σ − = ∫ (14) h i i ( ) H f i h f f i d b i (14) 4) where fsyn is the synchronization rate. Hsyn( f) is the transfer function and can be written as: where syn f is the synchronization rate. ( ) syn H f is the transfer function and can be written as: Hsyn( f) = 1 exp(−jπ fτsys) cos(π fτsys) (15) 1 ( )= exp( )cos( ) syn sys sys H f j f f π τ π τ − (15) Hsyn( f) = 1 exp(−jπ fτsys) cos(π fτsys) (1 1 ( )= exp( )cos( ) syn sys sys H f j f f π τ π τ − (15) (15) 5) Using the error model mentioned above, the theoretical bounds are given in Figure 7. Using the error model mentioned above, the theoretical bounds are given in Figure 7. Figure 7. The standard deviation (STD) of phase errors versus signal-noise ratio (SNR). Therefore, as shown in Figure 7, if we want the SNR of the phase synchronization signal to be around 30 dB, the phase accuracy is within 1°. 10 15 20 25 30 35 40 45 SNR (dB) 10 -1 10 0 10 1 Phase (deg.) Figure 7. The standard deviation (STD) of phase errors versus signal-noise ratio (SNR). Therefore, as shown in Figure 7, if we want the SNR of the phase synchronization signal to be around 30 dB, the phase accuracy is within 1◦. Phase (deg.) Figure 7. The standard deviation (STD) of phase errors versus signal-noise ratio (SNR). Figure 7. The standard deviation (STD) of phase errors versus signal-noise ratio (SNR). Therefore, as shown in Figure 7, if we want the SNR of the phase synchronization signal to be around 30 dB, the phase accuracy is within 1°. Therefore, as shown in Figure 7, if we want the SNR of the phase synchronization signal to be around 30 dB, the phase accuracy is within 1◦. 4.2. Results of Test Experiment Figure 8a shows that the primary satellite works spontaneously, its phase basically does not change with time, while the signal received by the slave satellite is sent by the primary satellite, and its phase changes with azimuth time. In the ground test system experiment, the system works in the BiSAR mode. Further, the primary satellite and slave satellite respectively use two sets of GNSS disciplined oscillators and reference frequency sources. The GNSS disciplined oscillator employs a disciplined rubidium clock to provide a time frequency signal for the reference frequency source module, and the multiple working frequency signals are generated by the reference frequency source. The primary satellite is self-transmitting and self-receiving, and since there is no synchronization error, the phase change of the recorded radar signals is mainly introduced by the system instrument, which can be ignored. The slave satellite is only-receiving, and the radar echo signals’ phase change is introduced by the GNSS disciplined oscillator module. Because the ground GNSS disciplined oscillator has added the function of correcting phase every 10 s, the radar echo phase of the slave satellite is periodically calibrated, as shown in the blue curve in Figure 8a. The primary satellite synchronization phase path and slave satellite synchronization phase path is shown in Figure 8b. The LFM-phase synchronization scheme, the radar signal phases and the synchronization phases of the two satellites are shown in Figure 8c,d, respectively. Sensors 2020, 20, x FOR PEER REVIEW 11 of 16 module, and the multiple working frequency signals are generated by the reference frequency source. The primary satellite is self‐transmitting and self‐receiving, and since there is no synchronization error, the phase change of the recorded radar signals is mainly introduced by the system instrument, which can be ignored. The slave satellite is only‐receiving, and the radar echo signals’ phase change is introduced by the GNSS disciplined oscillator module. Because the ground GNSS disciplined oscillator has added the function of correcting phase every 10 seconds, the radar echo phase of the slave satellite is periodically calibrated, as shown in the blue curve in Figure 8a. The primary satellite synchronization phase path and slave satellite synchronization phase path is shown in Figure 8b. The LFM‐phase synchronization scheme, the radar signal phases and the synchronization phases of the two satellites are shown in Figure 8c and 8d, respectively. 4.2. Results of Test Experiment o a e i e a e o i igu e a , e pe i e y Figure 8. The echo phase and synchronization phase of the two satellites. (a) pulsed signal at carrier frequency (PSCF)‐the radar signal phases of the two satellites; (b) PSCF‐the synchronization phases of the two satellites; (c) linear frequency modulation (LFM)‐the radar signal phases of the two satellites; (d) LFM‐the synchronization phases of the two satellites. Figure 9 shows the echo phase compensation phase of the two satellites and the compensation 0 20 40 60 80 100 120 Time (s) (a) -100 -80 -60 -40 -20 0 20 40 60 PSCF-Primary satellite echo phase path PSCF-Slave satellite echo phase path 0 20 40 60 80 100 120 Time (s) (b) -200 -150 -100 -50 0 50 100 150 PSCF-Primary synchronization phase path PSCF-Slave synchronization phase path 0 20 40 60 80 100 120 Time (s) (c) -60 -40 -20 0 20 40 60 80 100 120 140 LFM-Primary satellite echo phase path LFM-Slave satellite echo phase path 0 20 40 60 80 100 120 Time (s) (d) -50 0 50 100 150 200 250 300 350 400 Phase (degree) LFM-Primary synchronization phase path LFM-Slave synchronization phase path Figure 8. The echo phase and synchronization phase of the two satellites. (a) pulsed signal at carrier frequency (PSCF)-the radar signal phases of the two satellites; (b) PSCF-the synchronization phases of the two satellites; (c) linear frequency modulation (LFM)-the radar signal phases of the two satellites; (d) LFM-the synchronization phases of the two satellites. 0 20 40 60 80 100 120 Time (s) (b) -200 -150 -100 -50 0 50 100 150 PSCF-Primary synchronization phase path PSCF-Slave synchronization phase path 0 20 40 60 80 100 120 Time (s) (a) -100 -80 -60 -40 -20 0 20 40 60 PSCF-Primary satellite echo phase path PSCF-Slave satellite echo phase path 0 20 40 60 80 100 120 Time (s) (c) -60 -40 -20 0 20 40 60 80 100 120 140 LFM-Primary satellite echo phase path LFM-Slave satellite echo phase path 0 20 40 60 80 100 120 Time (s) (d) -50 0 50 100 150 200 250 300 350 400 Phase (degree) LFM-Primary synchronization phase path LFM-Slave synchronization phase path Figure 8. The echo phase and synchronization phase of the two satellites. 4.1. Theoretical Error Analysis Therefore, as shown in Figure 7, if we want the SNR of the phase synchronization signal to be around 30 dB, the phase accuracy is within 1°. Therefore, as shown in Figure 7, if we want the SNR of the phase synchronization signal to be around 30 dB, the phase accuracy is within 1◦. 10 of 14 Sensors 2020, 20, 3188 10 of 14 4.2. Results of Test Experiment (a) pulsed signal at carrier frequency (PSCF)‐the radar signal phases of the two satellites; (b) PSCF‐the synchronization phases of the two satellites; (c) linear frequency modulation (LFM)‐the radar signal phases of the two satellites; (d) LFM‐the synchronization phases of the two satellites. Figure 8. The echo phase and synchronization phase of the two satellites. (a) pulsed signal at carrier frequency (PSCF)-the radar signal phases of the two satellites; (b) PSCF-the synchronization phases of the two satellites; (c) linear frequency modulation (LFM)-the radar signal phases of the two satellites; (d) LFM-the synchronization phases of the two satellites. 11 of 14 Sensors 2020, 20, 3188 Figure 9 shows the echo phase compensation phase of the two satellites and the compensation phase path of the pulsed signal at carrier frequency (PSCF)-phase synchronization scheme. The compensation phase path is very close to the echo phase diﬀerence path. The residual phase is obtained after the compensation, which can be seen in Figure 9. Sensors 2020, 20, x FOR PEER REVIEW 12 of 16 Figure 9. The echo phase difference and compensation phase of the two satellites. Figure 9. The echo phase diﬀerence and compensation phase of the two satellites. Figure 9 The echo phase difference and compensation phase of the two satellites Figure 9. The echo phase diﬀerence and compensation phase of the two satellites. Figure 10 shows the echo phase difference and compensation phase of the two satellites. The LFM‐phase signal scheme residual phase is obtained after the compensation, which can be seen in Figure 10a, the standard deviation (STD) of the residual phase value is 0.978° and the peak–peak value is 0.524°. The pulsed signal at carrier frequency (PSCF) phase signal scheme residual phase is obtained after the compensation, which can be seen in Figure 10b, the standard deviation (STD) of the residual phase value is 0.780° and the peak–peak value is 1.139°. The comparison results of LFM‐phase signal and PSCF‐phase signal phase synchronization schemes are shown in Table 2. It can be seen that the PSCF‐phase signal is slightly worse than the linear frequency modulation during the short‐term startup of the BiSAR, but it can meet the requirements of accuracy. 4.2. Results of Test Experiment Moreover, the non‐interrupted PSCF at carrier frequency synchronization strategy has simple requirements for system design and high system reliability, which have guiding significance for the synchronization d i f bi i d l i i SAR Figure 10 shows the echo phase diﬀerence and compensation phase of the two satellites. The LFM-phase signal scheme residual phase is obtained after the compensation, which can be seen in Figure 10a, the standard deviation (STD) of the residual phase value is 0.978◦and the peak–peak value is 0.524◦. The pulsed signal at carrier frequency (PSCF) phase signal scheme residual phase is obtained after the compensation, which can be seen in Figure 10b, the standard deviation (STD) of the residual phase value is 0.780◦and the peak–peak value is 1.139◦. The comparison results of LFM-phase signal and PSCF-phase signal phase synchronization schemes are shown in Table 2. It can be seen that the PSCF-phase signal is slightly worse than the linear frequency modulation during the short-term startup of the BiSAR, but it can meet the requirements of accuracy. Moreover, the non-interrupted PSCF at carrier frequency synchronization strategy has simple requirements for system design and high system reliability, which have guiding signiﬁcance for the synchronization design of bistatic and multistatic SAR systems. y Table 2. The comparison results of two phase synchronization schemes. Device Peak‐Peak Standard (1σ LFM‐phase signal 0.978° 0.524° PSCF phase signal 1 139° 0 780° Table 2. The comparison results of two phase synchronization schemes. Device Peak-Peak Standard (1σ) LFM-phase signal 0.978◦ 0.524◦ PSCF-phase signal 1.139◦ 0.780◦ Table 2. The comparison results of two phase synchronization schemes. Table 2. The comparison results of two phase synchronization schemes. 12 of 14 3 of 16 Sensors 2020, 20, 3188 Sensors 2020, 20, Figure 10. The residual phase error after phase synchronization compensation. (a) Residual phase of LFM-phase signal scheme; (b) residual phase of pulsed signal at carrier frequency (PSCF)-phase signal scheme. 0 20 40 60 80 100 120 Time (s) (a) -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 Phase (degree) 0 20 40 60 80 100 120 Time (s) (b) -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 Phase (degree) Figure 10. The residual phase error after phase synchronization compensation. (a) Residual phase of LFM-phase signal scheme; (b) residual phase of pulsed signal at carrier frequency (PSCF)-phase signal scheme. 4.2. Results of Test Experiment 0 20 40 60 80 100 120 Time (s) (a) -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 Phase (degree) 0 20 40 60 80 100 120 Time (s) -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1 Phase (degree) Figure 10. The residual phase error after phase synchronization compensation. (a) Residual phase of LFM-phase signal scheme; (b) residual phase of pulsed signal at carrier frequency (PSCF)-phase signal scheme. Figure 10. The residual phase error after phase synchronization compensation. (a) Residual phase of LFM-phase signal scheme; (b) residual phase of pulsed signal at carrier frequency (PSCF)-phase signal scheme. References Moreira, A.; Prats-Iraola, P.; Younis, M.; Gerhard, K.; Irena, H.; Konstantinos, P.P. A tutorial on synthetic aperture radar. IEEE Geosci. Remote Sens. Mag. 2013, 1, 6–43. [CrossRef] . Willey, C.A. Synthetic aperture radars—A paradigm for technology evolution. IEEE Trans. Ae Electron. Syst. 1985, 21, 440–443. [CrossRef] 3. D’Errico, M. Distributed Space Missions for Earth System Monitoring; Springer: New York, NY, USA, 2012. 3. D’Errico, M. Distributed Space Missions for Earth System Monitoring; Springer: New York, NY, USA, 2012. 4. Sun, H.; Shimada, M.; Feng, X. 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Impact of Oscillator Noise in Bistatic and Multistatic SAR. IEEE Geosci. Remote Sens. Lett. 2006, 3, 424–428. [CrossRef] 8. Eineder, M. Ocillator clock drift compensation in bistatic interferometric SAR. In Proceedings of the IEEE International Geoscience & Remote Sensing Symposium, Toulouse, France, 21–25 July 2003. 8. Eineder, M. Ocillator clock drift compensation in bistatic interferometric SAR. In Proceedings of the IEEE International Geoscience & Remote Sensing Symposium, Toulouse, France, 21–25 July 2003. 9. Pinheiro, M.; Rodriguezcassola, M. Reconstruction methods of missing SAR data: Analysis in the frame of TanDEM-X synchronization link. In Proceedings of the European Conference on Synthetic Aperture Radar, Nuremberg, Germany, 23–26 April 2012. 9. Pinheiro, M.; Rodriguezcassola, M. Reconstruction methods of missing SAR data: Analysis in the frame of TanDEM-X synchronization link. In Proceedings of the European Conference on Synthetic Aperture Radar, Nuremberg, Germany, 23–26 April 2012. 10. Krieger, G.; De Zan, F. Relativistic Eﬀects in Bistatic SAR Processing and System Synchronization. In Proceedings of the European Conference on Synthetic Aperture Radar, Nuremberg, Germany, 23–26 April 2012. 11. 5. Conclusions In this paper, an improved scheme of non-interrupted synchronization pair alternates based on pulsed signal at carrier frequency phase synchronization signal is implemented and veriﬁed in the LuTan-1 ground test system. The feasibility of using a pulse signal at a carrier frequency as a phase synchronization signal and using an FFT algorithm to rapidly extract the synchronization error of two satellites and the phase synchronization in imaging are analyzed. In addition, the performance of 13 of 14 Sensors 2020, 20, 3188 the proposed scheme is veriﬁed by test results of the ground validation system. In order to further improve the BiSAR system reliability and reduce system complexity, compared with the LFM-phase synchronization scheme, the PSCF-phase synchronization scheme has an independent synchronization path to correct phase changes during the synchronization operation. Finally, the PSCF-phase synchronization system can fulﬁl the synchronization accuracy requirement for the BiSAR system. the proposed scheme is veriﬁed by test results of the ground validation system. In order to further improve the BiSAR system reliability and reduce system complexity, compared with the LFM-phase synchronization scheme, the PSCF-phase synchronization scheme has an independent synchronization path to correct phase changes during the synchronization operation. Finally, the PSCF-phase synchronization system can fulﬁl the synchronization accuracy requirement for the BiSAR system. Author Contributions: Conceptualization, K.L., Y.C. and R.W.; methodology, Y.C., K.L. and D.L. (Da Liang); software, D.L. (Da Liang), H.Y. and X.W.; validation, Y.C., K.L. and D.L. (Dacheng Liu); writing—original draft preparation, Y.C. and D.L. (Da Liang); writing—review and editing, Y.C., D.L. (Da Liang), D.L. (Dacheng Liu), Y.J. and H.Z.; visualization, D.L. (Da Liang) and H.Z.; project administration, R.W.; funding acquisition, R.W. All authors have read and agreed to the published version of the manuscript. ding: This research was supported in part by the National Science Fund for Distinguished Young Scholars. Funding: This research was supported in part by the National Science Fund for Distinguished Young Scholars. Conﬂicts of Interest: The authors declare no conﬂict of interest. Abbreviations The following abbreviations are used in this manuscript: SAR Synthetic aperture radar BiSAR Bistatic synthetic aperture radar LFM Linear frequency modulation PRT Pulse repetition time PRF Pulse repetition frequency GNSS Global navigation satellite system FFT Fast Fourier transform DEM Digital elevation models PSCF Pulsed signal at carrier frequency STD Standard deviation SNR Signal-noise ratio References Lopez-Dekker, P.; Mallorqui, J.J.; Serra-Morales, P.; Sanz-Marcos, J. Phase Synchronization and Doppler Centroid Estimation in Fixed Receiver Bistatic SAR Systems. IEEE Trans. Geosci. Remote Sens. 2008, 46, 3459–3471. [CrossRef] Sensors 2020, 20, 3188 14 of 14 14 of 14 12. Pinheiro, M.; Rodriguez-Cassola, M.; Prats-Iraola, P.; Andreas, R.; Gerhard, K.; Alberto, M. Reconstruction of Coherent Pairs of Synthetic Aperture Radar Data Acquired in Interrupted Mode. IEEE Trans. Geosci. Remote Sens. 2015, 53, 1876–1893. [CrossRef] 3. 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In Proceedings of the IEEE International Geoscience & Remote Sensing Symposium, Anchorage, AK, USA, 20–24 September 2004. 18. Breit, H.; Younis, M.; Niedermeier, A.; Grigorov, C.; Hueso-Gonzalez, J.; Krieger, G.; Eineder, M.; Fritz, T. Bistatic Synchronization and Processing of TanDEM-X Data. In Proceedings of the IEEE International Geoscience & Remote Sensing Symposium, Vancouver, BC, Canada, 24–29 July 2011. g J g Bistatic Synchronization and Processing of TanDEM-X Data. In Proceedings of the IEEE International Geoscience & Remote Sensing Symposium, Vancouver, BC, Canada, 24–29 July 2011. 19. Brautigam, B.; Gonzalez, J.H.; Schwerdt, M.; Bachmann, M. TerraSAR-X Instrument Calibration Results and Extension for TanDEM-X. IEEE Trans. Geosci. Remote Sens. 2010, 48, 702–715. [CrossRef] 20. Krieger, G.; Zink, M.; Bachmann, M.; Benjamin, B.; Daniel, S.; Michele, M.; Paola, R.; Ulrich, S.; John Walter, A.; Francesco, D.Z. TanDEM-X: A Radar Interferometer with Two Formation Flying Satellites. 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https://openalex.org/W4281771073	https://opus.hs-furtwangen.de/files/8325/fmedt-04-920384.pdf	English	null	Novel Three-Dimensional and Biocompatible Lift-Off Method for Selective Metallization of a Scleral Contact Lens Electrode for Biopotential Detection	Frontiers in medical technology	2,022	cc-by	8,385	Correspondence: Volker Bucher Correspondence: Volker Bucher volker.bucher@hs-furtwangen.de Specialty section: This article was submitted to Diagnostic and Therapeutic Devices, a section of the journal Frontiers in Medical Technology Specialty section: This article was submitted to Diagnostic and Therapeutic Devices, a section of the journal Frontiers in Medical Technology Received: 14 April 2022 Accepted: 23 May 2022 Published: 10 June 2022 June 2022 \| Volume 4 \| Article 920384 ORIGINAL RESEARCH published: 10 June 2022 doi: 10.3389/fmedt.2022.920384 ORIGINAL RESEARCH published: 10 June 2022 doi: 10.3389/fmedt.2022.920384 ORIGINAL RESEARCH published: 10 June 2022 doi: 10.3389/fmedt.2022.920384 Sven Schumayer 1,2, Nicolai Simon 1,3, Benjamin Sittkus 1,3, Sandra Wagner 2, Volker Bucher 1* and Torsten Strasser 2,4 Edited by: Shiming Zhang, The University of Hong Kong, Hong Kong SAR, China Edited by: Shiming Zhang, The University of Hong Kong, Hong Kong SAR, China 1 Institute of Microsystems Technology, Furtwangen University, Furtwangen, Germany, 2 Institute for Ophthalmic Research, University of Tuebingen, Tuebingen, Germany, 3 IMTEK—Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany, 4 University Eye Hospital Tuebingen, Tuebingen, Germany Reviewed by: Hyeran Noh, Seoul National University of Science and Technology, South Korea Binbin Ying, Massachusetts Institute of Technology, United States Yangzhi Zhu, Terasaki Institute for Biomedical Innovation, United States Presbyopia describes the eye’s physiological loss of the ability to see close objects clearly. The adaptation to different viewing distances, termed accommodation, is achieved by a change in the curvature of the eye lens induced by the ciliary muscle. A possible approach to correct presbyopia could be to detect the ciliary muscle’s neuromuscular signals during accommodation and transfer these signals electronically to a biomimetic, micro-optical system to provide the necessary refractive power. As a preliminary step toward such a described system, a novel three-dimensional and biocompatible lift-off method was developed. In addition, the inﬂuence of the distance between the electrically conducting surfaces of the lens on the accommodated signal amplitudes was investigated. Compared to the conventional masking methods, this process has the advantage that three-dimensional surfaces can be masked with biocompatible gelling sugar by utilizing a direct writing process with a dispensing robot. Since gelling sugar can be used at room temperature and is water-soluble, the process presented is suitable for materials that should not be exposed to organic solvents or excessively high temperatures. Apart from investigating the shrinkage behavior of the gelling sugar during the physical vapor deposition (PVD) coating process, this paper also describes the approaches used to partially coat a commercial scleral contact lens with an electrically conductive material. It was shown that gelling sugar withstands the conditions during the PVD processes and a successful lift-off was performed. To investigate the inﬂuence of the spacing between the electrically conductive regions of the contact lens on the measured signals, three simpliﬁed electrode conﬁgurations with different distances were fabricated using a 3D printer. By testing these in an experimental setup, it could be demonstrated that the distance between the conductive surfaces has a signiﬁcant inﬂuence on the INTRODUCTION Unlike agar-agar, which has a melting point between 51 and 54◦C (22), or gelatin, which turns into a solution above 35◦C (30), preliminary tests showed that gelling sugar retains its shape even at a temperature of 100◦C. This Accommodation describes the change of refractive power of the eye’s crystalline lens to receive a sharp image of objects at diﬀerent viewing distances (1). Over human life, this ability fades gradually until ﬁnally, at the age of about 65, only depth of focus remains. This process is called presbyopia and becomes noticeable around the age of 40 years (2). The reasons for presbyopia are still being discussed today but can possibly be attributed to the physiological decrease in lens plasticity (3). Uncorrected presbyopia already leads to a worldwide productivity loss of about 11 billion US- Dollars at a working age of 50 years. Assuming a working age of 65 in the future, the global productivity loss would increase even further to 25.4 billion US-Dollars. This is caused by the diﬃculties people experience while doing general tasks requiring near vision such as reading, writing, and driving (4). It is expected that presbyopia will aﬀect around 2.1 billion people worldwide in 2030 (5). Summarizing these facts together, it is plausible that further approaches to correct presbyopia should be developed. pp p y p p Scleral contact lenses belong to the group of therapeutic contact lenses designed to protect or assist in the healing process of areas of the ocular surface consisting of the cornea, sclera, and associated epithelial layers (6). A scleral lens is used here because it has an increased diameter of up to 25 mm compared to conventional contact lenses (7). It thereby spans the entire cornea, which is the most sensitive part of the anterior eye, and only touches the eye in the area of the scleral conjunctiva (8). In the past, scleral contact lenses have also been applied as a carrier for electrodes to detect the neuromuscular signals of the ciliary muscle during accommodation (9–13). These so-called biopotentials are induced by cellular activities that directly lead to a shift in extracellular potential. The signals measured in this way are of great importance in diagnostics, as they can provide information about abnormalities of muscles, nerves, or the brain via the signal characteristics (14). INTRODUCTION be achieved either by a subtractive method like etching or an additive lift-oﬀprocedure (16). However, etchants used in the subtractive method could harm the substrate (17). In the lift- oﬀprocess, the required segmentation is achieved by masking the areas which should not be coated before depositing the electrically conductive material (16–19). After deposition, the masking material can be dissolved and the unwanted metal is “lifted oﬀ” to reveal the patterned conductive surfaces (16– 19). The photoresists commonly used for this purpose are removed by organic solvents (e.g., acetone) after metal coating (20). Consequently, such processes are not compatible with chemically sensitive contact lens materials such as ﬂuorosilicone methacrylate copolymer. These polymers, being tailored to the physiology of the eye, are highly sensitive to the organic solvents, and any contact with, for example, acetone, isopropanol, or ethanol would lead to the loss of the lens transparency. Instead of a photosensitive coating, alternative materials for sacriﬁcial layers such as gelatin (21), agar-agar (21, 22), shellac (23), paraﬃn wax (24), sugar (25, 26), or even ice (27) are available. These are already widely used to manufacture diﬀerent microstructures, for instance, brain implants (26) or various microﬂuidic systems (22, 24, 27). Because of their biocompatibility, gelatin (28–31), agarose (29, 30, 32–35), and sugar (33, 34) are widely used for tissue engineering such as matrix materials for bioprinting. The ﬁrst two are used as carrier materials in hydrogel-based bioinks containing the living cells and act as a type of scaﬀold to support the mechanical integrity of the printed parts (29, 31). There are several techniques for bioprinting, with the main applications being inkjet, extrusion, or laser-assisted printing (31). Compared to conventionally utilized materials in a lift- oﬀprocess, sugar has positive aspects such as environmental friendliness and cost eﬃciency (25). Thus, a two-dimensional lift-oﬀprocess was already carried out using maltose (25). Utilizing commercially available 3D printing ﬁlament and an extrusion 3D printer, it was additionally demonstrated that a lift-oﬀprocess is possible on three-dimensional structures in order to produce, for instance, three-dimensional, selectively metallized high-frequency circuits (36). In this work, a three- axis dispensing robot was combined with the biocompatible and room temperature printable sacriﬁcial material, gelling sugar. The gelling sugar, which withstands the conditions during the metallization process, was successfully used to mask a scleral contact lens in three dimensions and to perform a lift-oﬀ after metallization. Citation: Schumayer S, Simon N, Sittkus B, Wagner S, Bucher V and Strasser T (2022) Novel Three-Dimensional and Biocompatible Lift-Off Method for Selective Metallization of a Scleral Contact Lens Electrode for Biopotential Detection. Front. Med. Technol. 4:920384. doi: 10.3389/fmedt.2022.920384 June 2022 \| Volume 4 \| Article 920384 Frontiers in Medical Technology \| www.frontiersin.org 1 Schumayer et al. Three-Dimensional and Biocompatible Lift-Off amplitude. Regarding the described lift-off process using gelling sugar, it was found that the dispensing ﬂow rate has a direct inﬂuence on the line uniformity. Future work should address the inﬂuence of the viscosity of the gelling sugar as well as the diameter of the cannula. It is assumed that they are the prevailing limitations for the lateral resolution. Keywords: three-dimensional direct writing, lift-off technology, gelling sugar, contact lens electrode, biocompatibility, electrode conﬁguration Keywords: three-dimensional direct writing, lift-off technology, gelling sugar, contact lens electrode, biocompatibility, electrode conﬁguration Frontiers in Medical Technology \| www.frontiersin.org Determination of the Printing Parameters Masking g To prepare the high-viscosity gelling sugar, the manufacturer’s recommended ratio of 10 g of granulated gelling sugar (2 plus 1, Suedzucker AG, Mannheim, Germany) was mixed with 10 ml of distilled water in a 100 ml beaker and then boiled on a hot plate (US150, Stuart, England) for 2 min. Before the solution could be dispensed by the dispensing robot (Figure 1A), it was ﬁlled in a 5 cc dispenser cartridge using a 10 ml disposable syringe. Manual ﬁlling of the cartridge with the disposable syringe was intended to avoid air inclusions, as this allowed the gelling sugar to be distributed in the cartridge in a more controlled manner. Any air inclusions would lead to an inconsistent volume ﬂow, resulting in interrupted dispensing or reduced linewidth. A dosing cannula (JP Kummer Semiconductor Technology GmbH, Augsburg, Germany) with an inner diameter of 0.33 mm was attached to the cartridge before it was inserted into the volumetric dispenser (Dispenser PreciFluid, France), as shown in Figure 1B. The dispensing unit was then secured in the holder of the dispensing robot (JR3303, Janome, Hachioji, Japan). For determining the printing parameters, a ﬂat 1.5 mm thick polycarbonate (PC) substrate was used. The theoretically expected electrical resistance (R) was calculated considering the speciﬁc gold resistance (ρ), the respective average circumference of the individual electrode (l), and their expected cross-sectional areas (A), as shown in the following equation. R = ρ l A [] (1) (1) For the inner conductive area, a mean diameter of 14.5 mm and a cross-sectional area of 1.5 × 10−4 mm² were assumed, while for the outer conductive area, a mean diameter of 18.5 mm and a cross-sectional area of 2.25 × 10−4 mm² were supposed. These underlying assumptions were made concerning the feasibility of the manufacturing process. Thus, the electrical resistance of 6.7 Ωwas calculated for the inner electrode surface and a resistance of 5.7 Ωfor the outer electrode surface. Before substrate was attached to the positioning table with Kapton, the PC was plasma-activated to increase the surface energy. For this purpose, the material was ﬁrst cleaned with distilled water and dried under a nitrogen ﬂow. The substrate was manually activated in a serpentine-like movement, maintaining a minimum distance of 3.0 mm between the substrate and the tip of the plasma activation unit (kINPen Neoplascontrol, Germany). Coating temperature stability, together with the fact that no organic solvents are required due to the water solubility, makes gelling sugar more advantageous compared to paraﬃn wax, with a melting point of 65◦C (24) or shellac, with a glass transition temperature of 43◦C (23). Since gelling sugar is a food product, it can be assumed that any residues are biocompatible and would not aﬀect the physiology of the eye. g The titanium-gold layer was deposited using an evaporation and sputtering system (AUTO 306, HHV Ltd, England). The base pressure (evacuation level before processing) was set in the range of 2–4 × 10−6 mbar. First, the surface was plasma cleaned with a glow discharge. For this purpose, an argon plasma was ignited at 3 kV at 50 mA and applied for 7 min at a chamber pressure of 8 × 10−2 mbar. This was followed by sputtering of the titanium adhesion promoter layer at 3 × 10−3 mbar and a plasma power of 100 W. To keep the pressure constant, a needle valve was used for manual readjustment. After deposition of the 20 nm thick titanium layer (deposition rate of 0.95 Å/s), the needle valve was closed until the base pressure was reached again. Then the evaporation process of the gold started. This was done by thermal evaporation with a boat current of about 140 A. To keep the radiant heat exposure on the contact lens low during the boat heating process, the power was ramped up with the shutter closed. To increase homogeneity, the target was rotated throughout the coating process and the ﬁlm thickness was controlled in situ with a calibrated quartz crystal. The gold deposition was stopped at a thickness of 150 nm (deposition rate of 7.5 Å/s). INTRODUCTION As one example, contact lens electrodes are used for recording the electroretinogram (ERG) to provide diagnostic information about the retina (15). Even though contact lens electrodes for ERGs are already in practical application, there is no commercially available scleral contact lens electrode for detection of ciliary muscle signals. The reliable detection and further processing of the ciliary muscle’s signals could give a better understanding of the accommodation process and lead to possible new corrective approaches. To detect electrical potentials of the ciliary muscle during accommodation at the ocular surface with a commercially available contact lens, certain areas of the polymer lens must be made electrically conductive. The required segmentation can June 2022 \| Volume 4 \| Article 920384 Frontiers in Medical Technology \| www.frontiersin.org 2 Three-Dimensional and Biocompatible Lift-Off Schumayer et al. Determination of the Printing Parameters Masking Subsequently, the distance between the dispensing needle and the attached PC substrate was calibrated to 0.3 mm. The dispensing- test program for the dispensing robot was written using the JR C-Point II V8 software (Janome, Hachioji, Japan) and a volume ﬂow of 1.1 µl/s was set on the dispensing control unit (Dispenser PreciFluid, France). As part of the test program, three rows consisting of eight lines, each 15 mm long, were dispensed at diﬀerent feed rates (2.5–20 mm/s) at a room temperature of 20◦C. The gelling sugar was then dried for 60 min in a climatic chamber (Binder MKF 115 E1.3, Germany) at 80◦C. Post-processing During post-processing, the PC substrate was immersed in an ultrasonic bath (RK 100H, Bandelin, Germany) containing distilled water. This dissolved the gelling sugar and the test structures remained. Testing the Electrode Conﬁguration Literature values for neuromuscular voltages emitted by the ciliary muscle and detected by the contact lens electrodes range from few micro- to millivolts (9–13). Hence, it appeals to optimize the contact lens electrode so that it detects as large amplitude values as possible. For this purpose, the inﬂuence of the distance between the two concentric conductive surfaces of the lens electrode on the amplitude level was investigated. For the fabrication of the test electrodes, circular holders with diﬀerent distances of concentric guide rails were printed using the 3D printing FDM method. A conductor wire (switch wire YV; 1.1 mm diameter; Conrad Electronics, Berlin, Germany), Measuring To determine the shrinkage behavior of the gelling sugar during the coating process, the sample was measured before and after metallization. For this purpose, the linewidths of the diﬀerent feed rates were measured at three diﬀerent locations, each using a two-point measurement of the digital microscope (VHX-700F, Keyence, Osaka, Japan) at a magniﬁcation factor of 50. June 2022 \| Volume 4 \| Article 920384 Frontiers in Medical Technology \| www.frontiersin.org 3 Three-Dimensional and Biocompatible Lift-Off Schumayer et al. FIGURE 1 \| (A) The upper part (1) of the dispensing robot moves in y- and z-direction while the positioning table (2) moves in x-direction. (B) The dosing unit consists of the volumetric dispenser with the cartridge and a dosing cannula. (C) The stepwise travel path of the dispensing unit deﬁned in the JR C-Point II V8 software for masking the surfaces (green) of the lens, which should not be coated. FIGURE 1 \| (A) The upper part (1) of the dispensing robot moves in y- and z-direction while the positioning table (2) moves in x-direction. (B) The dosing unit consists of the volumetric dispenser with the cartridge and a dosing cannula. (C) The stepwise travel path of the dispensing unit deﬁned in the JR C-Point II V8 software for masking the surfaces (green) of the lens, which should not be coated. stripped to the appropriate length, was then inserted into the provided holes of the holders along the guide rails, starting from the backside. A rendering of the resulting test electrodes and their dimensions can be seen in the following CAD illustration in Figure 2. FIGURE 2 \| Variant 1 is an electrode conﬁguration with a reduced distance between the two conductors, while variant 2 is the ideal arrangement that considers the physiology of the ciliary muscle. Variant 3 is an electrode conﬁguration with a maximum spacing of the detecting electrodes. A greater distance could result in the inner electrode interfering with the user’s ﬁeld of view in low light conditions. For testing, an experimental setup consisting of a frequency generator (4080 Function Generator, PeakTech, Ahrensburg, Germany), an electrical voltage emitting base plate, and an oscilloscope (TDS 210, Tektronix, Beaverton, OR, United States) was arranged. The baseplate shown in Figure 3 was 3D-printed via FDM and stripped conductor wires were added in the same manner as the fabricated testing electrodes. Frontiers in Medical Technology \| www.frontiersin.org Measuring The spacing of the corresponding concentrically emitting conductor wires considers the muscle physiology of the ciliary muscle. Within the ﬁrst 2.0 mm posterior to the scleral spur, the greatest change in ciliary muscle thickness occurs during contraction and decreases laterally (37). Hence, one assumption is that the contraction gradient is accompanied by the electrical sum potential of the ciliary muscle. The scleral spur running concentrically around the lens axis has an average distance of about 6.0 mm to the axis [cf. (38)]. Thus, the average radius of the inner conducting wire is 7.0 mm. The radius for the outer conductor wire was set to 9.5 mm. These electrode diameters were also considered when determining the diameter of the electrode variant 2 (Figure 2) as well as for the ﬁnally fabricated contact lens electrode. FIGURE 2 \| Variant 1 is an electrode conﬁguration with a reduced distance between the two conductors, while variant 2 is the ideal arrangement that considers the physiology of the ciliary muscle. Variant 3 is an electrode conﬁguration with a maximum spacing of the detecting electrodes. A greater distance could result in the inner electrode interfering with the user’s ﬁeld of view in low light conditions. A 10.0 mm thick synthetic resin foam soaked in a saline solution was placed on the emitting electrodes to simulate the contact resistance of the eye, which is mainly dominated by the impedance of the cornea [Z = 8–16 kcm² (39)]. the transmitting electrodes with the high level in the range of 3.9–4.7 V, a low level at 0 V and a frequency of 0.5 Hz to be able to measure signals in the expected millivolt range at The voltage measurements with the three detecting electrode conﬁgurations were performed on the described experimental setup. A voltmeter was used to set a square wave voltage at June 2022 \| Volume 4 \| Article 920384 4 Three-Dimensional and Biocompatible Lift-Off Schumayer et al. then twisted around a 0.8 mm diameter needle and positioned in the drilling holes. The positioned wire ends were then glued into the drill holes using a conductive biocompatible adhesive (EPO-TEK MED H20S, Epoxy Technology Inc, Billerica, MA, USA). A handheld dispenser (THE-200; TAEHA Corporation, Namyangju-Si, Korea) set to a pressure of 351 kPa and a time of 0.024 s was used for this purpose. Post-processing After the coating procedure (Figures 5A–D), the sample was immersed in an ultrasonic bath with distilled water. While the gelling sugar dissolved in the water bath, the partially coated scleral contact lens electrode was revealed (Figure 5E). The exposed contacts on the convex side of the lens were then sealed with PDMS (SILPURAN 4200; Wacker Chemie AG, Munich, Germany) (Figure 5F). The PDMS applied in this way had to cure for 30 min on a hot plate (US150; Stuart, England) at 80◦C. To establish a connection between the lens electrode and a measuring unit, two DIN connectors (1.5 mm) had to be soldered on at 220◦C in a further step. The two solder joints were then insulated with heat-shrink tubing. Measuring FIGURE 3 \| (A) The left side shows a simpliﬁed technical drawing of the electrode plate, while the right side (B) shows the wired plate next to the soaked foam. After curing the conductive adhesive, the Kapton tape was removed. This was followed by further plasma activation of the concave lens side and re-masking with gelling sugar. The masked contact lens was then dried for 3 h in a climate chamber (MKF 115 E1.3, Binder GmbH, Tuttlingen, Germany) at 80◦C and <5% humidity. After drying, the masked scleral lens was coated with a biocompatible metal ﬁlm consisting of titanium as the adhesive layer and gold as the electrode material. FIGURE 3 \| (A) The left side shows a simpliﬁed technical drawing of the electrode plate, while the right side (B) shows the wired plate next to the soaked foam. the test electrodes (12). Each of the electrode conﬁgurations was measured in two measurement series with 10 repetitions. Between the series of measurements, the resin foam was soaked again in the saturated saline solution and the square wave voltage was recalibrated. Contacting and Masking g g The high-viscosity gelling sugar, used to mask the 20 mm contact lens (ASF-120, Woehlk Contactlinsen GmbH, Schönkirchen, Germany), was prepared in the same manner as described in “Masking” section. Due to the lens geometry with diﬀerent radii, a cannula (JP Kummer Semiconductor Technology GmbH, Augsburg, Germany) with a larger diameter of 0.61 mm was used to reduce the travel distance for masking diﬀerent lens segments. Prior to the insertion of the scleral contact lens into a custom-developed 3D printed holder on the positioning table of the dispensing robot, the lens substrate was prepared. Thereby, the lens was ﬁrst rinsed with distilled water, wiped dry with a cleaning cloth, and ﬁnally, blown oﬀwith nitrogen. To increase the wettability of the lens polymer, a plasma activation was performed in the same procedure as the test substrate. Then, an oﬀset between the dispensing cannula and the lens which was ﬁxed in a 3D-printed holder was set to 0.3 mm. The program for the dispensing robot was written using the JR C-Point II V8 software (Janome, Hachioji, Japan) and the deﬁned areas (Figure 1C) were then masked with gelling sugar at a ﬂow rate of 10 mm/s and a volume ﬂow of 4 µl/s set on the dispensing control unit. Afterwards, the gelling sugar was dried in a climatic chamber at 80◦C for 20 min. This ﬁrst application of the gelling sugar served to deﬁne the connection points of the contacts of the later deposited electrically conductive surfaces holes. The individual work steps are schematically shown in Figure 4. Two holes, each with a diameter of 1.0 mm, were drilled (Micromot TBM 220, Proxxon, Hetzerath, Germany) on the convex lens surface for electrical contact before the gelling sugar was subsequently washed oﬀ. The drill holes on the concave side were taped with Kapton tape. The ends of the two insulated gold wires, about 10 cm long and 0.1 mm thick, were ﬁrst stripped with a lighter ﬂame, Coating The coating was done in the same procedure as described in detail in “Coating” section. Frontiers in Medical Technology \| www.frontiersin.org Printing Parameter To accurately mask the contact lens, the possible dissolution and shrinkage behavior must be considered. Figure 6A shows the coated PC-test substrate, demonstrating that droplet-like masking occurs at the beginning of dispensing and that the correct linewidth becomes visible only as dispensing progresses. As expected, a thinner linewidth can be achieved by keeping the volume ﬂow constant and increasing the feed rate. The lines with a feed rate between 10 and 15 mm/s show the highest uniformity, while linewidths above this range become narrower, but edge ﬁdelity decreases. This could also be observed in the other two-test series. Figure 6B shows the PC substrate masked at 12.5 mm/s, and Figure 6C shows the same line after the coating and lift-oﬀprocess. While the edge ﬁdelity is high at this feed rate, signiﬁcantly poorer edge ﬁdelity values were obtained at lower feed rates (2.5–7.5 mm/s) as well as at higher feed rates (17.5–20.0 mm/s) (cf. Figure 6A). The calculated shrinkage rate (linewidth before/linewidth after) of 1.05 for the structure width shown in Figures 6B,C is even below the range of the average shrinkage rate of 1.28 (SD = 0.101), as depicted in Figure 7. Figure 6B shows the PC substrate masked at 12.5 mm/s, and Figure 6C shows the same line after the coating and lift-oﬀprocess. While the edge ﬁdelity is high at this feed rate, signiﬁcantly poorer edge ﬁdelity values were obtained at lower feed rates (2.5–7.5 mm/s) as well as at higher feed rates (17.5–20.0 mm/s) (cf. Figure 6A). The calculated shrinkage rate (linewidth before/linewidth after) of 1.05 for the structure width shown in Figures 6B,C is even below the range of the average shrinkage rate of 1.28 (SD = 0.101), as depicted in Figure 7. June 2022 \| Volume 4 \| Article 920384 5 Schumayer et al. Three-Dimensional and Biocompatible Lift-Off FIGURE 4 \| An illustration of the individual work steps. (A) The gelling sugar (blue) is used as a drilling template (B) and can then be dissolved in water. (C) The concave side is then covered with Kapton® tape (orange) to protect the lens from adhesive ﬂowing in. (D) The centering of the gold wires in the middle of the drill holes can then take place (E) before the drilling holes are ﬁlled with the biocompatible and electrically conductive adhesive. DISCUSSION Here we presented a new 3D masking process and its successful application for partial coating of a scleral contact lens electrode. Areas of the three-dimensional substrate could be masked with gelling sugar and removed with water after the coating process without leaving any residue. This approach was inspired by the contributions of Byford et al., (36) and Zhang et al., (25). Whereas Zhang et al. showed that two-dimensional lift-oﬀis possible using maltose as a water-soluble masking material, Byford et al. demonstrated that three-dimensional printing is capable of creating lift-oﬀmasks using commercial 3D printing ﬁlament. The conditions prevailing during the PVD processes used, such as temperature and vacuum, showed no eﬀect at the interfaces to the gelling sugar. The electrical resistance of 6 for the inner conducting surface and about 5 for the outer surface is within the range of previous theoretical predictions (cf. “Coating”). Electrode Conﬁguration behavior of the sugar with the printing parameters used for the lenses. Further, no partial delamination of the conductive surfaces was evident on the lens after the ultrasonic bath. The electrical resistance measured with the two-wire measurement method was about 6 for the inner conducting surface and about 5 for the outer surface. The following combination of a line graph and boxplot (Figure 8) shows the relationship between the distance from the inner to the outer conductive surface and the determined voltage amplitude in millivolts, ﬁtted with a linear regression (blue dot line). The orange lines show the median of the twenty individual measurements of the respective conﬁgurations. With a reduced distance between the two conductive surfaces (variant 1), the average measured voltage is 18.6 mV, while with the physiologically motivated layout (variant 2) it is 26.3 mV, respectively. With an increased distance between the two conductive surfaces (variant 3) it is 39.0 mV. Based on the physiologically motivated layout, this means that a reduction in the distance of 44.4% between the conductive surfaces results in a reduction of the amplitude level by almost 30%, while an amplitude increment of 50% can be achieved by increasing the distance by 155.5%. Printing Parameter (F) After the adhesive has cured, the Kapton® can be peeled off (G) and the concave side of the lens can be masked a second time with gelling sugar, leaving the electrode sites unmasked. (H) This is followed by the metal coating of the lens. For this purpose, titanium is ﬁrst sputtered as an adhesion promoter before a gold layer is evaporated directly on it without vacuum breakage. (I) Afterwards, the gelling sugar mask is removed with water as solvent. FIGURE 4 \| An illustration of the individual work steps. (A) The gelling sugar (blue) is used as a drilling template (B) and can then be dissolved in water. (C) The concave side is then covered with Kapton® tape (orange) to protect the lens from adhesive ﬂowing in. (D) The centering of the gold wires in the middle of the drill holes can then take place (E) before the drilling holes are ﬁlled with the biocompatible and electrically conductive adhesive. (F) After the adhesive has cured, the Kapton® can be peeled off (G) and the concave side of the lens can be masked a second time with gelling sugar, leaving the electrode sites unmasked. (H) This is followed by the metal coating of the lens. For this purpose, titanium is ﬁrst sputtered as an adhesion promoter before a gold layer is evaporated directly on it without vacuum breakage. (I) Afterwards, the gelling sugar mask is removed with water as solvent. Manufacturing of the Scleral Contact Lens Electrode The gelling sugar was successfully applied selectively to the lens using the robot dispenser. The linewidth could be varied by changing the volume ﬂow and the cannula diameter. Furthermore, Figure 9 shows that the gelling sugar also withstands the coating process (left) and could easily be removed (right) within a minute using water in an ultrasonic bath. Measuring the linewidths unveiled a negligible shrinking While conventional masking methods require organic solvents to remove the masking layer after the coating process (20), this is not necessary with this newly described method using June 2022 \| Volume 4 \| Article 920384 Frontiers in Medical Technology \| www.frontiersin.org 6 Schumayer et al. Three-Dimensional and Biocompatible Lift-Off FIGURE 5 \| Schematic representation of the steps of the lift-off procedure. (A) After the biocompatible, conductive adhesive has cured in the drill hole, the Kapton® can be peeled off. (B) Masking of the substrate on the concave side with gelling sugar is performed (C), followed by sputtering of the titanium layer with a thickness of 20 nm. (D) Immediately afterwards, the gold layer with a thickness of 150 nm is deposited. (E) The lift-off step follows, in which the gelling sugar is dissolved with distilled water in an ultrasonic bath. (F) Finally, the contact points of the convex side of the lens are insulated with silicone polydimethylsiloxane (PDMS). FIGURE 5 \| Schematic representation of the steps of the lift-off procedure. (A) After the biocompatible, conductive adhesive has cured in the drill hole, the Kapton® can be peeled off. (B) Masking of the substrate on the concave side with gelling sugar is performed (C), followed by sputtering of the titanium layer with a thickness of 20 nm. (D) Immediately afterwards, the gold layer with a thickness of 150 nm is deposited. (E) The lift-off step follows, in which the gelling sugar is dissolved with distilled water in an ultrasonic bath. (F) Finally, the contact points of the convex side of the lens are insulated with silicone polydimethylsiloxane (PDMS). Until the dispenser robot has reached the new starting point for a dispensing process, excess gelling sugar can accumulate at the tip of the cannula. This can be reduced by various actions: for instance, a retraction can be set in the dispensing unit after each dispensing process. Alternatively, the dispensing needle can also be cleaned manually. Frontiers in Medical Technology \| www.frontiersin.org Manufacturing of the Scleral Contact Lens Electrode When masking the contact lens, no drop shape was seen at the starting point of the dispensing robot. This can be attributed to the fact that closing the dispensing ring homogeneously distributed the gelling sugar. The investigated shrinkage behavior during the line testing suggests, that higher dimensional accuracy is achieved with smaller structure widths respectively at higher feed rates. Possibly, this can be attributed to a disproportionate decrease in volume compared to the gelling sugar surface area. gelling sugar. In comparison to alternative masking methods using paraﬃn wax (24), agarose gel (22), or shellac (23), it was demonstrated that gelling sugar exhibited a higher temperature resistance of up to 100◦C. Moreover, gelling sugar can be applied at room temperature, which simpliﬁes the equipment needed. The use of a three-axis dispensing robot enables a direct three-dimensional partial masking, without further need of a mask or development steps compared to conventional masking methods (e.g., spin coating, spray coating, or dip coating). The results of the printing parameter feed rate at a constant volume ﬂow of 1.1 µl/s indicate an ideal range (10–15 mm/s) in which a high uniformity of the structure can be achieved. Feed rates above or below this range show a reduction in uniformity. If the feed rate is too slow, too much gelling sugar is applied to the substrate, resulting in a non-uniform distribution. The resulting backlog causes the gelling sugar to bulge to all sides of the cannula. If the feed rate is too high, not enough gelling sugar is applied to the substrate. The high viscosity of the gelling sugar and the outlet area limited by the inner diameter result in a drop-shaped structure at the start of the dosing process. The analysis of the inﬂuence of the distance between the conductive surfaces on the detectable signal amplitude suggests that with increasing distance between the concentric surfaces a larger voltage is detected. The varying high level of the square wave voltage (3.9–4.7 V) within the measurements is probably due to the diﬀerent saturation of the saline-soaked June 2022 \| Volume 4 \| Article 920384 7 Schumayer et al. Three-Dimensional and Biocompatible Lift-Off FIGURE 6 \| (A) Image of the coated test substrate with different feed rates after the lift-off process. The different feed rates are written above the lines. The image was post-processed by increasing the brightness to get a better edge contrast. Manufacturing of the Scleral Contact Lens Electrode The average shrinkage ratios of the different feed rates are represented as red dots, whereas the linear regression of the shrinkage ratio is represented by the red line. The shrinkage ratio decreases with higher feed rates. Note that the linewidth axis starts at 500 µm and the shrinkage ratio axis starts at 0.6. The arrows present the afﬁliation of the graphs to the axes. FIGURE 8 \| Linear correlation (blue dot line) between the measured voltage amplitude and the different electrode variants, whose conductive surface distance differs by 1.25 mm in each case. The orange lines show the median of the single measurements, which can be seen with the corresponding boxplot. Each conﬁguration was measured 20 times. Note that the y-axis starts at an amplitude voltage of 10 mV. FIGURE 8 \| Linear correlation (blue dot line) between the measured voltage amplitude and the different electrode variants, whose conductive surface distance differs by 1.25 mm in each case. The orange lines show the median of the single measurements, which can be seen with the corresponding boxplot. Each conﬁguration was measured 20 times. Note that the y-axis starts at an amplitude voltage of 10 mV. FIGURE 7 \| Averaged values (AVG) which had been measured before (blue dash-dot line) and after (green dash line) the metallization of the test substrate. The average shrinkage ratios of the different feed rates are represented as red dots, whereas the linear regression of the shrinkage ratio is represented by the red line. The shrinkage ratio decreases with higher feed rates. Note that the linewidth axis starts at 500 µm and the shrinkage ratio axis starts at 0.6. The arrows present the afﬁliation of the graphs to the axes. FIGURE 7 \| Averaged values (AVG) which had been measured before (blue dash-dot line) and after (green dash line) the metallization of the test substrate. The average shrinkage ratios of the different feed rates are represented as red dots, whereas the linear regression of the shrinkage ratio is represented by the red line. The shrinkage ratio decreases with higher feed rates. Note that the linewidth axis starts at 500 µm and the shrinkage ratio axis starts at 0.6. The arrows present the afﬁliation of the graphs to the axes. Manufacturing of the Scleral Contact Lens Electrode (B) The measured line width at a feed rate of 12.5 mm/s, before the metallization and the same line after (C) the lift-off. The pink dashed lines in (B) were post-processed to visualize the edge of the gelling sugar. FIGURE 6 \| (A) Image of the coated test substrate with different feed rates after the lift-off process. The different feed rates are written above the lines. The image was post-processed by increasing the brightness to get a better edge contrast. (B) The measured line width at a feed rate of 12.5 mm/s, before the metallization and the same line after (C) the lift-off. The pink dashed lines in (B) were post-processed to visualize the edge of the gelling sugar. FIGURE 6 \| (A) Image of the coated test substrate with different feed rates after the lift-off process. The different feed rates are written above the lines. The image was post-processed by increasing the brightness to get a better edge contrast. (B) The measured line width at a feed rate of 12.5 mm/s, before the metallization and the same line after (C) the lift-off. The pink dashed lines in (B) were post-processed to visualize the edge of the gelling sugar. FIGURE 7 \| Averaged values (AVG) which had been measured before (blue dash-dot line) and after (green dash line) the metallization of the test substrate. The average shrinkage ratios of the different feed rates are represented as red dots, whereas the linear regression of the shrinkage ratio is represented by the red line. The shrinkage ratio decreases with higher feed rates. Note that the linewidth axis starts at 500 µm and the shrinkage ratio axis starts at 0.6. The arrows present the afﬁliation of the graphs to the axes. FIGURE 8 \| Linear correlation (blue dot line) between the measured voltage amplitude and the different electrode variants, whose conductive surface distance differs by 1.25 mm in each case. The orange lines show the median of the single measurements, which can be seen with the corresponding boxplot. Each conﬁguration was measured 20 times. Note that the y-axis starts at an amplitude voltage of 10 mV. FIGURE 7 \| Averaged values (AVG) which had been measured before (blue dash-dot line) and after (green dash line) the metallization of the test substrate. Manufacturing of the Scleral Contact Lens Electrode FIGURE 8 \| Linear correlation (blue dot line) between the measured voltage amplitude and the different electrode variants, whose conductive surface distance differs by 1.25 mm in each case. The orange lines show the median of the single measurements, which can be seen with the corresponding boxplot. Each conﬁguration was measured 20 times. Note that the y-axis starts at an amplitude voltage of 10 mV. FIGURE 8 \| Linear correlation (blue dot line) between the measured voltage amplitude and the different electrode variants, whose conductive surface distance differs by 1.25 mm in each case. The orange lines show the median of the single measurements, which can be seen with the corresponding boxplot. Each conﬁguration was measured 20 times. Note that the y-axis starts at an amplitude voltage of 10 mV. circular conductive ring area, and by considering the positioning of the conductive surfaces with respect to the anatomical changes of the ciliary muscle during accommodation [cf. (37)]. Considering these improvements as well as the measurements on synthetic resin foam and the associated free ions. The results indicate that increasing the distance between the two conductive surfaces of the contact lens increases the amplitude of the measured biopotentials. This could be realized by reducing the June 2022 \| Volume 4 \| Article 920384 Frontiers in Medical Technology \| www.frontiersin.org 8 Three-Dimensional and Biocompatible Lift-Off Schumayer et al. taken into account. Even ﬁner structure widths would be possible if the viscosity of the gelling agent could be reduced, for example by heating during the metering process. Future investigations will focus on this and the production-speciﬁc boundary conditions of the maximum permissible process temperatures. These ﬁndings could open up further applications for this new masking method, e.g., for the production of brain electrodes or structuring microﬂuidic systems. FIGURE 9 \| The partially coated scleral contact lens electrode before (left) and after (right) the lift-off procedure. FUNDING This project was funded by the Carl Zeiss Foundation Breakthroughs at Universities 2020: Intelligent Solutions for an Aging Society, the University of Tuebingen, the Faculty of Medicine of the University of Tuebingen, and the Center for Ophthalmology at the University of Tuebingen. REFERENCES 9. Alpern M, Ellen P, Goldsmith R. The electrical response of the human eye in far-to-near accommodation. AMA Arch Ophthalmol. (1958) 60:592– 602. doi: 10.1001/archopht.1958.00940080612007 1. Kandel ER, Schwartz JH, Jessell TM, Siegelbaum S, Hudspeth AJ, Mack S. Principles of Neural Science. Vol 4. New York, NY: McGraw-hill (2000). 10. Hagiwara H, Ishikawa S. The action potential of the ciliary muscle. Ophthalmologica. (1962) 144:323–40. doi: 10.1159/000304372 2. Plainis S, Charman WN, Pallikaris IG. The Physiologic Mechanism of Accommodation. Cataract & Refractive Surgery Today Europe (2014) 4:23–9. 11. Jacobson JH, Romaine HH, Halberg GP, Stephens G. 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Aktionspotentiale des M. ciliaris beim Menschen. Albrecht Von Graefes Arch Klin Exp Ophthalmol. (1955) 157:116– 21. doi: 10.1007/BF00682518 14. Naït-Ali A. Advanced Biosignal Processing. Berlin: Springer Science & Business Media (2009). doi: 10.1007/978-3-540-89506-0 6. Astin CLK. Therapeutic contact lenses—an overview of some lens types. J Br Contact Lens Assoc. (1991) 14:129–33. doi: 10.1016/0141-7037(91)8 0005-7 15. Levin LA, Adler FH. Adler’s Physiology of the Eye. 11th ed. Edingburg: Saunders/Elsevier (2011). 7. van der Worp E, Bornman D, Ferreira DL, Faria-Ribeiro M, Garcia-Porta N, González-Meijome JM. Modern scleral contact lenses: a review. Cont Lens Anterior Eye. (2014) 37:240–50. doi: 10.1016/j.clae.2014.02.002 16. Yin W, Fillmore W, Dempsey KJ. Optimization of the lithographic performance for lift-oﬀprocessing. Advances in Resist Technology and Processing XVI. International Society for Optics and Photonics (1999). 3678:713–20. doi: 10.1117/12.350258 8. Michaud L, Lipson M, Kramer E, Walker M. The oﬃcial guide to scleral lens terminology. Cont Lens Anterior Eye. (2020) 43:529–34. doi: 10.1016/j.clae.2019. 09.006 17. Grebe K, Ames I, Ginzberg A. Masking of deposited thin ﬁlms by means of an aluminum-photoresist composite. J Vac Sci Technol. (1974) 11:458. DATA AVAILABILITY STATEMENT FIGURE 9 \| The partially coated scleral contact lens electrode before (left) and after (right) the lift-off procedure. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the experimental setup, we expect that the idealized contact lens electrode will measure in the millivolt range. Furthermore, the bipolar electrode arrangement and the diﬀerential measurement should minimize artifacts, for example caused by eye movements (10, 12). We could successfully show that even substrates not suitable for high temperature or sensitive for organic solvents can be masked with this new masking method based on gelling sugar. It can be concluded that gelling sugar does not adversely aﬀect the eye because sugar-based biomaterials have the advantage of being biocompatible and due to its temperature resistance, certain edge ﬁdelity can be expected. New ﬁndings were obtained which should help improve the process described. For example, new handling equipment will help to simplify the process. Since the holes are drilled by hand, there will be a permanent limitation in terms of accuracy, which will limit the distance between the two conductive surfaces. Still some open questions remain: a too high dispensing feed rate has a negative impact on the uniformity of the structure width, nevertheless, the inﬂuence of the viscosity of the gelling sugar and the thinner cannula diameters was not AUTHOR CONTRIBUTIONS SS, SW, and TS contributed to the conception of the diﬀerent electrode layouts and test-setup. NS and BS contributed by optimizing the PVD process and producing test samples. SS and VB contributed by researching alternative methods and sacriﬁcial layers as well as developing the three-dimensional lift- oﬀ. SS contributed by analyzing the data and writing the ﬁrst draft of the manuscript. SS and SW wrote the introduction and SW proofread the ﬁnal manuscript. All authors contributed to manuscript revision, read, and approved the submitted version. REFERENCES Anterior chamber lens sizing: comparison of white-to-white and scleral spur-to-scleral spur methods. J Cataract Refract Surg. (2020) 46:95–101. doi: 10.1016/j.jcrs.2019.08.043 25. Zhang C, Guo X, Yang X, Luo K, Zhu H, Leng H. Using sugar as a mask material (SAAMM) in micro-nano fabrication: a high- performance, green and simple method. J Micromech Microeng. (2021) 31:095001. doi: 10.1088/1361-6439/ac12a2 39. Klyce SD, Crosson CE. Transport processes across the rabbit corneal epithelium: a review. Curr Eye Res. (1985) 4:323– 31. doi: 10.3109/02713688509025145 26. Zhang EN, Clément JP, Alameri A, Ng A, Kennedy TE, Juncker D, et al. Mechanically matched silicone brain implants reduce brain foreign body response. Adv Mater Technol. (2020) 6:2000909. doi: 10.1002/admt.202000909 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. 27. Koesdjojo MT, Tennico YH, Remcho VT. Fabrication of a microﬂuidic system for capillary electrophoresis using a two-stage embossing technique and solvent welding on poly(methyl methacrylate) with water as a sacriﬁcial layer. Anal Chem. (2008) 80:2311–8. doi: 10.1021/ac7021647 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. 28. Zhang T, Yan KC, Ouyang L, Sun W. Mechanical characterization of bioprinted in vitro soft tissue models. Biofabrication. (2013) 5:045010. doi: 10.1088/1758-5082/5/4/045010 29. Selcan Gungor-Ozkerim P, Inci I, Zhang YS, Khademhosseini A, Dokmeci MR. Bioinks for 3D bioprinting: an overview. Biomater Sci. (2018) 6:915. doi: 10.1039/C7BM00765E Copyright © 2022 Schumayer, Simon, Sittkus, Wagner, Bucher and Strasser. 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. 30. Gasperini L, Mano JF, Reis RL. Natural polymers for the microencapsulation of cells. J R Soc Interface. (2014) 11:20140817. doi: 10.1098/rsif.2014.0817 31. Murphy SV, Atala A. 3D bioprinting of tissues and organs. Nat Biotechnol. REFERENCES doi: 10.1116/1.1318654 June 2022 \| Volume 4 \| Article 920384 Frontiers in Medical Technology \| www.frontiersin.org 9 Three-Dimensional and Biocompatible Lift-Off Schumayer et al. submerged in a hydrophobic high-density ﬂuid. Biofabrication. (2012) 5:015003. doi: 10.1088/1758-5082/5/1/015003 submerged in a hydrophobic high-density ﬂuid. Biofabrication. (2012) 5:015003. doi: 10.1088/1758-5082/5/1/015003 18. Moritz G. Optical single layer lift-oﬀprocess. IEEE Trans Electron Devices. (1985) 32:672–76. doi: 10.1109/T-ED.1985.21997 19. Jones SK, Chapman RC, Pavelchek EK. A practical approach to lift-oﬀ. Advances in Resist Technology and Processing IV. International Society for Optics and Photonics (1987). p. 231–43. doi: 10.1117/12.940329 33. Bégin-Drolet A, Dussault MA, Fernandez SA, Larose-Dutil J, Leask RL, Hoesli CA, et al. Design of a 3D printer head for additive manufacturing of sugar glass for tissue engineering applications. Addit Manuf. (2017) 15:29– 39. doi: 10.1016/j.addma.2017.03.006 20. Koch C, Rinke TJ. Fotolithograﬁe: Grundlagen der Mikrostrukturierung. MicroChemicals GmbH (2017). 34. Miller JS, Stevens KR, Yang MT, Baker BM, Nguyen DHT, Cohen DM, et al. Rapid casting of patterned vascular networks for perfusable engineered three- dimensional tissues. Nat Mater. (2012) 11:768–74. doi: 10.1038/nmat3357 21. Kärnfelt C, Zelenchuk D, Sinou M, Gallée F, Douglas P. Sacriﬁcial volume materials for small hole generation in low-temperature co-ﬁred ceramics. Electronics. (2020) 9:1–17. doi: 10.3390/electronics9122168 35. Wüst S, Müller R, Hofmann S. Functional biomaterials controlled positioning of cells in biomaterials-approaches towards 3D tissue printing. J Funct Biomater. (2011) 2:119–54. doi: 10.3390/jfb2030119 22. Gan Z, Zhang L, Chen G. Solvent bonding of poly (methyl methacrylate) microﬂuidic chip using phase-changing agar hydrogel as a sacriﬁcial layer. Electrophoresis. (2011) 32:3319–23. doi: 10.1002/elps.201100436 36. Byford JA, Ghazali MIM, Karuppuswami S, Wright BL, Chahal P. Demonstration of RF and microwave passive circuits through 3-D printing and selective metalization. IEEE Trans Compon Packag Manuf Technol. (2017) 7:463–71. doi: 10.1109/TCPMT.2017.2651645 23. Barz F, Lausecker R, Wallrabe U, Ruther P, Paul O. Wafer-level shellac- based interconnection process for ultrathin silicon chips of arbitrary shape. In: Proceedings of the IEEE International Conference on Micro Electro Mechanical Systems (MEMS). Institute of Electrical and Electronics Engineers Inc. Piscataway, NJ (2016). p. 520–3. doi: 10.1109/MEMSYS.2016.7421676 37. Wagner S, Zrenner E, Strasser T. Ciliary muscle thickness proﬁles derived from optical coherence tomography images. Biomed Opt Express. (2018) 9:5100. doi: 10.1364/BOE.9.005100 24. Fuentes HV, Woolley AT. Phase-changing sacriﬁcial layer fabrication of multilayer polymer microﬂuidic devices. Anal Chem. 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https://openalex.org/W2964500829	http://www.scielo.br/pdf/pcp/v39/1982-3703-pcp-39-e185662.pdf	Portuguese	null	O Perdão sob a Perspectiva do Ofensor: uma Revisão da Sistemática	Psicologia	2,019	cc-by	11,699	O Perdão sob a Perspectiva do Ofensor: uma Revisão da Sistemática Thalita Fernandes Alencar Universidade Federal da Paraíba, PB, Brasil. Eloá Losano Abreu Universidade Federal da Paraíba, PB, Brasil. Resumo: Neste estudo foram realizadas revisões sistemáticas sobre as atitudes para receber o perdão e para perdoar a si mesmo. Considerando a escassez de estudos e relevância dos temas, o objetivo foi conhecer a forma como essas atitudes foram investigadas nas publicações recentes. O levantamento dos artigos foi realizado no portal Periódicos Capes, sendo selecionados estudos publicados entre 2006 e 2016. Na revisão sobre receber o perdão foram utilizados os descritores: “receber o perdão” e “buscar o perdão”, em português e inglês. Após a aplicação dos critérios de inclusão e exclusão foram selecionados 12 artigos. Destacou-se a predominância do interesse na investigação da motivação para receber o perdão, sendo este um dos objetivos da maioria dos estudos revisados. Estes estudos contribuíram para o conhecimento da atitude para receber o perdão ao estabelecer os fatores, condições ou variáveis que favorecem a motivação para o pedido de desculpas ou para buscar receber o perdão. No que se refere ao autoperdão, foi utilizado o descritor “autoperdão”, em português e inglês. Foram selecionados 54 artigos após a aplicação dos critérios de inclusão e exclusão. Parte considerável dos estudos buscou delimitar e diferenciar os processos de autoperdão genuíno e de pseudo-autoperdão. Estes estudos procuraram estabelecer como é possível se perdoar genuinamente e exploraram as variáveis que tem um papel significativo nesse processo. Considera-se que as revisões realizadas poderão auxiliar estudos e atuações que busquem incentivar as atitudes para receber o perdão e para perdoar a si mesmo, possibilitando conhecimentos para que as atuações sejam mais efetivas. Palavras-chave: Receber o Perdão, Autoperdão, Revisão Sistemática. Psicologia: Ciência e Profissão 2019 v. 39, e185662, 1-17. https://doi.org/10.1590/1982-3703003185662 Psicologia: Ciência e Profissão 2019 v. 39, e185662, 1-17. https://doi.org/10.1590/1982-3703003185662 Artigo Artigo El Perdón bajo la Perspectiva del Ofensor: una Revisión de la Sistemática Resumen: En este estudio se realizaron revisiones sistemáticas sobre las actitudes para recibir el perdón y para perdonar a sí mismo. Considerando la escasez de estudios y la relevancia de los temas, el objetivo fue conocer la forma en que estas actitudes fueron investigadas en las publicaciones recientes. El levantamiento de los artículos fue realizado a través del portal Periódicos Capes, siendo seleccionados estudios publicados entre 2006 y 2016. En la revisión sobre recibir el perdón se utilizaron los descriptores: “recibir el perdón”, y “buscar el perdón”, en portugués e inglés. Después de la aplicación de los criterios de inclusión y exclusión se seleccionaron 12 artículos. Se destacó el predominio del interés en la investigación de la motivación para recibir el perdón, siendo éste uno de los objetivos de la mayoría de los estudios revisados. Estos estudios contribuyeron al conocimiento de la actitud para recibir el perdón al establecer los factores, condiciones o variables que favorecen la motivación para la disculpa o para buscar recibir el perdón. Con respecto al auto-perdón, se utiliza el descriptor de “auto- perdón” en portugués y en inglés. Se seleccionaron 54 artículos después de la aplicación de los criterios de inclusión y exclusión. Una parte considerable de los estudios trató de delimitar y diferenciar los procesos de auto-perdón genuino y pseudo-auto-perdón. Estos estudios buscaron establecer cómo es posible auto-perdonar genuinamente y explotaron las variables que tienen un papel significativo en ese proceso. Se considera que las revisiones realizadas podrán auxiliar estudios y actuaciones que busquen incentivar las actitudes para recibir el perdón y para perdonar a sí mismo, posibilitando conocimientos para que las actuaciones sean más efectivas. Parte considerable de los estudios buscó delimitar y diferenciar los procesos de auto-perdón genuino y de pseudo-auto-perdón. Palabras clave: Recibir el Perdón, Autoperdón, Revisión de la Sistemática. Este trabalho consiste em uma revisão da litera- tura sobre as atitudes para receber o perdão e perdoar a si mesmo. O perdão interpessoal é compreendido como uma atitude moral que contribui para o bem- -estar físico e emocional das vítimas e ofensores, sendo, inclusive, importante para a continuidade das relações sociais e a manutenção dos vínculos afetivos entre membros de uma família, amigos, colegas em ambientes de trabalho e mesmo entre grupos sociais com história de violência e preconceitos (Enright, & Fitzgibbons, 2015). Forgiveness from the Offender’s Perspective: A Review of Systematics Abstract: This study carried out systematic reviews on the attitudes to receiving forgiveness and self-forgiveness. Considering the lack of studies and the relevance of the themes, the aim was to understand how these attitudes have been investigated in recent publications. The articles were collected through the Periódicos Capes portal, and studies published between 2006 and 2016 were selected. In the receiving forgiveness’ review, the following descriptors were used: “receiving forgiveness” and “seeking forgiveness”, in Portuguese and English. After applying the inclusion and exclusion criteria, 12 articles were selected. It was highlighted the predominance of interest in the investigation of the motivation to receiving forgiveness, being this one of the objectives of most of the studies reviewed. These studies contributed to the knowledge of the attitude to receive forgiveness by establishing the factors, conditions or variables that favor the motivation to apologize or to seek forgiveness. Regarding self-forgiveness, the descriptor “self-forgiveness”, in Portuguese and English, was used. We selected 54 articles after applying the inclusion and exclusion criteria. A considerable part of the studies sought to delimit and differentiate the processes of genuine self-forgiveness and pseudo self-forgiveness. These studies sought to establish how it is possible to genuinely self-forgive and explore the variables that play a significant role in this process. It is considered that the revisions made may help studies and actions that seek to encourage attitudes to receive forgiveness and self-forgiveness, allowing knowledge to make the actions more effective. Keywords: Receiving Forgiveness, Self-forgiveness, Systematic Review. Disponível em www.scielo.br/pcp Disponível em www.scielo.br/pcp Psicologia: Ciência e Profissão 2019 v. 39, e185662,1-17. El Perdón bajo la Perspectiva del Ofensor: una Revisión de la Sistemática O interesse pela temática do per- dão na Psicologia tem crescido ao longo dos anos, e Robert Enright é um dos teóricos que se destaca nesse cenário com propostas teóricas e metodológi- cas. Enright e o Grupo de Estudos sobre o Desenvol- vimento Humano (1996) propõem o estudo do per- dão em uma tríade: (1) oferecer o perdão – que adota a perspectiva das vítimas, (2) receber o perdão e (3) autoperdão – que adotam a perspectiva dos ofensores. A atitude para oferecer o perdão tem sido privilegiada nas pesquisas em Psicologia, havendo maior concen- tração de estudos baseados na perspectiva da vítima. No entanto, sendo o perdão definido como uma ati- tude que envolve a qualidade da relação passada e presente entre vítimas e ofensores, é igualmente importante ampliar os conhecimentos sobre as atitu- des para receber o perdão e para perdoar a si mesmo.i A atitude para receber o perdão pode ser defi- nida como: “uma orientação para reduzir a culpa e o remorso gerados por uma injustiça cometida e o desejo de ser reconhecido como digno de perdão; sentir a dor da vítima e aceitar seus julgamentos, afetos e compor- tamentos negativos, enquanto espera por julgamentos, afetos e comportamentos positivos” (Alencar, 2017). De acordo com Enright (1996), uma atitude genuína para receber o perdão envolve, por parte dos ofensores, reconhecer a injustiça cometida, sen- tir remorso pelo comportamento injusto e respeitar a vítima, compreendendo que a decisão sobre o per- dão pertence a ela e que ela pode necessitar de tempo para considerar se perdoa ou não. É importante que o ofensor compreenda e tenha empatia pela vítima, que sofre por consequência da injustiça cometida por ele. No tocante ao autoperdão, segundo Holmgren (1998), para que ele ocorra genuinamente é fundamen- tal que o ofensor reconheça e sinta culpa pela ofensa 2 Alencar, T. F., & Abreu, E. L. (2019). O Perdão sob a Perspectiva do Ofensor. cometida, aceite seu erro e supere os sentimentos nega- tivos, tendo como base o respeito a si mesmo e pela vítima. É necessário que o ofensor vivencie sentimen- tos negativos decorrentes da ofensa e, em seguida, ela- bore estratégias para não cometer erros semelhantes, assumindo um compromisso moral consigo mesmo. Perspectivas teóricas dos estudos sobre receber o perdão Esta sessão tinha o propósito de apresentar as defi- nições de receber o perdão e as abordagens teóricas ado- tadas nos estudos revisados. No entanto, verificou-se que não houve uma descrição clara da definição de receber o perdão ou da perspectiva teórica adotada nos estudos revisados, que se fundamentaram majoritariamente em resultados de estudos empíricos anteriores. Destaca-se, entretanto, que Worthington foi o autor mais citado nos estudos, porém de forma pontual, sem fazer referên- cia direta a sua teoria sobre o perdão (Bassett, Bassett, Lloyd, & Johnson, 2006; Bassett et al., 2011; Chiaramello, Sastre, & Mullet, 2008; Exline, Prince-Paul, Root, Peere- boom, & Worthington Jr., 2012; Howell, Dopko, Turowski & Buro, 2011; Howell, Turowski, & Buro, 2012; Paleari, Regalia, & Fincham, 2011; Riek, 2010; Stouten, & Tripp, 2009; Wallace, Exline, & Baumeister, 2008). Worthing- ton, assim como Enright, concebe o perdão como um processo, porém enfatiza sua constituição emocional e motivacional, ressaltando a importância do vínculo entre esses dois componentes. Método e procedimentos O levantamento dos artigos foi realizado através do portal “Periódicos Capes” em abril de 2016, utili- zando como descritores os termos “receber o perdão”, “buscar o perdão”, “receiving forgiveness” e “seeking forgiveness”. A revisão englobou estudos teóricos e empíricos, sendo considerados teóricos aqueles que fundamentaram e discutiram a teoria sem realizar pesquisas empíricas; ao passo que foram considera- El Perdón bajo la Perspectiva del Ofensor: una Revisión de la Sistemática Nessa direção, Enright (1996) definiram o autoper- dão como: “uma orientação para abrandar a culpa e o remorso após o reconhecimento do erro, favorecendo a compaixão, generosidade e amor direcionados ao self” (p. 116, tradução nossa). Perdoar a si mesmo envolve refletir e reconhecer o mal causado ao outro, assumir a responsabilidade pela dor do outro e se arrepender da ação injusta para, a partir disto, buscar uma reconci- liação com o self e, possivelmente, com o outro que foi ofendido injustamente. dos estudos empíricos aqueles que se basearam em teorias para verificar relações entre variáveis ou tes- tar hipóteses. Os critérios de inclusão para seleção dos artigos foram: ser um estudo revisado por pares e publicado entre os anos de 2006 e 2016. Foram lista- dos 33 trabalhos, dos quais 23 eram artigos revisados por pares. Após a leitura dos resumos foram adotados os seguintes critérios de exclusão: (1) não apresentar a atitude para receber o perdão entre as temáticas principais; (2) repetição da publicação; e (3) impos- sibilidade de acesso ao texto completo. Ao fim deste processo restaram 12 artigos indexados nas bases de dados: Science Direct (n = 6), Academic OneFile (n = 3), EBSCOhost (n = 1), Scopus (n = 1) e PubMed/MEDLINE (n = 1). Estes 12 estudos foram descritos e analisados considerando as perspectivas teóricas, os objetivos, as metodologias e os resultados encontrados. Considerando a escassez de estudos e a relevância de se compreender as atitudes para receber o perdão e perdoar a si mesmo, a proposta desse artigo é fazer uma revisão da literatura sobre os temas como um ponto de partida para que avanços possam ocorrer nessas áreas de pesquisa na Psicologia. A revisão tem o objetivo de conhecer a forma como os estudos recentes vêm sendo desenvolvidos na literatura científica nacional e inter- nacional, e assim agregar conhecimentos à compreen- são da atitude moral do perdão. É importante esclare- cer que, como se trata de um estudo de exploração de temáticas pouco trabalhadas, não foram estabelecidos critérios rígidos na seleção dos artigos revisados. A pre- tensão é de sistematizar os estudos existentes e discutir suas características, e para isso, foi delimitada apenas a necessidade de tratar o perdão sob o olhar da Psico- logia. Para facilitar a análise sobre cada atitude, as pro- duções sobre receber o perdão e perdoar a si mesmo foram analisadas separadamente. Metodologias utilizadas nos estudos sobre receber o perdão sendo este um dos objetivos principais de oito dos 12 estudos revisados. Cinco deles focaram na moti- vação para receber o perdão sem tratar de relações interpessoais específicas (Bassett et al., 2006; 2011; Chiaramello et al., 2008; Neto, Mullet, Chiaramello, & Suwartono, 2013; Riek, 2010), enquanto três estudos trataram especificamente da motivação para pedir desculpas, considerando este comportamento como um componente importante para receber o perdão, sendo um possível precursor do mesmo (Howell et al., 2011; 2012; Leunissen, De Cremer, & Folmer, 2012). Esses estudos buscaram estabelecer quais fatores, condições ou variáveis favorecem a motivação para o pedido de desculpas ou para buscar receber o perdão. Com relação às metodologias utilizadas nos estu- dos revisados, observou-se que todos foram estudos empíricos, sendo as medidas realizadas em um único período de tempo ou em períodos muitos próximos (por exemplo, Bassett et al., 2006). Dentre esses estu- dos, verificou-se que apenas três trabalhos utilizaram delineamento experimental (Leunissen et al., 2012; Stouten, & Tripp, 2009; Wallace et al., 2008). Os demais realizaram exclusivamente pesquisas de levanta- mento sem manipulação de variáveis (Bassett et al., 2006; 2011; Chiaramello et al., 2008; Exline et al., 2012; Howell et al., 2011; 2012; Neto et al., 2013; Paleari et al., 2011; Riek, 2010). Além disso, alguns desses estudos analisaram de forma específica o papel das emoções morais, como culpa, vergonha, tristeza, raiva etc., na motivação para pedir desculpas e para receber o perdão (Bassett et al., 2006; 2011; Chiaramello et al., 2008; Howell et al., 2012; Riek, 2010). Essas emoções foram avaliadas como res- postas situacionais experienciadas em uma transgres- são específica e também como disposição de resposta do participante em situações de transgressão. Quanto aos participantes, percebe-se que na última década as pesquisas sobre receber o perdão trabalharam majoritariamente com estudantes uni- versitários. Apenas duas das pesquisas descritas pro- puseram um estudo com uma população específica: familiares de pessoas portadoras de transtorno men- tal (Exline et al., 2012) e casais (Paleari et al., 2011). É importante destacar, no entanto, que os estu- dos não limitaram seu interesse ao fator Motivação. Paleari et al. (2011), por exemplo, basearam-se na teo- ria da equidade para investigar o efeito da iniquidade ou desequilíbrio entre oferecer o perdão e receber o perdão no casamento, analisando como esse desequi- líbrio afetaria o bem-estar psicológico e emocional do casal. Metodologias utilizadas nos estudos sobre receber o perdão Ainda tendo como suporte a teoria da equidade, o estudo de Stouten e Tripp (2009) buscou examinar como membros de um grupo reagem quando a norma de equidade é violada e alguém contribui menos do que os outros. O objetivo dos autores foi analisar o efeito do pedido de perdão na reação das pessoas à violação, avaliando se a importância do pedido seria dependente do status do ofensor. Objetivos dos estudos sobre receber o perdão No que se refere aos objetivos dos estudos revi- sados, percebeu-se uma predominância do interesse na investigação da motivação para receber o perdão, 3 Psicologia: Ciência e Profissão 2019 v. 39, e185662,1-17. Resultados dos estudos sobre receber o perdão Conforme relatado anteriormente, apenas um estudo investigou o perdão sem distinguir entre as suas diferentes atitudes: oferecer o perdão, receber o perdão e perdoar a si mesmo (Exline et al., 2012). Esse estudo encontrou uma relação positiva entre ofensas não resolvidas e sintomas depressivos nos participantes, e mesmo sem tratar da atitude para receber o perdão de forma específica, esse resultado contribuiu para a rea- firmação da relação entre perdão e depressão, tradicio- nalmente apontada pela literatura da área. Nos demais estudos revisados, a atitude para receber o perdão foi tratada de forma clara, sendo distinta das outras atitudes do perdão ou de qualquer outra variável. Entre os estudos que tiveram como objetivo a motivação para receber o perdão, mere- cem destaque os trabalhos de Bassett et al. (2006) e Chiaramello et al. (2008), que avaliaram a estrutura fatorial dessa motivação e encontraram uma estru- tura de três fatores na disposição para buscar receber o perdão. Os fatores encontrados, em cada um dos estudos, representam diferentes graus de motivação e podem ser considerados similares, de forma que: Dureza no coração (Bassett et al., 2006) e Inabilidade Também foi objetivo de um dos estudos revisa- dos a análise das consequências de receber o perdão, testando se o mesmo desencorajaria ou incentivaria a repetição das transgressões (Wallace et al., 2008). E houve, ainda, um único caso em que a atitude para receber foi investigada em conjunto com as demais atitudes do perdão: oferecer o perdão e perdoar a si mesmo (Exline et al., 2012). Esse estudo teve como objetivo examinar a relevância do perdão pra familia- res de portadores de transtorno mental, porém sem fazer distinção entre os seus diferentes componentes. 4 Alencar, T. F., & Abreu, E. L. (2019). O Perdão sob a Perspectiva do Ofensor. de procurar o perdão (Chiaramello et al., 2008) refe- rem-se ao estado em que há menor motivação para buscar o perdão; Busca pela verdade (Bassett et al., 2006) e Sensibilidade às circunstâncias (Chiaramello et al., 2008) representam o estado em que o ofensor já considera a possibilidade, mas está avaliando as con- dições para decidir se deve ou não buscar o perdão; e, por fim, Busca pelo perdão (Bassett et al., 2006) e Procura incondicional do perdão (Chiaramello et al., 2008) referem-se ao estado em que há forte motivação para buscar o perdão, independentemente de qual- quer condição. Resultados dos estudos sobre receber o perdão Essa estrutura trifatorial foi testada e corroborada por Neto et al. (2013), que confirmaram a confiabilidade do modelo em diferentes culturas (Por- tugal, Brasil, Indonésia, Angola e Moçambique). mediadas pelo sentimento de culpa, reafirmando o papel positivo dessa emoção na promoção da atitude para receber o perdão. Em contrapartida, as corre- lações negativas englobaram: raiva (Chiaramello et al., 2008; Riek, 2010), ansiedade, cinismo, tendên- cias paranoicas, tendências autopunitivas e neurose (Chiaramello et al., 2008). Ainda, foi analisado o papel da orientação temporal, verificando que pessoas vol- tadas para o futuro têm maior motivação para buscar receber o perdão do que aquelas que são presas ao passado (Chiaramello et al., 2008). Ainda na temática da motivação, o estudo de Leu- nissen et al. (2012) propôs a investigação da chamada “motivação instrumental” para receber o perdão, que condiciona a motivação do ofensor à percepção da probabilidade de disposição da vítima para perdoar o mesmo. Os resultados mostraram uma motivação sig- nificativamente maior para pedir desculpas quando a chance da vítima desculpar era percebida como alta. Essa concepção da motivação para receber o perdão se diferencia dos demais estudos e na seção seguinte discutir-se-á o porquê de ela ser incompatível com a visão de uma atitude genuína. Ainda na temática dos fatores relacionados à motivação para pedir desculpas e para receber o per- dão, verificou-se que a culpa foi a emoção que rece- beu maior destaque nos estudos que investigaram o papel das emoções morais, e foi possível observar que os autores também se interessaram por compa- rá-la com outras emoções (Bassett et al., 2006; 2011; Howell et al., 2012). Apesar das distintas formas de análise, ressalta-se nesta revisão o resultado comum encontrado nos estudos acima citados: a vergonha como uma emoção que dificulta a procura do perdão, e a culpa como uma emoção que a facilita. Os estu- dos reiteraram a concepção de que a vergonha é uma emoção autocentrada, em que o sentido do Eu é alte- rado e a pessoa se sente sem valor, enquanto a culpa é uma emoção que volta a atenção para um compor- tamento específico, e não há uma desvalorização de si mesmo, pois apenas a ação é considerada repreen- sível. Assim, enquanto a vergonha levaria ao desejo de fuga, a culpa levaria ao desejo de reparação. Este resultado se manteve tanto em termos situacionais como em termos disposicionais. Resultados dos estudos sobre receber o perdão Entre os estudos que se basearam na teoria da equidade, há o estudo de Paleari et al. (2011) que bus- cou verificar o efeito da iniquidade entre oferecer o perdão e receber o perdão no casamento. Os resulta- dos mostraram que os homens perdoaram mais do que foram perdoados, o que representa uma iniqui- dade entre o casal. Porém, com relação ao impacto desta iniquidade para a satisfação com o casamento e bem-estar emocional, este estudo revelou que a per- cepção de iniquidade afetou negativamente o bem- -estar emocional e relacional das esposas, relação esta que não se estabeleceu com os maridos. No estudo de Stouten e Tripp (2009), por sua vez, foram utilizados dilemas sociais para analisar o efeito do pedido de perdão na reação de membros de um grupo à violação da norma de equidade, avaliando se haveriam diferenças quando o líder ou outro membro do grupo (considerado um igual) era o responsável pela violação de contribuir menos do que os demais membros. Com seus resultados, os autores consta- taram que a importância do pedido de desculpas foi dependente do status do ofensor. Entre os iguais, o pedido de desculpas foi necessário e eficiente para atenuar as emoções negativas e aumentar a possibi- lidade de perdão da vítima, ou seja, a probabilidade de receber o perdão. Entretanto, quando o violador Outras variáveis também foram apontadas pelos estudos como relacionadas à motivação para buscar receber o perdão e para se desculpar. As correlações positivas incluíram: amabilidade (Chiaramello et al., 2008; Howell et al., 2011), nível intelectual (Chiara- mello et al., 2008), arrependimento e preocupação (Bassett et al., 2006), índices de funcionamento adap- tativo, como compaixão, bem-estar, autoestima e aceitação (Howell et al., 2011), e variáveis situacionais, como responsabilidade sobre a ofensa, severidade da ofensa, proximidade da relação e ruminação (Riek, 2010). As relações com as variáveis situacionais foram 5 Psicologia: Ciência e Profissão 2019 v. 39, e185662,1-17. relação não pode ser confirmada devido à ausência de delimitação do referencial teórico dos estudos.i era um líder, o papel do pedido de desculpas não foi igualmente importante. Os líderes foram perdoados mais facilmente e foram menos percebidos como vio- ladores, sendo possível inferir que, devido a sua posi- ção de poder, tenham sido considerados como deten- tores do direito de contribuir menos. ii Ressalta-se, porém, que especificamente no estudo de Leunissen et al. Resultados dos estudos sobre receber o perdão (2012) a motivação o para receber o perdão foi abordada de forma diferente e contraditória. A denominada “motivação instrumen- tal” parte de um forte interesse do ofensor em prote- ger a si mesmo e só procurar o perdão quando este se mostra “seguro”, isto é, buscar o perdão quando nota que a vítima está disposta a oferecê-lo. Entretanto, quando o perdão é compreendido e vivenciado como uma atitude moral, ele não está condicionado a con- sequências externas. A genuína procura pelo perdão é uma atitude em que a pessoa escolhe reconhecer o erro e tenta repará-lo, independentemente da res- posta do outro (Enright, & Fitzgibbons, 2015). Por fim, para verificar as consequências de rece- ber o perdão no incentivo a repetição das transgres- sões, Wallace et al. (2008) utilizaram medidas compor- tamentais e de autorrelato, buscando com isso uma análise mais completa dessa relação. Em seus resul- tados, receber o perdão foi preditor de uma maior motivação para o arrependimento, diminuindo a dis- posição para ofender novamente a mesma pessoa, de maneira que a maioria dos participantes se mostrou menos propensa a repetir as ofensas quando havia recebido o perdão da vítima. Estudos como o de Paleari et al. (2011) e Stouten e Tripp (2009) também merecem ser destacados, pois chamam atenção para o aspecto relacional do per- dão ao tratarem, respectivamente, dos efeitos da ini- quidade nas relações conjugais e do status de poder do ofensor. Pode-se dizer que esses estudos reafir- maram a importância de sempre considerar todos os personagens envolvidos ao se trabalhar com o per- dão, pois a forma como cada um reage trará implica- ções para as vivências individuais dos processos e é preciso considerar o possível impacto dos diferentes níveis de relacionamento. Método e Procedimentos Os artigos desta revisão foram pesquisados atra- vés do portal Periódicos Capes em abril de 2016 e foram utilizados como descritores os termos “autoperdão” e “self forgiveness”. Assim como na revisão anterior, foram contemplados estudos teóricos e empíricos. Foram elencados 113 artigos que atenderam aos cri- térios de inclusão de ser um estudo revisado por pares e publicado entre os anos de 2006 e 2016. Em seguida foi realizada a leitura dos resumos, sendo aplicados os seguintes critérios de exclusão: (1) não apresentar a atitude para o autoperdão entre as temáticas princi- pais, (2) não ter relação com a Psicologia, (3) repetição da publicação e (4) impossibilidade de acesso ao texto completo. Ao fim deste processo restaram 54 artigos que foram revisados e analisados neste estudo. Estes estavam indexados nas bases de dados: Academic OneFile (n = 13), EBSCOhost (n = 12), SprigerLink (n = 8), Science Direct (n = 8), Sage Journals (4), Scopus (n = 1) e Ovid MEDLINE (n = 1). Sete artigos foram, ainda, obtidos através da Wiley Online Library que permitiu o acesso direto a revista na qual se encontra. As descrições e análises dos estudos foram realizadas considerando as perspectivas teóricas, os objetivos, as metodologias e os resultados encontrados. i Apesar da diversidade de definições e da ausên- cia de concordância sobre o que é o autoperdão, já existe na literatura um maior consenso na delimita- ção daquilo que ele não é. Houve uma clara distin- ção entre autoperdão e outros comportamentos de defesa, como negar, minimizar ou justificar a ofensa. Esses comportamentos podem ser denominados de pseudo-autoperdão, uma vez que não há o reconheci- mento do erro e a aceitação da responsabilidade, que são fundamentais para o autoperdão genuíno que, por sua vez, requer tempo e esforço (Cornish, & Wade, 2015; Exline et al., 2011; Fisher, & Exline, 2006; 2010; Kim, & Enright, 2014; Robb III, 2007; Ursúa & Eche- goyen, 2015; vanOyen-Witvliet et al., 2011; Wenzel et al., 2012; Wohl & McLaughlin, 2014; Woodyatt & Wen- zel, 2013a, 2013b, 2014). Discussão (2019). O Perdão sob a Perspectiva do Ofensor. conhecimentos sobre essa atitude, não só ampliando a quantidade de estudos, mas também a investigando com maior diversidade de delineamentos e popula- ções, pois isso tornará mais abrangente e representa- tivo o que se sabe sobre receber o perdão. ção de autoperdão adotada, sendo grande a variedade de conceitos e teorias. A definição de Enright (1996) foi a mais prevalente (Day, Gerace, Wilson, & Howells, 2008; Dixon, Earl, Lutz-Zois, Goodnight, & Peatee, 2014; Exline, Root, Yadavalli, Martin, & Fischer, 2011; Fisher, & Exline, 2010; Friedman et al., 2007; Kim, & Enright, 2014; Ursúa, & Echegoyen, 2015; Wenzel, Woodyatt, & Hedrick, 2012), seguida pela definição de Hall e Fin- cham (2005), que concebem o autoperdão como a diminuição da motivação para autopunição e retalia- ção contra si mesmo, bem como o aumento da motiva- ção para benevolência em relação a si (Hall, & Fincham, 2008; Krause, 2015; McConnell, & Dixon, 2012; Rang- ganadhan, & Todorov, 2010; Wohl, Pychyl, & Bennett, 2010; Woodyatt, & Wenzel, 2013a). Houve, ainda, a pro- posição de uma definição própria para o autoperdão no estudo de Cornish e Wade (2015), que criticaram as definições anteriores por considerar que são centradas apenas no ofensor e propuseram incluir na definição de autoperdão componentes voltados para vítima, a saber, a reparação da ofensa e a redução da probabili- dade de cometer ofensa semelhante. Nos demais estu- dos, a visão conceitual dos autores foi explicitada a par- tir de informações obtidas em estudos anteriores, sem delimitar uma perspectiva teórica específica. Por fim, é importante salientar a ausência de pesquisas brasileiras entre os estudos revisados. Veri- fica-se que a temática do perdão não é amplamente explorada no Brasil e o foco dos estudos têm sido as vítimas. Mostra-se, pois, relevante o desenvolvimento de pesquisas que considerem os ofensores, para ava- liar como essas relações se estabelecem com a popu- lação brasileira. Discussão A revisão da literatura permitiu constatar que os estudos recentes sobre receber o perdão, além de escassos, não apresentam claramente o conceito dos autores sobre essa variável, tendo pouca relação com abordagens teóricas. Considera-se que essa caracte- rística pode ser associada ao fato de todos os estudos serem empíricos, podendo-se inferir uma maior pre- ocupação dos autores com questões metodológicas e com a discussão de resultados em detrimento do esclarecimento teórico. No entanto, essa escolha é considerada problemática, pois a concepção teórica do perdão influencia substancialmente a forma de estudá-lo, sendo essa delimitação muito importante tanto para o autor, ao realizar seu estudo empírico, quanto para o leitor, na compreensão e análise crí- tica dos estudos. Ainda, destaca-se o estudo de Wallace et al. (2008), que avaliou as consequências de receber o perdão. Essa temática é bastante discutida por defensores e críticos do perdão que discordam sobre seu papel em reiterar ou não o comportamento ofensivo (Enright & Fitzgibbons, 2015). O resultado encontrado por Wallace et al. (2008) mostra que rece- ber o perdão pode ter consequências positivas não só para o ofensor, mas também para a vítima e para a relação entre eles, desmistificando, assim, a ideia de que perdoar favorece a repetição da ofensa. Informa- ções como esta estimulam o estudo sobre receber o perdão e corroboram a importância de trabalhos que busquem incentivá-lo.i No que se refere aos objetivos dos estudos revi- sados, destaca-se a prevalência do interesse pelo fator Motivação. Considera-se que tais investigações são de grande valia para estruturação epistemológica da ati- tude para receber o perdão e, sobretudo, para a pro- moção da mesma, pois elas possibilitam estabelecer os aspectos que devem ser foco de atenção no traba- lho com ofensores. Ainda, é preciso considerar que, mesmo que de forma indireta, Worthington foi o autor mais referenciado nos estudos revisados. Sendo este um teórico que valoriza a constituição motivacional do perdão, é possível que haja relação com o fato de a motivação ter constituído o interesse predominante nos estudos sobre receber o perdão. No entanto, essa Diante dos resultados desta revisão, verifica-se que a predominância do interesse das pesquisas pelo fator Motivação trouxe importantes contribui- ções para o conhecimento da atitude para receber o perdão. Porém, existem ainda muitos caminhos a serem explorados, especialmente por esta ser uma área recente de estudos. É importante expandir os 6 Alencar, T. F., & Abreu, E. L. Método e Procedimentos A aceitação da responsabilidade foi, inclusive, citada nos estudos revisados como um dos compo- nentes fundamentais para que o autoperdão seja genuíno (Cornish & Wade, 2015; Exline et al., 2011; Fisher & Exline, 2006, 2010; Griffin, Lavelock & Wor- thington Jr., 2014; Kim, & Enright, 2014; McConnell, Dixon, & Finch, 2012; Robb III, 2007; Ursúa, & Eche- goyen, 2015; Oyen-Witvliet et al., 2011; Wenzel et al., Resultados Perspectivas teóricas dos estudos sobre autoperdão No que diz respeito às perspectivas teóricas uti- lizadas para o estudo do autoperdão, destaca-se que apenas 15 estudos estabeleceram claramente a defini- 7 Psicologia: Ciência e Profissão 2019 v. 39, e185662,1-17. Cukrowicz, & Hirsch, 2013), submissão à violência íntima (Davidson, Lozano, Cole, & Gervais, 2015), pro- crastinação (Wohl et al., 2010), religião (Griffin et al., 2014; McConnell, & Dixon, 2012) etc. 2012; Wohl, & McLaughlin, 2014; Wohl et al., 2010; Woodyatt, & Wenzel, 2013a;b; 2014; Worthington, & Langberg, 2012). Afirmou-se nesses estudos que é preciso, inicialmente, reconhecer a injustiça come- tida, assumir a responsabilidade e vivenciar as emo- ções negativas oriundas dela, para em seguida elabo- rar essa ofensa e reconquistar a autoaceitação, apesar do erro cometido. Entre os estudos revisados também houve inte- resse nos possíveis efeitos do autoperdão para a saúde física (Webb, Phillips, Bumgarner, & Conwa- y-Williams, 2013a; Wilson, Milosevic, Carroll, Hart, & Hibbard, 2008) e mental (Fisher; & Exline, 2006; Krause, 2015; Macaskill, 2012; Nsamenang et al., 2013; Romero et al., 2006; Webb et al., 2013a; Webb, Hirsch, Conway-Williams, & Brewer, 2013b) do ofensor. Embora essa distinção seja clara na teoria, os estudos empíricos têm apontado a dificuldade em diferenciar o autoperdão genuíno e o pseudo-auto- perdão (Cornish, & Wade, 2015; Fisher, & Exline, 2006; 2010; Ursúa; & Echegoyen, 2015; Wenzel et al., 2012; Woodyatt & Wenzel, 2013a;b). Os autores atribuíram essa limitação ao fato de os estudos medirem o auto- perdão apenas pelo estado final do processo. Como o pseudo-autoperdão e o autoperdão genuíno levam ao mesmo resultado, que se caracteriza pela diminuição da autopunição e o aumento do sentimento positivo em relação a si mesmo, não é possível diferenciá-los focando apenas no resultado. Essa imprecisão jus- tificaria os resultados de estudos, como o de Strelan (2007a), que associam o autoperdão a pessoas narci- sistas e egocêntricas, que não sentem culpa ou arre- pendimento pelo ato injusto cometido. Os estudos revisados centraram-se, majoritaria- mente, nos aspectos positivos do autoperdão. Entre- tanto, três deles voltaram-se para um possível aspecto negativo do autoperdão, focando nas situações em que o mesmo poderia não ser apropriado (Squires, Sztainert, Gillen, Caouette, & Wohl, 2012; Wohl, & McLaughlin, 2014; Wohl, & Thompson, 2011). Resultados Ainda, há o estudo de Vitz e Meade (2011) que realizaram uma análise crítica sobre os potenciais problemas do autoperdão, enquanto o estudo de Kim e Enright (2014) apresenta uma defesa teológica e psicológica do autoperdão, respondendo críticas contra o mesmo, incluindo as realizadas por Vitz e Meade (2011). Metodologias utilizadas nos estudos sobre autoperdão j p Considerando que é no processo que o autoper- dão genuíno e o pseudo-autoperdão se diferenciam, dez estudos tiveram como objetivo a investigação e a delimitação desses processos (Cornish, & Wade, 2015; Fisher, & Exline, 2006; 2010; Hall, & Fincham, 2008; Kim, & Enright, 2014; McConnell et al., 2012; Ursúa, & Echegoyen, 2015; Wenzel et al., 2012; Woodyatt, & Wenzel, 2013a;b; 2014). Estes estudos buscaram esta- belecer como é possível se perdoar genuinamente e exploraram as variáveis que tem um papel significa- tivo ao longo desse processo. De forma mais especí- fica, destaca-se o estudo de Cornish e Wade (2015), que teve como objetivo a proposição de um novo modelo de processo terapêutico para o autoperdão genuíno, e o estudo Woodyatt e Wenzel (2013a), que propôs um modelo que diferenciou as possíveis res- postas do ofensor após a injustiça. No que se refere às metodologias aplicadas nos estudos, percebe-se que, dos 54 artigos revisados, 11 foram de natureza teórica (Cornish, & Wade, 2015; Day et al., 2008; Fisher, & Exline, 2010; Griffin et al., 2014; Hong, & Jacinto, 2012; Kim, & Enright, 2014; Robb III, 2007; Ursúa, & Echegoyen, 2015; Vitz, & Meade, 2011; Wohl, & McLaughlin, 2014; Worthington, & Langberg, 2012), sendo dominante a pesquisa empírica nos estudos recentes sobre autoperdão. Nota-se que entre os estudos empíricos analisados, dois foram estudos de caso (Hong, & Jacinto, 2012; Lander, 2012) e cinco foram longitudinais, porém a maioria destes compre- endeu períodos que podem ser considerados restri- tos, sendo o ofensor acompanhado por 11 dias (Wen- zel et al., 2012; Woodyatt & Wenzel, 2013a;b) ou sete semanas (Hall, & Fincham, 2008) após a ocorrência da ofensa. Apenas o estudo de Krause (2015) compreen- deu período maior, realizando rodadas de entrevista entre os anos de 2007 e 2008. Alguns estudos investigaram, ainda, a relação específica entre o autoperdão e determinadas vari- áveis, como ideação e/ou comportamento suicida (Cheavens, Cukrowicz, Hansen, & Mitchell, 2016; Hirsch, Webb, & Jeglic, 2011; 2012; Nsamenang, Webb, Ainda, prevaleceram entre os estudos empíricos revisados as pesquisas de levantamento, enquanto 8 Alencar, T. F., & Abreu, E. L. (2019). O Perdão sob a Perspectiva do Ofensor. Wenzel, 2013b). Os autores afirmaram que o pseudo- -autoperdão pode não ser uma atitude negativa deli- berada, mas sim uma resposta defensiva natural nos momentos em que o ofensor sente sua pertença ao grupo ameaçada devido à injustiça por ele cometida. Metodologias utilizadas nos estudos sobre autoperdão Para testar a hipótese, realizaram um estudo laborato- rial, em que manipularam a ameaça à pertença, e um estudo longitudinal, em que acompanharam os parti- cipantes 11 dias após ter sido cometida uma injustiça. Em seus resultados, o pseudo-autoperdão se rela- cionou positivamente com a percepção de respostas hostis da vítima, porém quando a resposta da vítima ocorreu de forma respeitosa, a relação com o pseudo- -autoperdão foi negativa. Apesar de buscarem enten- der o processo do pseudo-autoperdão, esses autores não defenderam que esta seja a forma mais adequada de lidar com a injustiça cometida, e salientaram que o processamento efetivo de uma ofensa deve envolver a aceitação de responsabilidade sobre ela. apenas cinco estudos fizeram uso de delineamento experimental (Exline et al., 2011; Oyen-Witvliet et al., 2011; Wenzel et al., 2012; Woodyatt, & Wenzel, 2013a; 2014). Em ambos os casos o autoperdão tem sido avaliado quase que exclusivamente através de medi- das explícitas de autorrelato. Considerando que este tipo de medida apresenta fraquezas pelo longo tempo de resposta que permite a interveniência de vieses, os estudos de Bast e Barnes-Holmes (2014; 2015a;b), Bast, Barnes-Holmes e Barnes-Holmes (2015) e Bast, Linares, Gomes, Kovac, e Barnes-Holmes (2016) obje- tivaram desenvolver e empregar medidas implícitas de autoperdão, utilizando o Procedimento Implí- cito de Avaliação Relacional1. As medidas implícitas demandam uma resposta rápida do participante e são apresentadas pelos autores como uteis para comple- mentar as medidas explícitas de autoperdão. Quanto aos participantes, observa-se que, assim como na revisão anterior, os estudos recentes sobre o autoperdão também têm trabalhado predominan- temente com estudantes universitários, até mesmo para tratar de questões específicas como problemas com álcool (Webb et al., 2013a), desordem alimentar (Feibelman, & Turner, 2015), sintomas depressivos (Hirsch et al., 2011), personalidade boderline (Law, & Chapman, 2015), problemas com jogo (Squires et al., 2012), vício em cigarros (Wohl, & Thompson, 2011) e vivência de violência íntima (Davidson et al., 2015). Apesar de em menor quantidade, foram encon- trados estudos que trabalharam com populações específicas e não universitárias, como idosos (Chea- vens et al., 2016; Ingersoll-Dayton, Torges, & Krause, 2010; Krause, 2015), mulheres com câncer de mama (Friedman et al., 2007; Romero et al., 2006), minorias sexuais LGBTQ (Greene, & Britton, 2013), adolescen- tes que praticam ou praticaram autoinjúria (Westers, Rehfuss, Olson, & Biron, 2012) e estudos de casos com pacientes com doença crônica (Hong, & Jacinto, 2012) e desordem alimentar (anorexia) (Lander, 2012). 1 Termo original na língua inglesa: Implicit Relational Assessment Procedure (IRAP). Metodologias utilizadas nos estudos sobre autoperdão Com relação aos estudos que se voltaram a com- preender o autoperdão genuíno, tem sido enfatizado o papel do autoperdão para a redução das emoções negativas associadas a ofensa como culpa, vergonha, remorso e/ou autocondenação. Porém, também foi avaliado o papel exercido por essas emoções nega- tivas no autoperdão, sendo estabelecidas relações similares às referidas nos estudos sobre receber o perdão (Cornish, & Wade, 2015; Davidson et al., 2015; Exline et al., 2011; Fisher, & Exline, 2006; 2010; Gre- ene, & Britton, 2013; Hall, & Fincham, 2008; Macaskill, 2012; Rangganadhan, & Todorov, 2010). Esses estu- dos mostraram que culpa e remorso desempenham papéis mais úteis para o autoperdão, pois têm como foco a ação cometida e a preocupação com a vítima, e por isso motivam tentativas de restauração, ao passo que a vergonha e a autocondenação são emoções globais que afetam a imagem que a pessoa tem de si mesma e são propensas a gerar respostas de fuga e/ ou de autoproteção. Uma visão diferenciada sobre a vergonha foi apresentada por Woodyatt e Wenzel (2014), que aler- taram sobre os perigos de não vivenciar essa emo- ção após cometer uma ofensa. De acordo com esses autores, a vergonha se associa a comportamentos de defesa apenas quando a ameaça de rejeição é grande e a reparação de sua causa é difícil. Sendo assim, eles alegam que quando a reparação do ato é possível, Resultados dos estudos sobre autoperdão Resultados dos estudos sobre autoperdão Entre os estudos que tiveram como objetivo a investigação e a delimitação dos processos de pseu- do-autoperdão e autoperdão genuíno, serão descritos, inicialmente, os resultados encontrados pelo único estudo que investigou diretamente o processo psico- lógico da atitude de pseudo-autoperdão (Woodyatt, & 9 Psicologia: Ciência e Profissão 2019 v. 39, e185662,1-17. (Exline et al., 2011; Fisher, & Exline, 2010; Ursúa, & Echegoyen, 2015), foram Cornish e Wade (2015) que, baseados nas discussões da literatura sobre o tema, propuseram um novo modelo de processo terapêutico para o autoperdão. Esse processo, de acordo com os autores, é formado por quatro componentes sequen- ciais: (1) aceitação da Responsabilidade; (2) vivência do Remorso; (3) Restauração através de comporta- mentos conciliatórios e reafirmação de valores; e (4) Renovação da visão de si mesmo, reestabelecendo a aceitação, o respeito, a compaixão e a bondade. Esse modelo traz como principal inovação a inclusão do componente Restauração, que é voltado para o outro e tem um papel central no processo desses autores, tornando-o menos intrapsíquico. a vergonha pode atuar como sua motivação, o que resultaria em um papel relevante para o autoperdão. A habilidade de manejar e de regular emo- ções vem sendo apontada na literatura como uma importante facilitadora do autoperdão (Hodgson, & Wertheim, 2007; Lander, 2012). Fisher e Exline (2010) defenderam que é preciso ajudar o ofensor a cami- nhar da vergonha para culpa, considerando que esta última desempenha um melhor papel para o auto- perdão. E no que se refere à culpa, Fisher e Exline (2010) e McConnell et al. (2012) ressaltaram a impor- tância de se trabalhar a diminuição da mesma, espe- cialmente por meio do incentivo a comportamentos conciliatórios, como pedir desculpas, confessar o erro ou buscar repará-lo. Woodyatt e Wenzel (2013a) também avaliaram o autoperdão genuíno, propondo um modelo que diferenciou três possíveis respostas dos ofensores: autopunição, pseudo-autoperdão e autoperdão genuíno. Seus resultados demonstraram que o auto- perdão genuíno foi a única resposta que, além de se associar a benefícios intrapessoais, como a melhora na autoestima e na autoconfiança, se associou tam- bém com benefícios interpessoais, ou seja, com respostas positivas também voltadas para a vítima, como maior empatia e desejo de reconciliação, e diminuição da evitação. Esses benefícios interpes- soais do autoperdão também foram encontrados em outros estudos (Cornish, & Wade, 2015; Day et al., 2008; Fisher, & Exline, 2006; 2010; Rangganadhan, & Todorov, 2010). Resultados dos estudos sobre autoperdão O papel dos comportamentos conciliatórios tam- bém foi investigado e analisado por outros estudos (Cornish, & Wade, 2015; Exline et al., 2011; Fisher, & Exline, 2006; Hall, & Fincham, 2008; McConnell et al., 2012; Oyen-Witvliet et al., 2011; Rangganadhan, & Todorov, 2010; Ursúa, & Echegoyen, 2015; Wenzel et al., 2012; Woodyatt, & Wenzel, 2013a; 2014) que corro- boraram sua influência positiva no autoperdão. Base- ados no estudo de Hall e Fincham (2008), em que foi confirmada a relação entre comportamentos conci- liatórios e autoperdão, McConnell et al. (2012) propu- seram um modelo alternativo em que acrescentaram a mediação da percepção do perdão da vítima para a explicação dessa relação. A ideia defendida foi de que os comportamentos conciliatórios do ofensor pode- riam favorecer o perdão da vítima, que uma vez per- cebido pelo ofensor, facilitaria também o autoperdão. O papel das variáveis situacionais também foi avaliado, mesmo que de forma superficial, em alguns estudos. Essas variáveis foram relacionadas direta- mente ao autoperdão ou a fatores importantes para o mesmo, como o remorso e os comportamentos conci- liatórios: 1) a Severidade da Ofensa foi a variável mais prevalente nos estudos, sendo apontada sua asso- ciação negativa com o autoperdão (Hall, & Fincham, 2008; McConnell et al., 2012) e sua associação positiva com o remorso e com os comportamentos concilia- tórios (Fisher, & Exline, 2006); 2) a Proximidade da Relação entre a vítima e o ofensor foi positivamente relacionada com o remorso (Fisher, & Exline, 2006) e com os comportamentos conciliatórios (Exline et al., 2011; Fisher, & Exline, 2006); e 3) o Tempo desde a Ocorrência da Ofensa se relacionou positivamente com o autoperdão (Hall, & Fincham, 2008) e negativa- mente com os comportamentos conciliatórios (Exline Ainda neste contexto, cabe destacar os estudos de Wenzel et al. (2012) e Woodyatt e Wenzel (2014) que explicaram a relação entre a aceitação da responsabi- lidade e o autoperdão por meio dos comportamentos conciliatórios e da sua relação com a reafirmação de valores. De acordo com os autores, ao cometer uma injustiça, o ofensor viola certos valores pessoais, e o reconhecimento do erro pode, inclusive, afetar nega- tivamente a identidade moral do ofensor. Por isso, há uma relação negativa entre a aceitação da responsa- bilidade e uma autoestima positiva. Resultados dos estudos sobre autoperdão Porém, quando o mesmo assume comportamentos conciliatórios, isso o leva a reafirmar seus valores anteriormente viola- dos e sua identidade moral, o que contribui para a melhora de sua autoimagem e facilita o autoperdão. Apesar de haver concordância com relação à percepção de um caminho ideal para o autoperdão 10 Alencar, T. F., & Abreu, E. L. (2019). O Perdão sob a Perspectiva do Ofensor. Ainda, é importante destacar os estudos que se voltaram para o possível lado negativo do autoper- dão, ressaltando as situações em que o mesmo pode- ria não ser apropriado (Squires et al., 2012; Wohl, & McLaughlin, 2014; Wohl, & Thompson, 2011). De acordo com esses autores, perdoar a si mesmo por um comportamento não saudável ou nocivo, como fumar ou apostar, pode ter um efeito prejudicial, como se associar a uma menor disposição para modificar tal comportamento. Wohl e McLaughlin (2014) ressalta- ram a importância de se perdoar no momento certo, quando o comportamento nocivo já estiver encerrado, para que assim não prejudique o desejo de modificar o comportamento. Exline et al. (2011) e Fisher e Exline (2010) também enfatizaram a importância de não incentivar o autoperdão cedo demais, considerando que isso poderia atrapalhar a busca de reparação por parte do ofensor. et al., 2011). Apesar do pouco destaque dado a essas variáveis, mostra-se importante considerar o valor das mesmas para o autoperdão. Os benefícios intrapessoais do autoperdão também foram apontados pelos artigos revisados. A melhora na autoimagem e na autoestima foram aspectos do autoperdão considerados por todos os estudos, mesmo que de forma indireta, uma vez que integram a própria definição do autoperdão, que é caracterizado pelo reestabelecimento do amor em relação a si mesmo, apesar do erro cometido. Além desse benefício, o autoperdão também se relacionou a melhorias para a saúde física (Webb et al., 2013a; Wilson et al., 2008), sendo, inclusive, indicados seus potenciais benefícios para o tratamento de câncer de mama (Friedman et al., 2007; Romero et al., 2006), alcoolismo (Webb et al., 2013b) e desordem alimentar (Feibelman, & Turner, 2015; Lander, 2012). Buscando responder as críticas tecidas em estu- dos como o de Vitz e Meade (2011), Kim e Enright (2014) elaboraram uma defesa do autoperdão, em que os autores ressaltaram a importância de dife- renciar pseudo-autoperdão do autoperdão genuíno, enfatizando o papel do reconhecimento do erro e da aceitação da responsabilidade. Resultados dos estudos sobre autoperdão Destacaram, ainda, a importância de se distinguir as três formas de perdão (autoperdão, perdão de Deus, perdão da vítima), con- siderando que cada uma se desenvolve de maneira singular e independente, embora uma possa facilitar a outra. O estudo de Hall e Fincham (2008) confirmou a associação positiva entre a percepção de perdão da vítima e de Deus e o autoperdão.i As melhorias para a saúde mental do ofensor tam- bém foram verificadas nos estudos (Fisher, & Exline, 2006; Krause, 2015; Macaskill, 2012; Nsamenang et al., 2013; Romero et al., 2006; Webb et al., 2013a;b), sendo apontada especificamente sua: associação negativa com depressão (Fisher, & Exline, 2006; Hirsch et al., 2011; Ingersoll-Dayton et al., 2010; Law, & Chapman, 2015; Nsamenang et al., 2013) e ansiedade (Fisher, & Exline, 2006; Krause, 2015; Macaskill, 2012); e asso- ciação positiva com a qualidade de vida (Friedman et al., 2007; Romero et al., 2006) e com a satisfação com a vida (Fisher & Exline, 2006; Krause, 2015; Macaskill, 2012). Entre os estudos que investigaram a relação espe- cífica entre o autoperdão e determinadas variáveis, os resultados mostraram que o autoperdão associa-se negativamente com: a ideação e/ou comportamento suicida (Cheavens et al., 2016; Hirsch et al., 2011; 2012; Nsamenang et al., 2013); a autoinjúria não sui- cida (Westers et al., 2012); a submissão à violência íntima (Davidson et al., 2015); a autocondenação em militares (Worthington, & Langberg, 2012); o perfec- cionismo (Dixon et al., 2014; Kim, Johnson, & Ripley, 2011); e a procrastinação (Wohl et al., 2010). Os resul- tados também apontaram as associações positivas do autoperdão com: a crença no mundo justo (Strelan, 2007b); a consciência plena2 (Webb et al., 2013a); a humildade (Krause, 2015); e a religião (Griffin et al., 2014; McConnell, & Dixon, 2012). Por fim, aponta-se a existência de estudos que enfatizaram que, enquanto a reconciliação é uma possibilidade para a atitude para oferecer o perdão, mas não uma condição, para o autoperdão a reconci- liação é imprescindível (Hall, & Fincham, 2008; Robb III, 2007; Ursúa, & Echegoyen, 2015). 2 Termo original na língua inglesa: mindfulness. Discussão A realização desta revisão evidenciou que o estudo do autoperdão tem crescido na Psicologia, o que denota fortalecimento desse campo de estudo e o reconhecimento da importância dessa atitude. O foco de parte dos estudos no autoperdão genuíno demons- tra a maturidade desse campo, que já discute e pro- cura solucionar problemas surgidos ao longo das aná- 11 11 Psicologia: Ciência e Profissão 2019 v. 39, e185662,1-17. dão da vítima e a tentativa de reparação. No entanto, é importante ter em mente que, ao se tratar do autoper- dão genuíno, a ideia de que o mesmo desencorajaria a mudança de comportamento e a preocupação com o outro não se sustenta, pois a pessoa se perdoa tendo consciência do erro que cometeu e de sua respon- sabilidade sobre ele, e não esquecendo, negando ou justificando o ato cometido. Além disso, a concepção de parte dos estudos revisados de que o autoperdão exige a reconciliação consigo mesmo é um impor- tante aspecto desta atitude, que fortalece a importân- cia dos comportamentos conciliatórios e da mudança de comportamento do ofensor como formas de recu- perar a confiança em si mesmo para que seja possível essa reconciliação. lises dessa atitude. As características que distinguem o autoperdão genuíno do pseudo-autoperdão, como o reconhecimento do erro e a aceitação da respon- sabilidade, legitimam o autoperdão como uma ati- tude moral, que apesar de auxiliar o ofensor em uma reconciliação consigo mesmo, não atua justificando seus atos e eximindo-o de sua responsabilidade. A assunção de responsabilidade gera emoções negativas que, por sua vez, possuem um papel com- plexo. Por um lado, essas emoções são essenciais para que o autoperdão seja genuíno, mas elas também são um obstáculo para o mesmo, pelo menos inicial- mente, pois quanto mais grave a ofensa e maiores as emoções negativas, maior será o esforço necessá- rio para que o autoperdão seja alcançado. Por esse motivo foi relevante que parte dos estudos revisados tenha analisado quais variáveis têm importância na transição desse processo e na explicação dessa rela- ção, ou seja, tenha buscado explicar como é possível perdoar a si mesmo de forma genuína. No que se refere aos delineamentos utilizados, percebe-se que os estudos longitudinais foram escas- sos e realizados em períodos muito curtos de tempo. Discussão Essa caraterística deve ser discutida e considerada em estudos futuros, pois como demonstraram Hall e Fin- cham (2008), as covariações com o autoperdão podem diferir ao longo do tempo e o foco momentâneo pode levar ao erro na análise do seu processo. i Outro elemento que chamou atenção nos estu- dos revisados foi a ênfase no papel positivo dos com- portamentos conciliatórios para o autoperdão. Faz-se importante ressaltar, no entanto, que esses comporta- mentos conciliatórios não devem ser encarados como meras compensações. Para que sejam moralmente elevados é preciso que esses comportamentos se ori- ginem do reconhecimento da responsabilidade e que se pautem primordialmente na preocupação com o outro, e não no desejo de se sentir melhor consigo mesmo. Defende-se que somente assim esses com- portamentos poderão ser genuínos, sendo viável a sua relação com a reafirmação de valores, conforme defendido nos estudos de Wenzel et al. (2012) e Woo- dyatt e Wenzel (2014). Além disso, verifica-se que, comparados com receber o perdão, os estudos com amostras popu- lacionais específicas estão mais desenvolvidos no autoperdão. No entanto, chama atenção a predomi- nância da participação de universitários, mesmo em estudos sobre assuntos específicos, como o alcoo- lismo e a desordem alimentar, por exemplo. Consi- dera-se que o estudo do autoperdão também merece maior diversidade de participantes e delineamen- tos, pois somente dessa maneira os conhecimentos sobre esta atitude poderão se tornar cada vez mais completos. Ainda, assim como na revisão anterior, a ausência de estudos brasileiros sobre autoperdão fortalece a visão de que as dimensões do perdão refe- rentes aos ofensores ainda precisam ser exploradas no nosso país. 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Developing an individualized implicit relational asses- sment procedure (IRAP) as a potential measure of self-forgiveness related to negative and positive behavior. The Psychological Record, 65(4), 717-730. https://doi.org/10.1007/s40732-015-0141-4 Bast, D. F., Linares, I. M. P., Gomes, C., Kovac, R., & Barnes-Holmes, D. (2016). The implicit relational assessment procedure (IRAP) as a measure of self-forgiveness: The impact of a training history in clinical behavior analysis. The Psychological Record, 66(1), 177-190. https://doi.org/10.1007/s40732-016-0162-7 Cheavens, J. S., Cukrowicz, K. C., Hansen, R., & Mitchell, S. M. (2016). Incorporating resilience factors into the interpersonal theory of suicide: The role of hope and self‐forgiveness in an older adult sample. Journal of Clinical Psychology, 72(1), 58-69. https://doi.org/10.1002/jclp.22230 Chiaramello, S., Sastre, M. T. M., & Mullet, E. (2008). Seeking forgiveness: Factor structure, and relationships with personality and forgivingness. Personality and Individual Differences, 45(5), 383-388. https://doi.org/10.1016/j. paid.2008.05.009 Cornish, M. A., & Wade, N. G. (2015). A therapeutic model of self forgiveness with intervention strategies for cou- nselors. Journal of Counseling & Development, 93(1), 96-104. https://doi.org/10.1002/j.1556-6676.2015.00185.x Day, A., Gerace, A., Wilson, C., & Howells, K. (2008). Promoting forgiveness in violent offenders: A more positive approach to offender rehabilitation? Aggression and Violent Behavior, 13(3), 195-200. https://doi.org/10.1016/j. avb.2008.03.004 13 Davidson, M. M., Lozano, N. M., Cole, B. P., & Gervais, S. J. (2015). Considerações Finais ç A análise, avaliação e integração da literatura publicada permitiu aprofundar o entendimento sobre as atitudes para receber o perdão e para perdoar a si mesmo. A ausência de estudos brasileiros na revisão realizada mostra como essas formas de perdão ainda são pouco exploradas no território nacional. As infor- mações da literatura internacional atestam a impor- tância de as dimensões receber o perdão e autoper- A revisão realizada também revelou a preocupa- ção de alguns estudiosos com o impacto negativo que o autoperdão poderia ter, caso seja alcançado cedo demais, pois isso poderia atrapalhar a busca pelo per- 12 Alencar, T. F., & Abreu, E. L. (2019). O Perdão sob a Perspectiva do Ofensor. dão, bem com os benefícios associados a elas. Desta maneira, este é um campo de estudo que se mostra promissor, sendo essencial avaliar até que ponto as teorias e modelos desenvolvidos se adequam à popu- lação nacional. estudos e atuações que busquem incentivar a atitude para receber o perdão e para perdoar a si mesmo, possibilitando conhecimentos para que a atuação prática seja mais efetiva. Sabe-se, no entanto, que existem limitações no alcance desta revisão de literatura. Todos os estudos revisados foram obtidos através do Portal “Periódi- cos Capes”, sendo provável a existência de produções científicas em bases de dados que não foram anali- sadas no presente estudo. Porém, considera-se que mesmo com limitações, este estudo realizou uma revisão da literatura relevante, que poderá ser uma ferramenta útil aos estudiosos do perdão. Além disso, também é importante partir para a aplicação do que já se sabe. Explorar teoricamente as dimensões receber o perdão e autoperdão tem grande valia, mas os estudos indicam que muitos ganhos podem ser advindos da promoção dessas ati- tudes, não somente para os ofensores, mas também para as vítimas e para as relações interpessoais de uma maneira geral. A presente revisão pode auxiliar Referências Relations between intimate partner violence and forgiveness among college women. Journal of Interpersonal Violence, 30(18), 3217-3243. https://doi. org/10.1177/0886260514555008 13 Psicologia: Ciência e Profissão 2019 v. 39, e185662,1-17. Dixon, L. J., Earl, K. A., Lutz-Zois, C. J., Goodnight, J. A., & Peatee, J. J. (2014). Explaining the link between perfec- tionism and self-forgiveness: The mediating roles of unconditional self-acceptance and rumination. Individual Differences Research, 12(3), 101-111. Enright, R. D. (1996). Counseling within the forgiveness triad: On forgiving, receiving forgiveness, and self-forgive- ness. Counseling and Values, 40(2), 107-126. https://doi.org/10.1002/j.2161-007X.1996.tb00844.x Enright, R. D., & Fitzgibbons, R. P. (2015). [Helping clients forgive] forgiveness therapy: An empirical guide for resol- ving anger and restoring hope. Washington, DC: American Psychological Association. Exline, J. J., Prince-Paul, M., Root, B. L., Peereboom, K. S., & Worthington Jr., E. L. (2012). 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Unforgiveness, rumination, and depressive symptoms among older adults. Aging & Mental Health, 14(4), 439-449. https://doi.org/10.1080/13607860903483136 14 Alencar, T. F., & Abreu, E. L. (2019). O Perdão sob a Perspectiva do Ofensor. Kim, J. J., & Enright, R. D. (2014). A theological and psychological defense of self-forgiveness: Implications for cou- nseling. Journal of Psychology & Theology, 42(3), 260-268. Kim, L. M., Johnson, J. L., & Ripley, J. (2011). A “perfect” storm: Perfectionism, forgiveness, and marital satisfaction. Individual Differences Research, 9(4), 199-209. Krause, N. (2015). Assessing the relationships among race, religion, humility, and self-forgiveness: A longitudinal investigation. Advances in Life Course Research, 24, 66-74. https://doi.org/10.1016/j.alcr.2015.02.003 Lander, I. (2012). Towards the incorporation of forgiveness therapy in healing the complex trauma of parental incarceration. Child and Adolescent Social Work Journal, 29(1), 1-19. https://doi.org/10.1007/s10560-011-0248-7 Law, K. C., & Chapman, A. L. (2015). Borderline personality features as a potential moderator of the effect of anger and depressive rumination on shame, self-blame, and self-forgiveness. Journal of Behavior Therapy and Experi- mental Psychiatry, 46, 27-34. https://doi.org/10.1016/j.jbtep.2014.07.008 Leunissen, J. M., De Cremer, D., & Folmer, C. P. R. (2012). Referências An instrumental perspective on apologizing in bargai- ning: The importance of forgiveness to apologize. Journal of Economic Psychology, 33(1), 215-222. https://doi. org/10.1016/j.joep.2011.10.004 Macaskill, A. (2012). Differentiating dispositional self-forgiveness from other-forgiveness: Associations with mental health and life satisfaction. Journal of Social and Clinical Psychology, 31(1), 28-50. https://doi.org/10.1521/jscp.2012.31.1.28 McConnell, J. M., & Dixon, D. N. (2012). Perceived forgiveness from god and self-forgiveness. Journal of Psychology and Christianity, 31(1), 31-39. McConnell, J. M., Dixon, D. N., & Finch, W. H. (2012). An alternative model of self-forgiveness. Journal of Social and Psychological Sciences , 5(2), 22-53. Neto, F., Mullet, E., Chiaramello, S., & Suwartono, C. (2013). Seeking forgiveness: Factor structure in samples from Latin America, Africa, Asia, and Southern Europe. Universitas Psychologica, 12(3), 663-669. https://doi. org/10.11144/Javeriana.UPSY12-3.sffs Nsamenang, S. A., Webb, J. R., Cukrowicz, K. C., & Hirsch, J. K. (2013). Depressive symptoms and interpersonal needs as mediators of forgiveness and suicidal behavior among rural primary care patients. Journal of Affective Disorders, 149(1-3), 282-290. https://doi.org/10.1016/j.jad.2013.01.042 Oyen-Witvliet, C., Hinman, N. G., Exline, J. J., & Brandt, T. (2011). Responding to our own transgressions: An experi- mental writing study of repentance, offense rumination, self-justification, and distraction. Journal of Psychology and Christianity, 30(3), 223-238. Paleari, F. G., Regalia, C., & Fincham, F. D. (2011). Inequity in forgiveness: Implications for personal and relational well-being. Journal of Social and Clinical Psychology, 30(3), 297-324. https://doi.org/10.1521/jscp.2011.30.3.297 Rangganadhan, A. R., & Todorov, N. (2010). Personality and self-forgiveness: The roles of shame, guilt, empathy and conciliatory behavior. Journal of Social and Clinical Psychology, 29(1), 1-22. https://doi.org/10.1521/jscp.2010.29.1.1 Riek, B. M. (2010). Transgressions, guilt, and forgiveness: A model of seeking forgiveness. Journal of Psychology and Theology, 38(4), 246-254. https://doi.org/10.1177/009164711003800402 Robb III, H. B. (2007). Treating anger with forgiveness may sometimes require reconciliation. Journal of Rational-E- motive & Cognitive-Behavior Therapy, 25(1), 65-75. https://doi.org/10.1007/s10942-006-0030-5 Romero, C., Friedman, L. C., Kalidas, M., Elledge, R., Chang, J., & Liscum, K. R. (2006). Self-forgiveness, spiritua- lity, and psychological adjustment in women with breast cancer. Journal of Behavioral Medicine, 29(1), 29-36. https://doi.org/10.1007/s10865-005-9038-z Squires, E. C., Sztainert, T., Gillen, N. R., Caouette, J., & Wohl, M. J. (2012). The problem with self-forgiveness: For- giving the self deters readiness to change among gamblers. Journal of Gambling Studies, 28(3), 337-350. https:// doi.org/10.1007/s10899-011-9272-y Stouten, J., & Tripp, T. M. (2009). Claiming more than equality: Should leaders ask for forgiveness? The Leadership Quarterly, 20(3), 287-298. https://doi.org/10.1016/j.leaqua.2009.03.002 Strelan, P. (2007b). Referências The prosocial, adaptive qualities of just world beliefs: Implications for the relationship between justice and forgiveness. Personality and Individual Differences, 43(4), 881-890. https://doi.org/10.1016/j. paid.2007.02.015 15 Psicologia: Ciência e Profissão 2019 v. 39, e185662,1-17. Strelan, P. (2007a). Who forgives others, themselves, and situations? The roles of narcissism, guilt, self-esteem, and agreeableness. Personality and Individual Differences, 42(2), 259-269. https://doi.org/10.1016/j.paid.2006.06.017 Ursúa, M. P., & Echegoyen, I. (2015). ¿Perdón a uno mismo, autoaceptación o restauraciónintrapersonal? Cuestio- nes abiertas em psicología del perdón. Papeles del psicólogo, 36(3), 230-237. Vitz, P. C., & Meade, J. M. (2011). Self-forgiveness in psychology and psychotherapy: A critique. Journal of Religion and Health, 50(2), 248-263. https://doi.org/10.1007/s10943-010-9343-x Wallace, H. M., Exline, J. J., & Baumeister, R. F. (2008). Interpersonal consequences of forgiveness: Does forgive- ness deter or encourage repeat offenses? Journal of Experimental Social Psychology, 44(2), 453-460. https://doi. org/10.1016/j.jesp.2007.02.012 Webb, J. R., Hirsch, J. K., Conway-Williams, E., & Brewer, K. G. (2013b). Forgiveness and alcohol problems: Indirect associations involving mental health and social support. Addiction Research & Theory, 21(2), 141-153. https:// doi.org/10.3109/16066359.2012.703267 Webb, J. R., Phillips, T. D., Bumgarner, D., & Conway-Williams, E. (2013a). Forgiveness, mindfulness, and health. Mindfulness, 4(3), 235-245. https://doi.org/10.1007/s12671-012-0119-0 Wenzel, M., Woodyatt, L., & Hedrick, K. (2012). No genuine self forgiveness without accepting responsibility: Value reaffirmation as a key to maintaining positive self‐regard. European Journal of Social Psychology, 42(5), 617-627. https://doi.org/10.1002/ejsp.1873 Westers, N. J., Rehfuss, M., Olson, L., & Biron, D. (2012). The role of forgiveness in adolescents who engage in nonsuicidal self-injury. The Journal of nervous and mental disease, 200(6), 535-541. https://doi.org/10.1097/ NMD.0b013e318257c837 Wilson, T., Milosevic, A., Carroll, M., Hart, K., & Hibbard, S. (2008). Physical health status in relation to self-forgive- ness and other-forgiveness in healthy college students. Journal of Health Psychology, 13(6), 798-803. https://doi. org/10.1177/1359105308093863 Wohl, M. J.,& McLaughlin, K. J. (2014). Self forgiveness: The good, the bad, and the ugly. Social and Personality Psy- chology Compass, 8(8), 422-435. https://doi.org/10.1111/spc3.12119 Wohl, M. J., Pychyl, T. A., & Bennett, S. H. (2010). I forgive myself, now I can study: How self-forgiveness for procras- tinating can reduce future procrastination. Personality and Individual Differences, 48(7), 803-808. https://doi. org/10.1016/j.paid.2010.01.029 Wohl, M. J., & Thompson, A. (2011). A dark side to self forgiveness: Forgiving the self and its association with chronic unhealthy behaviour. British Journal of Social Psychology, 50(2), 354-364. https://doi.org/10.1111/j. 2044-8309.2010.02010.x Woodyatt, L., & Wenzel, M. (2013a). Self-forgiveness and restoration of an offender following an interpersonal transgression. Thalita Fernandes Alencar Doutoranda em Psicologia pelo Programa de Pós-Graduação em Psicologia Social da Universidade Federal da Paraíba (UFPB), João Pessoa – PB. Brasil. Mestre em Psicologia Social pela UFPB. E-mail: thalitaalays@hotmail.com https://orcid.org/0000-0003-3898-6242 Cómo citar: Alencar, T. F., & Abreu, E. L. (2019). El perdón bajo la perspectiva del ofensor: Una revisión de la sistemática. Psicologia: Ciência e Profissão, 39, 1-17. https://doi.org/10.1590/1982-3703003185662 Como citar: Alencar, T. F., & Abreu, E. L. (2019). O perdão sob a perspectiva do ofensor: Uma revisão da sistemática. Psicologia: Ciência e Profissão, 39, 1-17. https://doi.org/10.1590/1982-3703003185662 Referências Journal of Social and Clinical Psychology, 32(2), 225. https://doi.org/10.1521/jscp.2013.32.2.225 Woodyatt, L., & Wenzel, M. (2013b). The psychological immune response in the face of transgressions: Pseudo self-forgiveness and threat to belonging. Journal of Experimental Social Psychology, 49(6), 951-958. https://doi. org/10.1016/j.jesp.2013.05.016 Woodyatt, L., & Wenzel, M. (2014). A needs-based perspective on self-forgiveness: Addressing threat to moral iden- tity as a means of encouraging interpersonal and intrapersonal restoration. Journal of Experimental Social Psy- chology, 50, 125-135. https://doi.org/10.1016/j.jesp.2013.09.012 Worthington, E. L., & Langberg, D. (2012). Religious considerations and self-forgiveness in treating complex trauma and moral injury in present and former soldiers. Journal of Psychology & Theology, 40(4), 274-288. https://doi. org/10.1177/009164711204000403 Thalita Fernandes Alencar Doutoranda em Psicologia pelo Programa de Pós-Graduação em Psicologia Social da Universidade Federal da Paraíba (UFPB), João Pessoa – PB. Brasil. Mestre em Psicologia Social pela UFPB. E-mail: thalitaalays@hotmail.com https://orcid.org/0000-0003-3898-6242 16 Alencar, T. F., & Abreu, E. L. (2019). O Perdão sob a Perspectiva do Ofensor. Eloá Losano Abreu Professora Adjunto do Departamento de Psicologia da Universidade Federal da Paraíba (UFPB). Email: eloalosano@gmail.com https://orcid.org/0000-0001-7712-6275 Endereço para envio de correspondência: Rua Antônio Vieira da Silva, n 400, ap 1404 C. Bairro Jardim São Paulo. Cep: 58053-175. João Pessoa PB. Brasil. Recebido 22/09/2017 Aceito 16/07/2018 Received 09/22/2017 Approved 07/16/2018 Recibido 22/09/2017 Aceptado 16/07/2018 Eloá Losano Abreu Professora Adjunto do Departamento de Psicologia da Universidade Federal da Paraíba (UFPB). Email: eloalosano@gmail.com https://orcid.org/0000-0001-7712-6275 Endereço para envio de correspondência: Rua Antônio Vieira da Silva, n 400, ap 1404 C. Bairro Jardim São Paulo. Cep: 58053-175. João Pessoa PB. Brasil. Recebido 22/09/2017 Aceito 16/07/2018 Received 09/22/2017 Approved 07/16/2018 Recibido 22/09/2017 Aceptado 16/07/2018 Endereço para envio de correspondência: Rua Antônio Vieira da Silva, n 400, ap 1404 C. Bairro Jardim São Paulo. Cep: 58053-175. João Pessoa PB. Brasil. Recebido 22/09/2017 Aceito 16/07/2018 Received 09/22/2017 Approved 07/16/2018 Recibido 22/09/2017 Aceptado 16/07/2018 Como citar: Alencar, T. F., & Abreu, E. L. (2019). O perdão sob a perspectiva do ofensor: Uma revisão da sistemática. Psicologia: Ciência e Profissão, 39, 1-17. https://doi.org/10.1590/1982-3703003185662 How to cite: Alencar, T. F., & Abreu, E. L. (2019). Forgiveness from the offender’s perspective: A review of systematics. Psicologia: Ciência e Profissão, 39, 1-17. https://doi.org/10.1590/1982-3703003185662 Cómo citar: Alencar, T. F., & Abreu, E. L. (2019). El perdón bajo la perspectiva del ofensor: Una revisión de la sistemática. Psicologia: Ciência e Profissão, 39, 1-17. https://doi.org/10.1590/1982-3703003185662 Cómo citar: Alencar, T. F., & Abreu, E. L. (2019). Alencar, T. F., & Abreu, E. L. (2019). O Perdão sob a Perspectiva do Ofensor. How to cite: Alencar, T. F., & Abreu, E. L. (2019). Forgiveness from the offender’s perspective: A review of systematics. Psicologia: Ciência e Profissão, 39, 1-17. https://doi.org/10.1590/1982-3703003185662 Referências El perdón bajo la perspectiva del ofensor: Una revisión de la sistemática. Psicologia: Ciência e Profissão, 39, 1-17. https://doi.org/10.1590/1982-3703003185662 17 17
https://openalex.org/W2971384749	https://europepmc.org/articles/pmc6767106?pdf=render	English	null	Navigation in Unknown Dynamic Environments Based on Deep Reinforcement Learning	Sensors	2,019	cc-by	11,179	Received: 4 August 2019; Accepted: 4 September 2019; Published: 5 September 2019 Abstract: In this paper, we propose a novel Deep Reinforcement Learning (DRL) algorithm which can navigate non-holonomic robots with continuous control in an unknown dynamic environment with moving obstacles. We call the approach MK-A3C (Memory and Knowledge-based Asynchronous Advantage Actor-Critic) for short. As its ﬁrst component, MK-A3C builds a GRU-based memory neural network to enhance the robot’s capability for temporal reasoning. Robots without it tend to suﬀer from a lack of rationality in face of incomplete and noisy estimations for complex environments. Additionally, robots with certain memory ability endowed by MK-A3C can avoid local minima traps by estimating the environmental model. Secondly, MK-A3C combines the domain knowledge-based reward function and the transfer learning-based training task architecture, which can solve the non-convergence policies problems caused by sparse reward. These improvements of MK-A3C can eﬃciently navigate robots in unknown dynamic environments, and satisfy kinetic constraints while handling moving objects. Simulation experiments show that compared with existing methods, MK-A3C can realize successful robotic navigation in unknown and challenging environments by outputting continuous acceleration commands. Keywords: autonomous navigation; unknown environments; deep reinforcement learning; continuous control Sensors 2019, 19, 3837; doi:10.3390/s19183837 Received: 4 August 2019; Accepted: 4 September 2019; Published: 5 September 2019 Article Navigation in Unknown Dynamic Environments Based on Deep Reinforcement Learning Junjie Zeng , Rusheng Ju , Long Qin , Yue Hu, Quanjun Yin and Cong Hu College of Systems Engineering, National University of Defense Technology, Changsha 410073, China Correspondence: jrscy@sina.com Junjie Zeng , Rusheng Ju , Long Qin , Yue Hu, Quanjun Yin and Cong Hu College of Systems Engineering, National University of Defense Technology, Changsha 410073, China Correspondence: jrscy@sina.com sensors sensors 1. Introduction Autonomous navigation plays an important role in the ﬁelds of video games [1] and robotics [2], to generate reasonable trajectories for Non-Player Characters (NPCs) and meet the fundamental needs of mobility for real-life robots. This paper focuses on the navigation problem of non-holonomic robots with continuous motion control in unknown dynamic environments. Existing approaches [3] can be divided into two categories: (1) those based on global environmental data; (2) those using only local environmental information. Algorithms from the ﬁrst category usually conduct eﬃcient path search in a heuristic or non-heuristic manner knowing precise environmental states. They include Simple Subgoal Graph (SSG) [4] and its variants [5], sampling-based path planning algorithms (such as Probabilistic RoadMaps (PRM) [6], Rapidly exploring Random Trees (RRT) [7]), etc. The sampling-based algorithms commonly used in robotics are complete and eﬃcient. However, these methods need a model of the entire map often represented by grids or topology maps. Methods in the second category use local environment data detected by sensors to plan motions for robots, which can avoid ﬁrst-move lags [8] and have the potential to realize real-time planning. These algorithms are classiﬁed into non-learning-based and learning-based methods. Typical non-learning-based algorithms, such as Artiﬁcial Potential Fields (APF) [9] and Velocity Obstacle (VO) [10], calculate motion behaviors at every time step following preset rules. Their generated reactive strategies usually lead to bad performance in local minima areas. Besides, some key parameters Sensors 2019, 19, 3837; doi:10.3390/s19183837 www.mdpi.com/journal/sensors www.mdpi.com/journal/sensors 2 of 18 Sensors 2019, 19, 3837 of the algorithms are diﬃcult to be manually adjusted, and the algorithms require some overly strict assumptions. Local learning-based navigation algorithms include Deep Learning (DL) and Deep Reinforcement Learning (DRL). Methods based on DL [11] use deep neural networks to extract patterns of reasonable navigation behavior from a large number of labelled expert data. However, it is time-consuming and energy-consuming to collect labelled samples for navigation in unknown environments, which prevents DL-based methods from being widely applied to solve the proposed problem. In contrast, DRL cannot learn from labelled data but experience generated from interactions between the agent and the environment [12]. Generally, they can be divided into two sorts, namely value-based and policy-based DRL. Compared with value-based DRL methods, the latter methods are more competent to deal with continuous action spaces. 2. Related Work This section introduces related works in terms of navigation approaches including non-learning-based methods, DL and DRL. Generally, APF and velocity-based methods are typical non-learning-based navigation methods that are not suitable to deal with changes in unknown environments. Learning-based methods such as DL and DRL can enhance the adaptability of mobile robots in unknown environments by accumulating navigation experience. While DL requires large-scale labelled data that are diﬃcult to collect, DRL merely needs data from interactions between agents and environments. 1. Introduction Among them, Asynchronous Advantage Actor-Critic (A3C) [13] is well-received in the ﬁeld of computer games [14,15] and robotics [16,17] owing to its high training eﬃciency. Nonetheless, applying A3C to solve the navigation problem in unknown dynamic environments is still facing the following challenges. First, in an unknown environment the robot can only collect incomplete and noisy environmental data within a certain range through its own sensors, which make the robot controlled by A3C fall into traps of local minima at areas of concave canyon, long corridors and other challenging terrains. Other DRL algorithms are also diﬃcult to avoid local minima. Second, for in most cases there is only one goal location, the rewards are sparsely distributed in the environment, which empirically greatly slows down the learning eﬃciency and even leads to non-convergence. To solve the above problems, this paper proposes MK-A3C, a novel DRL algorithm that improves on the A3C algorithm by using memory mechanism, domain knowledge and transfer learning. MK-A3C constructs a Gated Recurrent Unit (GRU)-based network [18] architecture to store abstract historical information in the internal state of the GRU layer. In addition, MK-A3C introduces domain knowledge by reward shaping [19] and transfer learning to increase learning eﬃciencies and enhance generality. To deal with incomplete states, MK-A3C constructs the belief state by combining the abstracted historical trajectories with the current observation. Then, to overcome the challenge of sparse reward, MK-A3C builds a domain knowledge-based reward function and a transfer learning-based training task framework [20], which can increase the density of reward signals and meanwhile realize the migration of knowledge from simple tasks to complex tasks. The rest of this paper is organized as follows: Section 2 discusses some related work about non-learning-based navigation methods, DL, and DRL. Section 3 presents our navigation approach in detail. In Section 4, the performance of the proposed method is evaluated through simulation experiments. Finally, Section 5 gives our conclusions. 2.1. Non-Learning-Based Methods It is diﬃcult for non-learning-based methods to deal with navigation in unknown dynamic environments, since these methods cannot address unpredictable situations and is not adaptive to environmental changes. This subsection focuses on two typical methods, VO and APF. VO [10] can generate reachable trajectories by computing a velocity space of a robot. However, it is ineﬃcient for real-time applications to compute maneuvers via the velocity space. Comparatively, APF [9] employs 3 of 18 Sensors 2019, 19, 3837 time-varying potential ﬁeld functions as a low-cost navigation technique in dynamic environments, which uses only local information near the agent as input. Unfortunately, potential ﬁeld functions, key to APF, are subject to several parameters, such as the relative strength between the repulsive and attractive potentials, which are generally determined by experience and easily incur local minima issues. In addition, non-learning-based methods generally make plans by current observations, thereby only ﬁnding reactive policies with poor adaptability to unknown and dynamic environments. 2.3. Deep Reinforcement Learning In contrast to DL-based methods, DRL can learn navigation policies by samples collected from interactions between the agent and the environment. Therefore, DRL can avoid collecting large-scale labelled data. In addition, unlike DL which extracts existing navigation patterns from data sets, DRL can explore unseen policies by actively interacting with the environment, which brings about further breakthroughs in autonomous navigation. Currently, much research is devoted to solving problems of navigation in static unknown environments. For example, Tai et al. [24] proposed asynchronous deep deterministic policy gradients to learn navigation policies under simple static unknown environments. Wang et al. [25] proposed a fast-recurrent deterministic policy gradient algorithm (fast-RDPG) to accomplish unmanned aerial vehicles’ navigation tasks in more complex environments. Zhelo et al. [26] proposed curiosity-driven exploration strategies to argument robots’ abilities to explore complex unknown environments. However, these works mainly focus on navigation in static environments, which are unpractical for most realistic applications since various kinds of moving objects are required to be taken into consideration. Compared with the above methods, MK-A3C can solve the navigation problem in unknown dynamic environments with moving obstacles by introducing memory mechanism and domain knowledge into its DRL framework. 3. The Methodology In this section, we ﬁrst introduce the navigation problem and related backgrounds, and then build a model for the crawler robot. Then, we illustrate the architecture of the proposed algorithm. Finally, the motion planner based on MK-A3C is described in detail. 2.2. Deep Learning DL is essentially a collection of models for non-linear function approximations through abstracting features hidden in inputs by deep neural networks and presently is widely used in robotic applications, such as robot manipulation, indoor navigation and so on [21–23]. Generally, navigation methods based on DL are included in two genres, i.e., data-driven control methods and Model-Predictive Control (MPC) methods. The ﬁrst kind directly maps raw sensory inputs to control commands by large-scale supervised learning. For example, Xu et al. [21] developed an end-to-end trainable architecture to learn a generic vehicle motion model based on a large set of crowd-sourced video data. Tai et al. [22] adopted a convolutional neural network (CNN) to learn indoor navigation policies by self-constructed indoor depth data sets. The second kind of approach uses deep neural networks to learn dynamics of controlled agents, and then calculates optimal control input based on the predicted future outputs. For instance, Agrawal et al. [23] took raw sensory images as input to predict the dynamic model of robotic arms for manipulation by deep neural networks. However, it is diﬃcult to collect a large set of labelled data for training deep neural networks in the scenario of navigation in unknown environments. 3.1. Problem Description The targeted navigation problem in this paper can be described as follows: In an initially unknown environment where terrains like dead corners and long corridors are widely distributed, a mobile robot only equipped with sparse local distance sensors is required to move safely to a given destination controlled by continuous control commands. Since the environment is initially unknown, the robot Sensors 2019, 19, 3837 polynomial-hard I thi 4 of 18 obot is can only build its own environmental model through exploration. Meanwhile, the environment with moving obstacles is dynamic and uncertain, which greatly increases the complexity. As proved in reference [27], navigation in dynamic environments is a non-deterministic polynomial-hard problem. illustrated in Figure 1 and its dynamics are given in Equation (1) and (2). We use a tuple (𝑥௥௢௕, 𝑦௥௢௕, 𝜃௥௢௕) to denote the position information of the mobile robot, where 𝑥௥௢௕ and 𝑦௥௢௕ are the robot’s global horizontal and vertical coordinate respectively, and 𝜃௥௢௕ is the angle between the orientation of the robot and the positive abscissa. To decrease the hardware requirements and take In this paper, we adopt a Cartesian coordinate system. The model of the mobile robot is illustrated in Figure 1 and its dynamics are given in Equations (1) and (2). We use a tuple (xrob, yrob, θrob) to denote the position information of the mobile robot, where xrob and yrob are the robot’s global horizontal and vertical coordinate respectively, and θrob is the angle between the orientation of the robot and the positive abscissa. To decrease the hardware requirements and take moving obstacles into consideration, the range sensor equipped with the robot has a 360-degree detection angle range, and the detected angular interval is 15 degrees, which means that 24-dimensional local obstacle distance information can be obtained. Its maximum detection distance of the sensor is 7 m. The mobile robot is a crawler robot that can be controlled by the left and right tracks. To achieve meticulous manipulations and meet kinetic constraints, continuous angular acceleration is used as control commands. The kinetic equation is described as follows: p q moving obstacles into consideration, the range sensor equipped with the robot has a 360-degree detection angle range, and the detected angular interval is 15 degrees, which means that 24- dimensional local obstacle distance information can be obtained. Its maximum detection distance of the sensor is 7 m. 3.1. Problem Description The mobile robot is a crawler robot that can be controlled by the left and right tracks. To achieve meticulous manipulations and meet kinetic constraints, continuous angular acceleration is used as control commands. The kinetic equation is described as follows: ቌ 𝑥ሶ௥௢௕ 𝑦ሶ௥௢௕ 𝜃ሶ௥௢௕ ቍ= 𝑅 ⎢ ⎢ ⎢ ⎢ ⎡𝑐𝑜𝑠𝜃௥௢௕ 2 𝑐𝑜𝑠𝜃௥௢௕ 2 𝑠𝑖𝑛𝜃௥௢௕ 2 𝑠𝑖𝑛𝜃௥௢௕ 2 1 1 ⎥ ⎥ ⎥ ⎥ ⎤ ቀ𝜔௟ 𝜔௥ቁ, (1)   .xrob .yrob . θrob   = R   cosθrob 2 cosθrob 2 sinθrob 2 sinθrob 2 1 lrob −1 lrob   ωl ωr ! , (1) .ωl .ωr ! =   ∂ωl ∂t ∂ωr ∂t  , (2) 𝜃௥௢௕ ⎣ ⎢ 1 𝑙௥௢௕ −1 𝑙௥௢௕ ⎦ ⎥ ൬𝜔ሶ௟ 𝜔ሶ௥൰= ൮ 𝜕𝜔௟ 𝜕𝑡 𝜕𝜔௥ 𝜕𝑡 ൲, (2) he d i i heel a d de ote the a ula elo itie of the left a d (1) .ωl .ωr ! =   ∂ωl ∂t ∂ωr ∂t  , (2) ൬𝜔ሶ௥൰= ൮𝜕𝜔௥ 𝜕𝑡 ൲, (2) h l d d h l l i i f h l f d (2) 2) where R is the radius of the driving wheel; ωl and ωr denote the angular velocities of the left and right driving wheels respectively (rad·s−1); .ωl and .ωr denote the angular accelerations of them (rad s−2); lrob denotes the distance between two tracks. where 𝑅 is the radius of the driving wheel; 𝜔௟ and 𝜔௥ denote the angular velocities of the left and right driving wheels respectively (rad· 𝑠ିଵ); 𝜔ሶ௟ and 𝜔ሶ௥ denote the angular accelerations of them (rad 𝑠ିଶ); 𝑙௥௢௕ denotes the distance between two tracks. where R is the radius of the driving wheel; ωl and ωr denote the angular velocities of the left and right driving wheels respectively (rad·s−1); .ωl and .ωr denote the angular accelerations of them (rad s−2); lrob denotes the distance between two tracks. where 𝑅 is the radius of the driving wheel; 𝜔௟ and 𝜔௥ denote the angular velocities of the left and right driving wheels respectively (rad· 𝑠ିଵ); 𝜔ሶ௟ and 𝜔ሶ௥ denote the angular accelerations of them (rad 𝑠ିଶ); 𝑙௥௢௕ denotes the distance between two tracks. Figure 1. Schematic diagram of the crawler robot. Figure 1. Schematic diagram of the crawler robot. 3 2 The Architecture of the Proposed Algorithm Figure 1. Schematic diagram of the crawler robot. Figure 1. Schematic diagram of the crawler robot. 3 2 The Architecture of the Proposed Algorithm 3.2. The Architecture of the Proposed Algorithm Next, to overcome the challenge of sparse reward, a reward function based on domain knowledge As shown in Figure 2, in the offline phase of the motion planner, the main task is to learn navigation policies in unknown dynamic environments. First, key information about the robot and the environment is extracted to construct a Partially Observable Markov Decision Process (POMDP) model, since in unknown environments the robot cannot directly obtain the complete environmental state to construct a general Markov Decision Process (MDP) model. Then, a neural network architecture with a memory mechanism is built to solve the POMDP model, which integrates historical data with the current observations to construct the robot’s own belief state as its cognition about environments. Next, to overcome the challenge of sparse reward, a reward function based on domain knowledge is constructed by reward shaping, which can increase the density of reward signals and accelerate the convergence of the training process. To further improve learning efficiencies, the architecture of training tasks is constructed by transfer learning. Finally, MK-A3C is used to learn the navigation policy. In the online phase, navigation policy learned in the offline phase takes the environmental information detected by the sensor and internal states of the robot as its input, and output continuous control commands to navigate the robot to a given goal location. As shown in Figure 2, in the oﬄine phase of the motion planner, the main task is to learn navigation policies in unknown dynamic environments. First, key information about the robot and the environment is extracted to construct a Partially Observable Markov Decision Process (POMDP) model, since in unknown environments the robot cannot directly obtain the complete environmental state to construct a general Markov Decision Process (MDP) model. Then, a neural network architecture with a memory mechanism is built to solve the POMDP model, which integrates historical data with the current observations to construct the robot’s own belief state as its cognition about environments. Next, to overcome the challenge of sparse reward, a reward function based on domain knowledge is constructed by reward shaping, which can increase the density of reward signals and accelerate the convergence of the training process. To further improve learning eﬃciencies, the architecture of training tasks is constructed by transfer learning. Finally, MK-A3C is used to learn the navigation policy. 3 2 The Architecture of the Proposed Algorithm 3.2. The Architecture of the Proposed Algorithm 3 2 The Architecture of the Proposed Algorithm 3.2. The Architecture of the Proposed Algorithm 3 2 The Architecture of the Proposed Algorithm 3.2. The Architecture of the Proposed Algorithm 3.2. The Architecture of the Proposed Algorithm In this paper, the proposed MK-A3C algorithm makes improvements on the original A3C algorithm by adding the memory mechanism and domain knowledge. In addition, MK-A3C can avoid the local minima trap caused by partial observability and the sparse reward challenge in unknown environments Figure 2 shows the architecture of the proposed algorithm In this paper, the proposed MK-A3C algorithm makes improvements on the original A3C algorithm by adding the memory mechanism and domain knowledge. In addition, MK-A3C can avoid the local minima trap caused by partial observability and the sparse reward challenge in unknown environments. Figure 2 shows the architecture of the proposed algorithm. 5 of 18 5 f 17 Sensors 2019, 19, 3837 Figure 2. Flowchart of the MK-A3C method. Figure 2. Flowchart of the MK-A3C method. Figure 2. Flowchart of the MK-A3C method. Figure 2. Flowchart of the MK-A3C method. As shown in Figure 2, in the offline phase of the motion planner, the main task is to learn navigation policies in unknown dynamic environments. First, key information about the robot and the environment is extracted to construct a Partially Observable Markov Decision Process (POMDP) model, since in unknown environments the robot cannot directly obtain the complete environmental state to construct a general Markov Decision Process (MDP) model. Then, a neural network architecture with a memory mechanism is built to solve the POMDP model, which integrates historical data with the current observations to construct the robot’s own belief state as its cognition about environments. Next, to overcome the challenge of sparse reward, a reward function based on As shown in Figure 2, in the oﬄine phase of the motion planner, the main task is to learn navigation policies in unknown dynamic environments. First, key information about the robot and the environment is extracted to construct a Partially Observable Markov Decision Process (POMDP) model, since in unknown environments the robot cannot directly obtain the complete environmental state to construct a general Markov Decision Process (MDP) model. Then, a neural network architecture with a memory mechanism is built to solve the POMDP model, which integrates historical data with the current observations to construct the robot’s own belief state as its cognition about environments. 3.3.1. Backgrounds and Concepts of POMDP Policy-based DRL algorithms, such as the Deep Deterministic Policy Gradient (DDPG) [28], A3C, and Distribute Proximal Policy Optimization (DPPO) [29,30], are powerful tools for solving MDP models. However, navigation in unknown environments cannot be formalized as an MDP model since the robot cannot obtain the complete state st. Speciﬁcally, the sensor mounted on the robot can only obtain distance information of nearby obstacles, and even cannot directly know velocities of moving obstacle and the global terrain. POMDPs, as an extension from the MDP models can describe uncertain interactions between robots and environments where only a limited range of area can be obtained. Therefore, we model the process of robots’ safe navigation in unknown dynamic environments as POMDPs, which can better capture the hidden state in the environment through the analysis of environmental observations. Due to the terminal conditions in the proposed navigation problem, we use a ﬁnite POMDP that can be formally described as a 6-tuple (S, A, P, R, O, Ω). Among the elements, S is a ﬁnite set of states, A is a ﬁnite set of actions, O is a ﬁnite set of observations and P denotes a state transition function being part of the environment model. P(s, a, s′) = P[st+1 = s′st = s, at = a], denotes the probability of transferring to the next state s′ when the action a is executed under the current state s. Besides, R(s, a, s′) = f(st = s, at = a, st+1 = s′) is a reward function to quantify the immediate feedback from the environment when the agent takes action a in the current state s, indicating an evaluation of the agent’s behavior at the current step [31]. Ω(o′, s, a) = P[ot+1 = o′st = s, at = a], an observation function, denotes the probability of receiving to the next observation o′ for the agent when the action a is executed in the current state s. Although in unknown environments the robot cannot directly obtain the complete environmental state, in the POMDP model, the robot’s inner representation of the environment can be constructed through historical data, i.e., the belief state Bs. Due to the powerful feature extraction capability of the neural network, we choose the recurrent neural network unit GRU to construct the belief state. 3 2 The Architecture of the Proposed Algorithm 3.2. The Architecture of the Proposed Algorithm In the online phase, navigation policy learned in the oﬄine phase takes the environmental information detected by the sensor and internal states of the robot as its input, and output continuous control commands to navigate the robot to a given goal location. MK-A3C can learn near-optimal policies to navigate robots in complex unknown environments and satisfy kinetics and task-specific constraints. The algorithm embraces two main features. The first is that it builds a novel DRL framework to address partial observable problems. Specifically, the memory-based neural network integrates historical information and current observations to solve the POMDP that formally describes the targeted navigation problem, which equips the robot with temporal reasoning ability. Additionally, the neural network based on GRU contains the robot’s cognition of the environment and can be used to avoid being trapped in local minima. MK-A3C can learn near-optimal policies to navigate robots in complex unknown environments and satisfy kinetics and task-speciﬁc constraints. The algorithm embraces two main features. The ﬁrst is that it builds a novel DRL framework to address partial observable problems. Speciﬁcally, the memory-based neural network integrates historical information and current observations to solve the POMDP that formally describes the targeted navigation problem, which equips the robot with temporal reasoning ability. Additionally, the neural network based on GRU contains the robot’s cognition of the environment and can be used to avoid being trapped in local minima. Sensors 2019, 19, 3837 6 of 18 Second, an innovative reward function and the novel architecture of training tasks are constructed to training the memory network. Speciﬁcally, we introduce domain knowledge into the reward function by reward shaping to solve the problem of sparse rewards. The shaped reward function can increase the destiny of reward signals to accelerate the training process. Besides, inspired by reference [17], an architecture of training tasks is built by transfer learning, which can improve training eﬃciencies by eﬀectively using accumulated knowledge from the previous learning task. In conclusion, improvements to reward function and training architecture are helpful to collect eﬀective training samples in unknown environments, thereby relieving the method from the problem of sparse reward. In addition, due to the generalization of MK-A3C, the motion planner can quickly adapt to new environments without retraining. 3.3.1. Backgrounds and Concepts of POMDP GRU takes the last belief state Bsa t−1, the current observation ot and last action at−1 as its input to estimate hidden environment states. 3.3. Motion Planner Based on MK-A3C This section introduces the motion planner based on MK-A3C in the following four parts: (1) backgrounds and concepts of POMDP; (2) description of the proposed POMDP; (3) basic concepts of policy-based DRL methods and A3C algorithm; and (4) a description of the MK-A3C algorithm. 3.3.2. Description of Navigation POMDP It is diﬃcult to construct models for unknown dynamic environments, which means that the state transition function P and the observation function Ωare unknown. The constructed POMDP model is presented in detail, including the observation space O, the action space A and the reward function R. (3) O = (S1, S2, . . . , S24, dtar, atar, θ, ωl, ωr) (3) O = (S1, S2, . . . , S24, dtar, atar, θ, ωl, ωr) Sensors 2019, 19, 3837 7 of 18 The observation space O consists of sensor data, the relative position between the target and the robot, and the internal state of the robot. The sensor data includes 24 dimensions distance information of nearby obstacles which cover 360-degree detection range at intervals of 15 degrees. To be speciﬁc, Si (i ∈(1, 2, 3 . . . , 24)) refers to the distance to the nearest obstacle located in the range from (i −1) ∗15 to i ∗15 degrees. If no obstacle is detected, the value of Si is the maximum detection distance. The value range of Si is [0,7], whose unit is meter. Since the environment is dynamic, we abandon the mobile robot’s absolute position (xrob, yrob, θrob), and use the relative position to describe the relationship between the target and the robot. The relative position (dtar, atar) includes the distance and orientation information between the target and the robot. dtar refers to the Euclidean distance between the robot and the destination and atar represents to the angle between the robot’s orientation and the line connecting the robot and the target. If the target is on the left hand side of the robot, the value of atar is set positive, otherwise negative. Its value ranges from [−π, π]. The internal information of the robot includes its orientation, angular velocities of its left and right driving wheels. θ refers to the orientation of the robot, and its value ranges from [0, 2π]. ωl and ωr refer to the angular velocity of the left and right driving wheels, ranging from [−0.5, 0.5] in rad·s−1. A = .ωl, .ωr (4) A = .ωl, .ωr A = .ωl, .ωr (4) The action space A includes the angular accelerations of left and right driving wheels in tracks, .ωl and .ωr. Range of their value is [−0.5, 0.5], and their unit is rad·s−2. 3.3.2. Description of Navigation POMDP In the problem of robot navigation, the output action is generally represented in the following two ways: (1) discrete actions, such as forward, left turn and right turn; (2) continuous velocities, such as linear velocity and steering velocity. To achieve more realistic and precise control, we take the continuous angular acceleration of the driving wheels as output. The reward function R is an instant evaluation of the executed action’s eﬀects in the current state. For a fully observable MDP model, the reward function can be generally set as obtaining 1 if realizing the goal and obtaining 0 if not. However, in the proposed partially observable problem, adopting the simple reward function deﬁnitely leads to sparse reward. Speciﬁcally, since the initial policy is randomly generated, the probability that the robot reaches the destination in an unknown dynamic environment with long corridors and dead corners is close to zero, which means that it is hard to collect eﬀective samples. Reward shaping [19], as a way to solve sparse rewards, can provide early rewards for agents to accelerate the convergence of training policies while ensuring policy invariance. We use reward shaping to embed the domain knowledge into the reward function. The shaped reward function is divided into two parts, i.e., non-terminal rewards and terminal rewards, respectively. Terminal rewards include rewards of reaching target and collision penalties. The ﬁrst part of terminal rewards is described as follows: (5) rarr = 10; if dt < dtol (5) where dtol refers to the tolerance distance to the target. where dtol refers to the tolerance distance to the target. g e equation of collision penalties is as follows: The equation of collision penalties is as follows: (6) rcol = −5; if Smin < dsa f (6) where Smin refers to the minimum obstacle distance; dsa f refers to the safety distance between the robot and the obstacle. The idea behind the collision penalty, essentially a negative reward, is that when the distance between the robot and the obstacle is less than the safe distance, the robot will be at the risk of colliding with another object and failing in the task. Sensors 2019, 19, 3837 8 of 18 Non-terminal rewards include rewards for safe orientation and closer distance to the target and penalties for collision danger and time consumption. Their equations are as follows: Non-terminal rewards include rewards for safe orientation and closer distance to the target and penalties for collision danger and time consumption. Their equations are as follows:  rsori = 0.002 rstep = 0.001 rdang = η ∗2ζdmin rd_gol = λ(dtart−1 −dtart)  (7) (7) Rewards for safe orientation denote that a smaller reward is gained when the minimum obstacle distance in the forward direction of the robot is greater than a given value. The design of this reward not only further ensures the safety of the navigation process, but also helps the robot to escape from the local minima trap. Rewards for closer distance to the target denote that when the robot gets closer to the target, it obtains a positive reward, otherwise obtains a negative reward. λ is a parameter used to control the strength of this reward. Additionally, when the robot is closer to the obstacle, it will get more negative rewards, known as danger penalties. The reward signal can provide the robot with strong safety awareness. Time penalty denotes that during the training process, the robot receives a smaller negative reward at every step. This reward can encourage the robot to reach the destination in less time. In addition, hyper-parameters of reward function slightly change as the diﬃculty of the training task increases. 3.3.3. A3C Method In DRL, V(s) refers to the long-term expected cumulative reward starting from the state s, which is usually used to evaluate the state s. Q(s, a) denotes the long-term expected cumulative reward starting from executing action a in state s. Π(s, a) refers to the probability of executing action a under state s [31]. In this paper, we improve A3C algorithm to solve the proposed navigation problem for two reasons: (1) compared with value-based DRL methods such as Deep Q-Network (DQN) [32] and its various improved versions [33,34], A3C can deal with continuous action space and generate excellent policies; (2) compared with other policy-based DRL methods such as DDPG and Trust Region Policy Optimization (TRPO) [35], A3C consumes less GPU computation resources and training time. Besides, it is convenient to debug and optimize A3C method thanks to its clear and elegant structure. Before proposing A3C, experience replay [32] was used to solve the non-convergence problem of combining deep neural network with traditional oﬀ-policy Reinforcement Learning (RL) methods. The experience replay method stores samples collected from interactions between the agent and the environment in the experience memory unit, and then randomly sample training data. However, this method is not suitable for on-policy RL methods, because samples used to train policies must be generated by this policy in this kind of methods. To combine on-policy RL methods with deep neural networks, Mnih et al. [13] proposed a simple lightweight framework that uses asynchronous gradient descent to optimize neural network controllers. The framework can be applied to the traditional on-policy Reinforcement Learning (RL) algorithm, actor-critic, known as A3C for short. In contrast to the experience replay method, the asynchronous framework reduces the correlation between training samples by asynchronously executing multiple agents simultaneously in multiple instances of the environment. As shown in Figure 3, the core idea of A3C algorithm is to asynchronously execute multiple replicas of the agent in multiple environmental instances. During the training process, each of them interacts with its own environmental instance, collects diﬀerent training samples, calculates the network’s gradient, uploads the gradient to update the global network, and ﬁnally downloads the latest network to the local. Please note that the master agent’s global network cannot be updated by multiple agent replicas’ uploaded gradients at the same time. 9 of 18 ated by Sensors 2019, 19, 3837 latest network to t l i l Figure 3. 3.3.3. A3C Method The diagram of Asynchronous Advantage Actor-Critic (A3C). Environment Instance 1 Environment Instance 2 Environment Instance n … Agent Replica 1 Agent Replica 2 Agent Replica n … Master Agent action obs action obs action obs Δθ θ Figure 3. The diagram of Asynchronous Advantage Actor-Critic (A3C). Master Agent Agent Replica 1 Agent Replica n Agent Replica 2 action action Environment Instance 2 Figure 3. The diagram of Asynchronous Advantage Actor-Critic (A3C). Figure 3. The diagram of Asynchronous Advantage Actor-Critic (A3C). Consistent with the network design of the AC algorithm, A3C uses the actor network and the critic network to parameterize the policy function Π(at st; θp) and the value function V(st; θv), respectively. Each agent replica calculates gradients of actor and critic networks in every ﬁxed step or at the end of the episode, and then uploads gradients asynchronously to update the global network. The gradient calculation formula of the critic network is as follows: dθv = ∂(A(st; θv))2/∂θv (8) (8) where A(st; θv) denotes an estimate of the advantage function given by: A(st; θv) = Xk−1 i=0 γirt+i + γkV(st+k; θv) −V(st; θv) (9) (9) where γ is the discount factor and k is the diﬀerence between the length of the sample sequence and the index of the current sample (coded from zero). where γ is the discount factor and k is the diﬀerence between the length of the sample sequence and the index of the current sample (coded from zero). p The gradient calculation equation of the actor network is as follows: The gradient calculation equation of the actor network is as follows: dθp = ∇θplogΠ at st; θp A(st; θv) + β∇θpH Π st; θp (10) (10) where H is the entropy of the policy Π. The hyper-parameter β controls the strength of the entropy regularization term. Adding entropy to the process of updating the policy function’s gradient can improve the agent’s exploration capability and prevent policies from converging to the local optimal solution [13]. where H is the entropy of the policy Π. The hyper-parameter β controls the strength of the entropy regularization term. Adding entropy to the process of updating the policy function’s gradient can improve the agent’s exploration capability and prevent policies from converging to the local optimal solution [13]. 3.3.4. MK-A3C Method Directly applying A3C to solve navigation problems in unknown dynamic environments faces challenges of sparse rewards and incomplete states. In Section 3.3.1, to solve the problem of non-convergence policies caused by sparse reward, we use reward shaping to construct reward functions with domain knowledge. In this section, to further overcome above challenges, improvements are made on A3C algorithm to: (1) construct a memory network architecture based on GRU; (2) and build a training task architecture based on transfer learning. Firstly, the GRU-based memory network architecture can avoid being trapped in local minima caused by partial observability of environments. A3C is used to solve MDPs, but MDPs can only describe fully observable problems such as Go, Chess, etc. Therefore, a POMDP model is built to formally describe the proposed problem. To solve the POMDP, this paper constructs a GRU-based memory Sensors 2019, 19, 3837 10 of 18 network architecture. The recurrent neural network unit GRU is used as a function approximator of the belief state Bs(ot), which takes abstract historical data, the current observation and last action as input. As shown in Figure 4, the observation ot ﬁrst is passed through two layers of Fully Connected (FC) layers, and the numbers of their hidden units are 256 and 64, respectively. Then, the GRU layer takes them as input that are extracted from 64 dimensions of observation features, the 32 dimensions of the last hidden state of the GRU and the two dimensions of the last action. The number of hidden units of the GRU layer is 32. The output layer is divided into two streams. One stream uses a FC layer to output the expectation of the Gaussian distribution of the action space, and the other stream outputs the variance. Instead of directly outputting actions, constructing the action distribution can improve the randomness of the action selection in the training process and increase explorations in unknown environments. Sensors 2019, 19, x FOR PEER REVIEW 10 of 17 Figure 4. The structure of actor network in MK-A3C. 𝑂௧ denotes the current observation. 𝐻௧ିଵ is the last hidden states of GRU layer. 𝐴௧ିଵ denotes last action. μ denotes expectation of the output action probability distribution, and σ denotes variance. The structure of critic network is similar to that of actor network, but replaces two streams of the output layer of the actor network with one stream and no activation function. 3.3.4. MK-A3C Method Ot 29 dim Fully Connect ReLU 256 Fully Connect ReLU 64 GRU ReLU 32 Fully Connect Tanh 2 Fully Connect Softplus 2 μ 2 dim σ 2 dim At-1 2 dim Ht-1 32 dim Figure 4. The structure of actor network in MK-A3C. Ot denotes the current observation. Ht−1 is the last hidden states of GRU layer. At−1 denotes last action. µ denotes expectation of the output action probability distribution, and σ denotes variance. The structure of critic network is similar to that of actor network, but replaces two streams of the output layer of the actor network with one stream and no activation function. Fully Connect Figure 4. The structure of actor network in MK-A3C. 𝑂௧ denotes the current observation. 𝐻௧ିଵ is the last hidden states of GRU layer. 𝐴௧ିଵ denotes last action. μ denotes expectation of the output action probability distribution, and σ denotes variance. The structure of critic network is similar to that of actor network, but replaces two streams of the output layer of the actor network with one stream and no activation function. Figure 4. The structure of actor network in MK-A3C. Ot denotes the current observation. Ht−1 is the last hidden states of GRU layer. At−1 denotes last action. µ denotes expectation of the output action probability distribution, and σ denotes variance. The structure of critic network is similar to that of actor network, but replaces two streams of the output layer of the actor network with one stream and no activation function. In the proposed network, the first two FC layers are used to extract features from the current observation. The GRU layer with recurrent mechanism is used to approximate the belief state by integrating features of the current observation with abstract historical trajectories. Finally, a FC layer is used to output the action distribution. The key to realizing memory and inference is to use the last hidden state of GRU layer as part of the current input of GRU layer, which means that GRU layer can use the historical trajectory as input to infer the current complete state (i.e., 𝑖𝑛𝑝𝑢𝑡௚௥௨ ௧ = 𝑜𝑢𝑡𝑝𝑢𝑡௚௥௨ ௧ିଵ+ 𝑎௧ିଵ+ 𝑜𝑢𝑡𝑝𝑢𝑡௙௖_ଶ ௧ , or 𝑖𝑛𝑝𝑢𝑡௚௥௨ ௧ = 𝑓(𝑜௧, 𝑎௧ିଵ, 𝑜௧ିଵ, … , 𝑜଴)). In the proposed network, the ﬁrst two FC layers are used to extract features from the current observation. 3.3.4. MK-A3C Method Inspired by 11 of 18 Sensors 2019, 19, 3837 reference [37], we propose an improved curriculum learning method to train GRU-based memory network architecture, which divides the whole learning task into two parts. The complexity of the ﬁrst part increases with training episodes, while the complexity of the second part is randomly determined, but the minimum value of its complexity is greater than the complexity of the ﬁrst part. In this paper, two indicators, i.e., the number of moving obstacles and the initial Euclidean distance between the robot and the destination, are used to quantify the complexity of the learning task. Key parameters of the ﬁrst part of learning tasks are presented in Table 1. Table 1. The ﬁrst part of learning task based on transfer learning. Training Episodes Distance Number of Obstacles 3000 3 1 4000 5 2 5000 9 2 3000 13 2 Table 1. The ﬁrst part of learning task based on transfer learning. In conclusion, MK-A3C solves problems of incomplete states and sparse reward by memory mechanism and domain knowledge, which can navigate the non-holonomic robots in unknown dynamic environments. 4. Experiments and Results To verify the eﬀectiveness of the MK-A3C algorithm, we implement mentioned improved components including the GRU-based memory network architecture, the shaped reward function with domain knowledge and the transfer learning-based training task architecture. To evaluate the performance of MK-A3C, we test it in unknown dynamic environments with moving obstacles. The motion pattern of the moving obstacles is the uniform linear motion, imitating common human movement behaviors. In the simulation environment, numerical methods are used to calculate trajectories of the robot by kinetic equations. Due to limitations of the hardware platform, the step of simulation time is set as 0.1 s by considering both eﬃciencies of calculation and accuracies of results. The hyper-parameters of MK-A3C are described as follows: the learning rates of actor network and critic network are 0.00003 and 0.0003, respectively. The unrolling step is 10. The discount factor γ is 0.99. The goal tolerance distance is 0.7. In the training phase, the action selection interval is 2 s, while in the test phase, it is 1 s. Experiments are carried out on a machine with 3.4-GHz Intel Core i7-6700 CPU and 16 GB of RAM. 3.3.4. MK-A3C Method The GRU layer with recurrent mechanism is used to approximate the belief state by integrating features of the current observation with abstract historical trajectories. Finally, a FC layer is used to output the action distribution. The key to realizing memory and inference is to use the last hidden state of GRU layer as part of the current input of GRU layer, which means that GRU layer can use the historical trajectory as input to infer the current complete state (i.e., inputt gru = outputt−1 gru + at−1 + outputt fc 2, or inputt gru = f(ot, at−1, ot−1, . . . , o0)). ௙_ ௚ Secondly, although reward shaping is used to introduce domain knowledge into the reward function, it is still difficult for the randomly initialized policy to collect valid samples in complex navigation tasks. To solve this problem, a training task architecture is constructed by transfer learning which can realize the migration of knowledge between different learning tasks. Specifically, this paper uses the curriculum learning method to build the architecture of training tasks whose complexity gradually increases. Curriculum learning [36] is a method that gradually increases the complexity of training samples to accelerate the training process. Although the method was originally proposed to solve problems of supervised learning, this method can also be applied to solve the challenge of sparse reward encountered by RL approaches. Since the network structure contains the recurrent network unit GRU, it is not appropriate to directly use the original curriculum learning. g g fc_2 g Secondly, although reward shaping is used to introduce domain knowledge into the reward function, it is still diﬃcult for the randomly initialized policy to collect valid samples in complex navigation tasks. To solve this problem, a training task architecture is constructed by transfer learning which can realize the migration of knowledge between diﬀerent learning tasks. Speciﬁcally, this paper uses the curriculum learning method to build the architecture of training tasks whose complexity gradually increases. Curriculum learning [36] is a method that gradually increases the complexity of training samples to accelerate the training process. Although the method was originally proposed to solve problems of supervised learning, this method can also be applied to solve the challenge of sparse reward encountered by RL approaches. Since the network structure contains the recurrent network unit GRU, it is not appropriate to directly use the original curriculum learning. 4.2. Evaluation of MK-A3C 4.2. Evaluation of MK-A3C To verify the effectiveness of the GRU-based memory network, we compare MK-A3C with A3C- R. A3C-R replaces the GRU layer in MK-A3C with a FC layer and removes the input of last action and abstract history data. The reward function and the training task architecture of A3C-R are To verify the eﬀectiveness of the GRU-based memory network, we compare MK-A3C with A3C-R. A3C-R replaces the GRU layer in MK-A3C with a FC layer and removes the input of last action and abstract history data. The reward function and the training task architecture of A3C-R are consistent with MK-A3C. The value function V(s) refers to the total long-term expected discount rewards starting from state s, which can be used to reﬂect the performance of the learned policy. The success rate denotes the probability that the robot safely reaches the goal point in the nearest 1000 episodes. As shown in Figure 6a, when the number of episodes is less than 3000, the performance of A3C-R is slightly better than that of MK-A3C. It shows that the A3C-R can handle simple navigation tasks well, and the learning eﬃciency of GRU-based memory network is lower than that of FC-based networks. When they collect experience from more episodes, until 12,000, the performance of MK-A3C is slightly better than that of A3C-R, and trends showed by the two curves are similar. When the number of episode is greater than 12,000, MK-A3C can have more successful navigation instances than its counterpart. This proves that the reactive navigation policies generated by A3C-R cannot deal with complex navigation tasks by only considering the current observation, while MK-A3C estimates hidden environmental states by combining historical trajectories and the current observation and is capable of ﬁnding more reasonable navigation policies. In addition, the training task architecture based on transfer learning is also more suitable to train the GRU-based network. In Figure 6b, their trends of V-value data in the training process are the same as that of success rate curves, which further proves the previous conclusions. To evaluate the validity of the domain knowledge-based reward function constructed by reward shaping, we remove the non-terminal rewards from MK-A3C’s reward function. However, MK-A3C without non-terminal rewards cannot converge. MK-A3C without the proposed training architecture cannot converge to useful policies, which demonstrates that the transfer learning-based training task architecture is necessary for MK-A3C to solve navigation in unknown environments. Figure 5. Training environment. Black areas are occupied by obstacles, and white areas are traversable for robots. Figure 5. Training environment. Black areas are occupied by obstacles, and white areas are traversable for robots. p p g Figure 5. Training environment. Black areas are occupied by obstacles, and white areas are traversable for robots. Figure 5. Training environment. Black areas are occupied by obstacles, and white areas are traversable for robots. Figure 5. Training environment. Black areas are occupied by obstacles, and white areas are traversable for robots. Figure 5. Training environment. Black areas are occupied by obstacles, and white areas are traversable for robots. 4.1. Training Environment Settings As shown in Figure 5, the training environment is a complex environment ﬁlled with long corridors and dead corners, the size of which is 20 by 20. In each episode, the starting point is randomly sampled from the lower-left corner of the map, and the goal point is randomly initialized within a certain range from the starting point. The initial motion states of moving obstacles are also randomly initialized in each episode. The range of velocities of moving obstacles is [0.06, 0.08]. To increase the diﬃcultly of navigation problem, the initial directions of moving obstacles are roughly toward the robot. If moving obstacles reach the border of the map or collide with static obstacles, they will be initialized again. When the robot reaches the goal or collides with obstacles, the current episode ends and the new episode begins. 12 of 18 y current Sensors 2019, 19, 3837 will be initialized i d d d t 4.2. Evaluation of MK-A3C 4.2. Evaluation of MK-A3C In conclusion, experiments on the reward function and the training task architecture show that improvements to these two components can solve the problem of sparse reward by increasing the density of reward signals and improving learning eﬃciencies. 13 of 18 Sensors 2019, 19, 3837 curves, which furth (a) (b) Figure 6. The performance of our method MK-A3C and A3C-R in the training phase. (a) The orange and black lines denote success rates of A3C-R and MK-A3C, respectively; (b) The light orange and gray lines represent data of V-value, and the overstriking orange and black lines denote the mean V- value of the last 1000 episodes. (a) Success rate; (b) V-value. Figure 6. The performance of our method MK-A3C and A3C-R in the training phase. (a) The orange and black lines denote success rates of A3C-R and MK-A3C, respectively; (b) The light orange and gray lines represent data of V-value, and the overstriking orange and black lines denote the mean V-value of the last 1000 episodes. (a) (b) Figure 6. The performance of our method MK-A3C and A3C-R in the training phase. (a) The orange and black lines denote success rates of A3C R and MK A3C respectively; (b) The light orange and Figure 6. The performance of our method MK-A3C and A3C-R in the training phase. (a) The orange and black lines denote success rates of A3C R and MK A3C respectively; (b) The light orange and gray (b) Figure 6. The performance of our method MK-A3C and A3C-R in the training phase. (a) The orange and black lines denote success rates of A3C-R and MK-A3C, respectively; (b) The light orange and gray lines represent data of V-value, and the overstriking orange and black lines denote the mean V- value of the last 1000 episodes. (a) Success rate; (b) V-value. Figure 6. The performance of our method MK-A3C and A3C-R in the training phase. (a) The orange and black lines denote success rates of A3C-R and MK-A3C, respectively; (b) The light orange and gray lines represent data of V-value, and the overstriking orange and black lines denote the mean V-value of the last 1000 episodes. 4.3. Performance on Unknown Dynamic Environments 4.3. Performance on Unknown Dynamic Environments In this section, to fully evaluate performance of the proposed method, we design more dynamic testing environments than that used for training, and their topologies are also very diﬀerent. Speciﬁcally, the distributions of traversable areas are diﬀerent among testing environments. The shapes and positions of static obstacles are randomly chosen. Besides, starting points are randomly sampled from the lower-left corner of the environment, and goal points are randomly distributed in the environment. To increase dynamics of testing environments, we add disturbance in the positions, orientations and velocities of moving obstacles. The moving obstacles are randomly distributed in environments and their orientations are randomly selected from 0 to 360 degrees. The velocities are randomly chosen from 0.06 to 0.08. The uniform linear motion is the motion pattern of moving obstacles. There are ﬁve moving obstacles in the testing environments, which is more than that in the training phase. The performance of the MK-A3C is evaluated on tasks of navigation in unknown dynamic environments. As shown in Figure 7, the MK-A3Cbased motion controller is capable of implementing eﬃcient and safe navigation in unknown challenging environments with moving obstacles. Besides, navigation policies generated by MK-A3C have powerful generalization capabilities, and can adapt to new unknown environments without retraining. Since A3C-R cannot complete all diﬃcult examples in Figure 7, we do not present paths generated by A3C-R. 14 of 18 difficult Sensors 2019, 19, 3837 to new unknown l i Fi (a) (b) (c) (d) Figure 7. Examples of the robot completing one navigation tasks in the training and testing environments. The light purple circle denotes the starting point and the red pentagram denotes the goal point. The dark green lines are the trajectories generated by MK-A3C. For clear presentation, we do not show trajectories of moving obstacles. (a) Training Environment; (b) Testing Environment 1; (c) Testing Environment 2; (d) Testing Environment 3. (a) (b) (b) (a) ( ) (d) (c) (d) (c) Figure 7. Examples of the robot completing one navigation tasks in the training and testing environments. The light purple circle denotes the starting point and the red pentagram denotes the goal point. The dark green lines are the trajectories generated by MK-A3C. For clear presentation, we do not show trajectories of moving obstacles. (a) Training Environment; (b) Testing Environment 1; (c) Testing Environment 2; (d) Testing Environment 3. 4.3. Performance on Unknown Dynamic Environments As shown in Figure 8a, MK-A3C achieves success rates of over 62.4% in all testing environments, whose highest success rate is 65.6% in testing environment 2. However, the success rates of A3C-R are lower than MK-A3C in all environments, especially in testing environment 1 only reaching 35.4%. Given that A3C-R plans motion behaviors only based on the current observation and ignores hidden states of the environment, it is diﬃcult for A3C-R to deal with complex terrains and moving obstacles. Comparatively, MK-A3C integrates historical trajectories within a certain length with the current observation to estimate the complete environmental state by the GRU-based memory network, which can avoid local minima areas and handle moving obstacles. In Figure 8b,c, compared with A3C-R, navigation policies generated by MK-A3C require longer path lengths and more time to complete navigation tasks, which shows that the robot controlled by MK-A3C are more cautious about unknown environments. 15 of 18 us about Sensors 2019, 19, 3837 navigation tasks unknown enviro nknown environments. (a) (b) (c) Figure 8. 1000 navigation tasks are generated in each testing environments. (a) Success rate denotes the probability of successfully arriving at the destination; (b) Average path length denotes the mean path lengths for all successful navigation; (c) Average execution time denotes the average value of execution time for all successful navigation. (a) (b) (c) (a) (a) (b) (b) (c) (c) Figure 8. 1000 navigation tasks are generated in each testing environments. (a) Success rate denotes the probability of successfully arriving at the destination; (b) Average path length denotes the mean path lengths for all successful navigation; (c) Average execution time denotes the average value of execution time for all successful navigation. 5. Conclusions and Q.Y.; Software, R.J.; Validation, J.Z.; Writing—original draft, J.Z.; Writing—review & editing, J.Z. and R.J. Funding: This work was sponsored by the National Science Foundation of Hunan Province (NSFHP) project 2017JJ3371, China. Funding: This work was sponsored by the National Science Foundation of Hunan Province (NSFHP) project 2017JJ3371, China. Acknowledgments: We appreciate the fruitful discussion with the Sim812 group: Qi Zhang and Yabing Zha. Conﬂicts of Interest: The authors declare no conﬂicts of interest. Conﬂicts of Interest: The authors declare no conﬂicts of interest. 5. Conclusions In this paper, we propose a novel DRL algorithm called MK-A3C, which can navigate the non-holonomic robot in unknown and complex environments with moving obstacles. To solve challenges of partially observable states and sparse reward, MK-A3C makes improvements to the original A3C with memory mechanism, domain knowledge and transfer learning. Firstly, MK-A3C takes the time dimension into consideration, and constructs a GRU-based memory network architecture to strength the temporal reasoning ability of the robot. Additionally, robots with certain memory ability endowed by MK-A3C can avoid local minima traps by estimating the environmental model. Secondly, MK-A3C combines the domain knowledge-based reward function and the transfer learning-based training task architecture, which can solve non-convergence problem for policies caused by sparse Sensors 2019, 19, 3837 16 of 18 16 of 18 reward. These improvements of MK-A3C enable controlled robots to eﬃciently navigate in unknown complex environments, meanwhile satisfying kinetic constraints and dealing with moving obstacles. In the simulation experiment, ﬁrst, the eﬀectiveness of improved components of MK-A3C is veriﬁed by ablation study. These experimental results show that the GRU-based memory network can improve the performance in complex navigation tasks. The domain knowledge-based reward function and the transfer learning-based learning task architecture are essential to train useful policies. Second, the performance of MK-A3C is evaluated in unknown challenging environments with moving obstacles. Compared with A3C-R which has no memory mechanism with the same other components as MK-A3C, our method can navigate the robot in new unknown environments with high success rates. In future work, we will consider more complex environments. Speciﬁcally, delayed actions, irregular moving obstacles and the like can be added into existing environments, which can realistically simulate a real world. In addition, this paper uses the domain knowledge-based reward function to overcome negative eﬀects of sparse rewards, but it is time-consuming to adjust parameters of the reward function and the design of this kind reward function depends on subjective experience. It is promising to use hierarchical reinforcement learning [38], auxiliary tasks [14] and intrinsic rewards [26] to replace domain knowledge-based reward functions, which can avoid heavy work of designing reward function and further improve the training eﬃciency. Author Contributions: Conceptualization, Y.H. and C.H.; Data curation, J.Z.; Formal analysis, L.Q. and C.H.; Funding acquisition, L.Q. and Q.Y.; Investigation, J.Z., Y.H. and C.H.; Methodology, J.Z., R.J. and Y.H.; Project administration, L.Q. and Q.Y.; Resources, R.J., L.Q. 2. Li, Y. 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https://openalex.org/W4389144561	http://journal.thamrin.ac.id/index.php/jtik/article/download/1315/pdf	Indonesian	null	Aplikasi Layanan Pengaduan Lampu Penerangan Jalan Umum	Jurnal Teknologi Informatika & Komputer	2,023	cc-by	1,910	p-ISSN 2656-9957; e-ISSN 2622-8475 p-ISSN 2656-9957; e-ISSN 2622-8475 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin Volume 9 No 1; Maret 2023 Abstract Complaint is any activity carried out to report an activity or event that will end with making a report. Reports are records that provide information about certain activities and the results are presented in the form of data. Street lighting is an important component on a road. Street lighting must provide road users with a sense of security and comfort, especially at night. This study aims to design and build a complaint service application for street lighting problems. In designing and building a complaint service application using the SDLC (Software Development Life cycle) method with the RAD (Rapid Application Development) model with the end result being an application that can help staff manage community reports. Keywords: Complaint Service, Software Development Life cycle, Rapid Application Development Abstrak Pengaduan adalah setiap kegiatan yang dilakukan untuk melaporkan suatu kegiatan atau kejadian yang akan diakhiri dengan pembuatan laporan. Laporan adalah catatan yang memberikan informasi tentang kegiatan tertentu dan hasilnya yang disampaikan berupa data. Penerangan Lampu jalan merupakan suatu komponen penting pada suatu ruas jalan. Penerangan lampu jalan harus memberikan rasa aman dan nyaman khususnya pada saat malam hari kepada pengguna jalan. Penelitian ini bertujuan untuk merancang dan membangun suatu aplikasi layanan pengaduan permasalahan lampu penerangan jalan. Dalam merancang dan membangun aplikasi layanan pengaduan menggunakan metode SDLC (Software Development Life cycle) dengan permodelan RAD (Rapid Aplication Development) dengan hasil akhir sebuah aplikasi yang dapat membantu staf mengelola laporan masyarakat. Kata Kunci: Layanan Pengaduan, software Development Life cycle, Rapid Aplication Development Lutfi Andrianto1)), Sita Muharni2) Lutfi Andrianto1)), Sita Muharni2) 1)2) Teknik Informatika, STMIK Dharmawacana Metro Correspondence author: zoidsblue@gmail.com, Lampung, Indonesia DOI: https://doi.org/10.37012/jtik.v9i1.1315 1)2) Teknik Informatika, STMIK Dharmawacana Metro Correspondence author: zoidsblue@gmail.com, Lampung, Indonesia DOI: https://doi.org/10.37012/jtik.v9i1.1315 http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf Jurnal Teknlogi Informatika dan Komputer MH. Thamrin Volume 9 No 1; Maret 2023 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin Volume 9 No 1; Maret 2023 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin Volume 9 No 1; Maret 2023 Penerangan Lampu jalan merupakan suatu komponen penting pada suatu ruas jalan. Penerangan lampu jalan harus memberikan rasa aman dan nyaman khususnya pada saat malam hari kepada pengguna jalan. Penerangan lampu jalan harus dapat memperjelas pandangan saat malam hari tanpa memberikan efek silau yang berlebihan pada pengguna jalan. Kualitas penerangan lampu jalan harus sesuai dengan perencanaan sistem penerangan jalan yang telah ditetapkan pada jenis lampu, tinggi dan jarak spasi pemasangan penerangan lampu jalan. Dinas Perumahan dan Kawasan Permukiman Kota Metro adalah salah satu instansi pemerintah yang terletak di Jl. Jend. A. H Nasution No.42 Yosorejo, Metro Timur, Kota Metro, Lampung. Dinas perumahan dan kawasan permukiman Kota Metro terdiri dari beberapa yaitu bidang perumahan, penataan kawasan permukiman dan pertanahan serta bidang pertamanan, infrastruktur areal pemakaman dan penerangan jalan umum. Berdasarkan latar belakang tersebut maka penelitian ini bertujuan untuk memperbaiki adanya kekurangan dari proses pelaporan yang sudah berjalan. Pada penelitian ini dibangun sebuah aplikasi untuk layanan pengaduan lampu penerangan jalan umum pada dinas perumahan dan kawasan permukiman Kota Metro. Diharapkan aplikasi ini dapat memudahkan staf mengelola data laporan masyarakat tentang permasalahan yang terjadi pada bidang penerangan jalan umum (PJU) dan dapat direspon serta ditangani dengan cepat oleh staf pegawai Dinas Perumahan dan Kawasan Permukiman Kota Metro khususnya petugas yang bertanggung jawab pada penerangan jalan umum (PJU). PENDAHULUAN Pelaporan adalah setiap kegiatan yang dilakukan, diakhiri dengan pembuatan laporan. Laporan adalah catatan yang memberikan informasi tentang kegiatan tertentu dan hasilnya yang disampaikan berupa data. Sedangkan mekanisme pelaporan kerusakan yang saat ini sudah berjalan adalah, user melaporkan kerusakan melalui telephone seluler maupun whatsapp, staf menerima dan memverifikasi laporan. Laporan yang diterima akan diproses, kemudian satgas menerima memo dari staf yang berisi jadwal kegiatan, dikerjakan oleh satgas dan dilaporkan kembali ke staf. Staf memberikan hasil laporan kepada kasi dan kabid untuk divalidasi lalu diarsipkan. http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf 352 p-ISSN 2656-9957; e-ISSN 2622-8475 http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf METODE http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf 353 Observasi adalah teknik pengamatan langsung di Dinas Perumahan dan Kawasan Permukiman, untuk mengamati langsung proses pelaporan yang sedang berjalan, mulai dari masyarakat melaporakan, staf menerima laporan, dan satgas melakukan perbaikan. Selain observasi, dilakukan juga wawancara. Wawancara adalah teknik pengumpulan data melalui tatap muka dan tanya jawab langsung dengan orang-orang yang berkompeten pada bidang Penerangan Jalan Umum. Dalam hal ini peneliti melakukan wawancara kepada bapak Budi Dwi Radiastanto, ST., MT. selaku Kepala Bidang Pertamanan Infrastruktur Pemakaman dan Penerangan Jalan Umum dan Kepala Seksi Penerangan Jalan Umum Bapak Ir. Heri Martianto untuk memvalidasi informasi yang didapat pada saat observasi. Studi Pustaka 353 p-ISSN 2656-9957; e-ISSN 2622-8475 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin Volume 9 No 1; Maret 2023 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin Volume 9 No 1; Maret 2023 dilakukan untuk mendapatkan literature yang serupa yang pernah dilakukan peneliti lain terdahulu terkait pada bidang laporan yang paling berpengaruh terhadap penelitian ini. Metode Pengembangan Perangkat Lunak yang digunakan dalam membangun sistem layanan pengaduan kerusakan lampu penerangan jalan umum pada Dinas Perumahan dan Kawasan Permukiman Kota Metro adalah metode Rapid Aplication Development (RAD). RAD adalah metode yang berfokus pada pengembangan aplikasi secara cepat, melalui pengulangan dan feedback berulang-ulang. Berikut ini adalah 5 langkah dari metode RAD: Gambar 1. Langkah-langkah metode Rapid Aplication Develompment (RAD) Gambar 1. Langkah-langkah metode Rapid Aplication Develompment (RAD) Pada fase Business Modeling peneliti membuat suatu use case yang menerangkan bagaimana sistem yang sedang berjalan. Dimulai dari masyarakat yang melapor melalui telephone selular maupun whatsapp, staf menerima laporan, kemudian menjadwalkan aktifitas perbaikan, lalu di konfirmasi oleh Kepala Seksi dan Kepala Bidang, kemudian baru diteruskan kepada satgas untuk mengerjakan perbaikan. Sistem yang dibuat nantinya akan berjalan mulai dari masyarakat sebagai user melapor dengan cara mengisi form laporan. Staf sebagai admin akan masuk kedalam sistem melalui login terlebih dahulu, setelah masuk staf dapat melihat daftar laporan, mengkonfirmasi, meng update, menghapus, mencetak laporan, melihat feedback dan riwayat laporan yang terdahulu. Use case tersebut akan dibangun berdasarkan use case scenario. Pada fase Data Modeling, semua informasi yang diperoleh dalam fase Business Modeling ditinjau, dianalisis dan dipisahkan menjadi kumpulan data yang dianggap penting bagi proses bisnis. Data yang di peroleh dari fase Business Modeling diantaranya: Data http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf 354 p-ISSN 2656-9957; e-ISSN 2622-8475 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin Volume 9 No 1; Maret 2023 Jurnal Teknlogi Informatika dan Komputer MH. METODE Thamrin Volume 9 No 1; Maret 2023 Admin terdiri dari data login username, password, data pribadi berisi nama, nip, jabatan, alamat dan nomor telephone. Pada fase Process Modeling, semua objek data yang dikumpulkan diubah menjadi informasi yang berguna dan diperlukan. Data yang didapat dari Dinas Perumahan dan Kawasan Permukiman kemudian oleh admin diproses meliputi proses input, update, dan delete. User masyarakat bisa melaporkan pengaduan melalui aplikasi yang dikembangkan. Pada fase Application Generation, peneliti mulai melakukan pengkodean program program menggunakan bahasa pemrograman HTML, PHP, dan database My SQL. Pada tahap Testing and Turnover, peneliti melakukan testing menggunakan metoda blackbox testing dengan cara menguji terhadap aktifitas user admin dan user masyarakat. Pada tahap ini peneliti melaporkan hasil testing menggunakan black box pada halaman laporan. http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf HASIL DAN PEMBAHASAN Use Case Diagram, menggambarkan secara ringkas siapa yang akan menggunakan sistem ini dan apa saja yang bisa dilakukan oleh pengguna. Gambar 2. Use Case Diagram Gambar 2. Use Case Diagram Implementasi Interface sangat berpengaruh dalam interaksi antara masyarakat dan sistem. Interface menjadi sarana komunikasi serta memberikan kemudahan bagi user dalam melakukan aktivitasnya. Berikut ini perancangan user interface yang telah dibuat, diantaranya: http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf 355 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin p-ISSN 2656-9957; e-ISSN 2622-8475 Volume 9 No 1; Maret 2023 Interface Login, adalah tampilan untuk login ke dalam sistem dengan cara memasukkan username dan password. Gambar 3. Tampilan Halaman Login p-ISSN 2656-9957; e-ISSN 2622-8475 Interface Login, adalah tampilan untuk login ke dalam sistem dengan cara memasukkan username dan password. Interface Login, adalah tampilan untuk login ke dalam sistem dengan cara memasukkan username dan password. Gambar 3. Tampilan Halaman Login Gambar 3. Tampilan Halaman Login Halaman beranda adalah halaman utama yang akan muncul setelah melakukan login sistem. Halaman beranda dapat dilihat pada gambar 4 berikut: Gambar 4. Tampilan Halaman Beranda Gambar 4. Tampilan Halaman Beranda http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf 356 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin Volume 9 No 1; Maret 2023 p-ISSN 2656-9957; e-ISSN 2622-8475 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin p-ISSN 2656-9957; e-ISSN 2622-8475 Volume 9 No 1; Maret 2023 Halaman Laporan adalah halaman yang berisi data laporan masyarakat mengenai kerusakan lampu penerangan jalan yang diinput oleh admin. Gambar 5. Tampilan Halaman Laporan Halaman input laporan berisi form input laporan meliputi: nama pelapor, no hp pelapor, lokasi laporan kerusakan, keterangan laporan dan foto kerusakan, yang di input oleh admin. Halaman Laporan adalah halaman yang berisi data laporan masyarakat mengenai kerusakan lampu penerangan jalan yang diinput oleh admin. Gambar 5. Tampilan Halaman Laporan Gambar 5. Tampilan Halaman Laporan Gambar 5. Tampilan Halaman Laporan Halaman input laporan berisi form input laporan meliputi: nama pelapor, no hp pelapor, lokasi laporan kerusakan, keterangan laporan dan foto kerusakan, yang di input oleh admin. http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf 357 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin p-ISSN 2656-9957; e-ISSN 2622-8475 Volume 9 No 1; Maret 2023 Gambar 6. Tampilan Halaman input Laporan Jurnal Teknlogi Informatika dan Komputer MH. Thamrin p-ISSN 2656-9957; e-ISSN 2622-8475 Volume 9 No 1; Maret 2023 p-ISSN 2656-9957; e-ISSN 2622-8475 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin Volume 9 No 1; Maret 2023 Gambar 6. Tampilan Halaman input Laporan bar 6. Tampilan Halaman input Laporan Gambar 6. Tampilan Halaman input Laporan Halaman Riwayat adalah halaman yang berisi data laporan yang telah masuk dan dikerjakan oleh admin Dinas Perumahan dan Kawasan Permukiman Kota Metro. http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf 358 p-ISSN 2656-9957; e-ISSN 2622-8475 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin Volume 9 No 1; Maret 2023 Volume 9 No 1; Maret 2023 Gambar 7. Tampilan Halaman riwayat Gambar 7. Tampilan Halaman riwayat http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf 359 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin p-ISSN 2656-9957; e-ISSN 2622-8475 Volume 9 No 1; Maret 2023 p-ISSN 2656-9957; e-ISSN 2622-8475 Jurnal Teknlogi Informatika dan Komputer MH. Thamrin p-ISSN 2656-9957; e-ISSN 2622-8475 Volume 9 No 1; Maret 2023 KESIMPULAN DAN REKOMENDASI Dari penelitian, analisis, perancangan dan pembangunan aplikasi di atas dapat diambil kesimpulan sebagai berikut : 1. Aplikasi yang di rancang dapat memudahkan admin selaku staff Dinas Perumahan dan Kawasan Permukiman Kota Metro mengelola data laporan masyarakat mengenai kerusakan lampu penerangan jalan umum. 1. Aplikasi yang di rancang dapat memudahkan admin selaku staff Dinas Perumahan dan Kawasan Permukiman Kota Metro mengelola data laporan masyarakat mengenai kerusakan lampu penerangan jalan umum. 2. Penggunaan metode Rapid Application Develompent (RAD) sudah cukup tepat untuk merancang dan membangun Aplikasi Layanan Pengaduan Lampu Penerangan Jalan Umum. 3. Masyarakat selaku pelapor di mudahkan untuk melaporkan kerusakan melalui aplikasi, melalui telefon atau Whatsapp. REFERENSI Hakim, D. K., (2018). Pembangunan Aplikasi Pelaporan Perijinan dengan Online Analytical Processing (OLAP). Jurnal Pengabdian Kepada Masyarakat Vol 2, No 2. Maulidiansyah R., Rakhman D. F., Ramdhani, M. A., (2017). Aplikasi Pelaporan Kerusakan Jalan Tol Menggunakan Layanan Web Service Berbasis Android. Jurusan Teknik Informatika, Fakultas Sains dan Teknologi, Universitas Islam Negeri Sunan Gunung Djati Bandung. Mahaldi, I., (2018). Perancangan Sistem Pelaporan Kerusakan Jalan Di Kota Medan. Program Studi D3 Teknik Informatika Departemen Matematika Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Sumatera Utara Medan. Turaina R., Putri, N. E., Anwar, N., (2018). Aplikasi Pelaporan Aktivitas Siswa kepada Orang Tua Berbasis Web dan SMS Gateway. Indonesian Journal of Computer Science Vol 7 No 1 April 2018 Issue. Alamsyah, A., (2020). Aplikasi Pelaporan Kerusakan Perlengkapan Kantor Menggunakan Metode Json Melalui Fungsi IP Camera. Jurnal INSTEK (Informatika Sains dan Teknologi) Vol 5, No 2. http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf http://journal.thamrin.ac.id/index.php/jtik/article/view/1315/pdf 360
https://openalex.org/W4304207502	https://zenodo.org/records/7184042/files/ZDTF2418.pdf	English	null	THE INFLUENCE OF PSYCHO-SOCIAL FACTORS IN THE DEVELOPMENT OF COMORBID CONDITIONS.	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	1,399	« Zamona izlanishla « Zamona izlanishla « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi THE INFLUENCE OF PSYCHO-SOCIAL FACTORS IN THE DEVELOPMENT OF COMORBID CONDITIONS. Xamraeva Gulnora Ilyosovna TMA Independent researcher +998950228889 https://doi.org/10.5281/zenodo.7184042 Abstract: It is known that chronic stress can lead to the development of various diseases or aggravate an existing disease. This article provides information about the influence of psychosocial factors in the development of comorbid conditions. Key words: Stress, cortisol immunity, visceral fat, Depression, Distress. Stress is the human body's response to excessive tension, negative emotions or simply boredom. During stress, the human body produces adrenaline, a hormone that prompts the search for a solution. A small amount of stress is necessary for everyone, because it encourages a person to think, to find a way out of a problem, without stress life would be boring. On the other hand, if there is too much stress, the body becomes weak, weak and loses its ability to solve problems. Stress plays a major role in the development of diseases such as obesity, heart disease, Alzheimer's disease, diabetes, depression, digestive problems and asthma. In fact, stress can be a very strong innate or acquired protective reaction of the body, during which the hormones adrenaline and cortisol are produced. It is these stress hormones that make the heart beat faster, constrict blood vessels, affect breathing, and improve blood flow to muscles, lungs, and other internal organs. This puts the body collectively in a "fight or flight" mode. If stress becomes chronic, these physiological changes can lead to health problems. How does stress affect health? Because the stress hormone cortisol inhibits immune cells, prolonged stress and excess cortisol can weaken the immune system, resulting in frequent colds in the body. Heart diseases. It has long been known that people with chronic stress and anxiety are more likely than others to develop cardiovascular disease (high blood pressure being the most common). Stress can increase heart rate and increase triglyceride cholesterol levels. It is known that stress is often associated with smoking and overweight, which indirectly increases the risk of cardiovascular diseases. Sudden emotional stress can lead to serious heart 63 63 « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi Asthma. Many studies have shown that stress can make asthma worse. Some evidence suggests that chronic parental stress increases the risk of asthma in children. THE INFLUENCE OF PSYCHO-SOCIAL FACTORS IN THE DEVELOPMENT OF COMORBID CONDITIONS. 64 « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi Depending on the result, the following types of stress are distinguished in psychology: Eustresses ("beneficial" stresses) We all need a certain amount of stress to live « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi Eustresses ("beneficial" stresses). We all need a certain amount of stress to live successfully. This is the main factor of our development. This condition can be called "awakening reaction". It is like waking up from sleep. To go to work in the morning, you must first get out of bed and wake up. To achieve work activity, you need a boost - a small dose of adrenaline. Eustress performs exactly these tasks. Distresses (harmful stresses) occur in acute stress. This type of stress represents the stress that everyone understands. Causes of stress. The cause of stress can actually be anything that affects and disturbs a person. For example, external causes can include anxiety caused by something (change of job, death of a relative). Internal causes of stress include life values and beliefs. This includes a personal assessment Stress and depression can occur in approximately equal proportions in women and men. However, each organism has its own characteristics. If you notice symptoms that may indicate stress, you should first identify the cause of the condition. It is much easier to eliminate the causes of stress than the consequences. It is not a lie that "all diseases are caused by nerves". Clinical observations have shown that small stresses are not harmful to the body, even beneficial. They encourage a person to find a way out of the current situation. Each of us should educate ourselves and develop our will so that depression does not turn into a more serious and long-term form. Many people are used to fight against stress with the help of antidepressants and alcohol. However, choosing such a path leads to getting used to them and not being able to give them up without expert help. Treatment and prevention of stress. As everyone knows, there is no better medicine than good sleep. Therefore, you should think about how you sleep. Here are some tips to help you sleep better. Regular exercise is a big help for normal sleep. THE INFLUENCE OF PSYCHO-SOCIAL FACTORS IN THE DEVELOPMENT OF COMORBID CONDITIONS. Overweight and obesity. Excess fat in the abdomen (visceral fat) is considered more dangerous than fat in the arms, legs or other parts of the body. Unfortunately, compared to healthy people, people with chronic stress suffer the most from abdominal (visceral) fat. Stress increases the level of cortisol, which indirectly contributes to the accumulation of fat around the organs. Type 2 diabetes. Stress can worsen diabetes in two ways. First, it increases the likelihood of consuming junk food and excessive amounts of alcohol. Second, the sensitivity of cells to insulin (a hormone released by the pancreas to lower blood sugar) decreases, and blood sugar levels rise. Headache. Stress is a common cause of headaches, not just headaches or migraines. Feelings of depression and anxiety. It's no surprise that chronic stress is linked to depression and anxiety. A recent study found that people who experience stress at work (such as feeling underpaid for the work they do) are 80% more likely to develop depression than those with low levels of stress. Digestive problems. It is known that stomach or duodenal ulcers can appear against the background of stress. Also, stress can lead to exacerbation of chronic diseases of the intestinal tract, gastritis, inflammation of the intestines, and reflux. Irritable bowel syndrome is more common in people with high levels of stress. Aging and death. There is evidence that stress can accelerate aging. People with chronic stress lose 9-17 years of life, age faster and die earlier. Managing and dealing with stress is great, but the hardest part is taking the first step. It is not always possible to remove the cause of stress from life, but you can try to change your attitude. Several scientific studies have been devoted to this problem. The mechanisms of stress have been studied in detail and are very complex: they are related to our hormonal, nervous and vascular systems. In some cases, the definition of "stress" refers to the trigger itself. For example, physical - this is severe cold or unbearable heat, decrease or increase in atmospheric pressure. Under the concept of chemical stress, the effect of all toxic substances is understood. Mental stress can be the result of extremely negative or positive emotions. Injuries, viral diseases, muscle overload are biological stress. THE INFLUENCE OF PSYCHO-SOCIAL FACTORS IN THE DEVELOPMENT OF COMORBID CONDITIONS. It is recommended to do them two hours before going to bed, in the open air. Before going to sleep, you can take a hot bath and listen to quiet music. Try to do this every day. In order for sleep to be normal and healthy, the body needs a hormone called melatonin. It increases after the intake of B group vitamins, and this vitamin is abundant in rice, wheat, barley, pistachio seeds, dried apricots. The bedroom should not be noisy and bright, these interfere with a peaceful sleep. will give. 65 p p g References. References. References. « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi 1. E. Ghaziyev "Psychology" book 2018. 2. Ziyo.Net 3.Khaidarov F., Joraev N. Is it easy to maintain stress or mental balance? -T., 1997. 4.Gamezo M.V., Domashenko I.A. Atlas of psychology. M., 2oo2 5. Psychology.uz « Zamonaviy dunyoda tabiiy fanlar: Nazariy va amaliy izlanishlar» nomli ilmiy, masofaviy, onlayn konferensiyasi
https://openalex.org/W4229674699	https://journals.rudn.ru/linguistics/article/download/20613/16692	Latin	null	null	Vestnik Rossijskogo universiteta družby narodov. Seriâ Lingvistika	2,019	cc-by	6,631	Abstract Peoples and individuals around the globe continuously develop their own communicative habits. With each generation, adjustments to changing circumstances are made — economic circumstances, natural circum- stances, and, for instance, mobility circumstances. The outcome of such transitions is cultural variation, which is visible in hierarchical social systems, belief systems, legal systems, traditions, attire, and all kinds of rituals. Communicative systems are part of culture, and they deserve a role in research focussing on language and communication. However, applying culture as a variable is a challenge, not only because of the cultural variation between peoples and individuals but also because the effects of culture on actual language utterances are hard to measure. Another issue is the dominance of Anglowestern cultural patterns in many analyses. This paper explains these issues and critically reviews the various criteria that well-known cultural models — like the one by Hofstede (1980), Lewis (1969), and Hall (1959, 1976) — use to categorise cultures. Examples of such criteria are: region, relationship with uncertainty, femininity/masculinity, and power relations. The paper concludes by giving a number of practical solutions to the challenge of treating culture as a variable in sociolinguistic research. These solutions are related to, amongst others, the reviewing process for journals, widespread norms of ‘good academic language’, author/editor selection, cross-cultural academic cooperation, and sharing of funds. Keywords: cultural models, sociolinguistics, Anglowestern bias, cultural criteria, solutions Cultural bias and Sociolinguistics Dick Smakman Leiden University, Centre for Linguistics PO Box 9515, 2300 RA, Leiden, Netherlands Russian Journal of Linguistics Вестник РУДН. Серия: ЛИНГВИСТИКА Russian Journal of Linguistics Вестник РУДН. Серия: ЛИНГВИСТИКА 2019 Vol. 23 No. 1 9—22 2019 Vol. 23 No. 1 9—22 http://journals.rudn.ru/linguistics СОЦИОЛИНГВИСТИКА DOI: 10.22363/2312-9182-2019-23-1-9-22 DOI: 10.22363/2312-9182-2019-23-1-9-22 Культурные предубеждения и социолингвистика Дик Смакман Лейденский университет, Центр лингвистики PO Box 9515, 2300 RA, Лейден, Нидерланды Люди во всем мире постоянно развивают свои собственные коммуникативные обычаи. Каждое поколение приспосабливает их к меняющимся обстоятельствам — экономическим, природ- ным, демографическим и т.д. В результате возникает культурное разнообразие, проявляющееся в социальной иерархии, верованиях, законотворчестве, традициях, одежде и разного рода ритуа- лах. Коммуникативные системы являются частью культуры и заслуживают внимания как объект лингвистических и коммуникативных исследований. Однако здесь возникают некоторые 9 SOCIOLINGUISTICS Дик Смакман. Вестник РУДН. Серия: ЛИНГВИСТИКА. 2019. Т. 23. № 1. С. 9—22 сложности не только из-за культурной вариативности, но и из-за того, что влияние культуры на язык трудно измерить. Проблема также состоит в том, что во многих исследованиях превали- рует западная англоязычная модель анализа. В данной статье дается критический обзор этих проблем, а также критериев, используемых в известных моделях анализа культуры Хофстеде (Hofstede 1980), Льюиса (Lewis 1969), Холла (Hall 1959, 1976), таких как отношение к неопре- деленности, фемининность/маскулинность, дистанция власти и др. В статье предлагается ряд прак- тических решений, способствующих эффективному рассмотрению культуры как переменной в социолингвистических исследованиях. Они, в частности, касаются процесса рецензирования для научных журналов, широко распространенных норм «хорошего научного языка», выбора авторов и редакторов, межкультурного научного сотрудничества и совместного использования научных фондов. Ключевые слова: модели исследования культуры, социолингвистика, англо-западные предубеж- дения, культурные критерии, решения 2. THE NATURE OF THE PROBLEM Coulmas (2013) rightly indicated that Variationist Sociolinguistics is predominantly a Western science. The same thing can be said about post-Variationist waves. Meyerhoff and Nagy (2008) laid out the realities of the mechanisms behind this bias by illustrating that linguistic situations in the United States received relatively much attention in two major international journals in the field of Sociolinguistics, namely the Journal of Socio- linguistics and Language Variation and Change. They considered this a good motivation to report more on non-western language situations and make them publically available. The issue the above authors pointed out is one of availability and accessibility of re- search, but another, perhaps equally serious issue is the bias in the interpretation of data. Introductory books on Sociolinguistics often place the Anglowestern way of thinking and forming categories at the basis of argumentation. Meyerhoff and Nagy (2008) as well as an important author like Jennifer Jenkins (2009) pointed out what everyone knows to be true, namely that most introductions into the field of Sociolinguistics themselves stem from an Anglophone cultural pattern and will interpret language situa- tions through that perspective. Situations that are different from what the Western authors are used to are tacitly presented as deviant. The treatment of standard language in the literature provides a good example to demonstrate the issue (Smakman & Barasa 2016). The most common point of departure in the literature dealing with the language norm is a monolingual culture (usually a nation state) with an obvious standard language (e.g., Swedish in Sweden and Spanish in Spain). This language is spoken in the media and in official and educational contexts. It is associated with the nation state’s history and identity and is broadly supported by the nation’s inhabitants, who consider it a neutral lingua franca (Smakman 2012). Educated speakers tend to speak this language. Indeed, in European countries like the United Kingdom, Denmark, France, and the Netherlands this situation is a reality. Language norms and the norm language in Africa and some Asian countries ill-fit the traditional standardness paradigm, and these themes — probably as a result of the bad fit — do not receive the sociolinguistic attention they deserve. It ill-fits because in some countries an old colonial language exists as a language norm besides a more autochthonous lan- guage. 1. INTRODUCTION Every society has historically developed under unique circumstances. Different natural and climatic conditions have led to different communities of people, each with their own systems of beliefs, ideologies, and morals, and each with their own system of wealth and power division. The different roles of women and men, in particular, have been influenced by these natural conditions; societies that were originally of the hunter- gatherer type nowadays tend to have different roles assigned to the sexes, compared to societies that were originally horticultural or, for instance, pastoral (Evans-Prit- chard 1951; Halliday 2001). The natural outcome of this variation are communicative habits that reflect the cultures in question. These are deeply engrained in societies and are passed on from generation to generation, and every generation adapts the passed- on system to their emerging practical and symbolical needs. The communication patterns of each member in each new generation are thus affected by the practices and beliefs instilled through their culture, by changing needs in this society, but also through contact with other societies as well as regional and global tendencies and technical progress. This paper seeks to treat culture as one of the key determinants of sociolinguistic systems (i.e. systems of interpersonal communication within societies); culture partly determines how people within societies communicate. It investigates how to treat culture as a sociolinguistic variable. The basic premise this paper departs from is that there is a certain overrepresentation of Anglowestern ideologies in the literature, and an under- representation of cultures that are not Anglowestern, including cultures that large groups of speakers adhere to. This is a fact, and this is a problem (Coulmas 2013; Meyerhoff & Nagy 2008; Smakman 2015) because this cultural bias has led to cultural assumptions. It has also led to assumptions as to whether and how to incorporate culture methodo- logically in research. A field like Sociolinguistics, in which human behavioural tenden- cies are an important factor, is particularly sensitive to this. First, the nature of the problem is described. Then, an outline is given of the criteria that are often used to define culture. Some suggestions are then presented that may lead to an improvement of the situation; i.e. a ‘globalising’ Sociolinguistics that represents a broad range of cultures rather than a predominantly Western cultural concept. 10 СОЦИОЛИНГВИСТИКА Dick Smakman. Russian Journal of Linguistics, 2019, 23 (1), 9—22 2. THE NATURE OF THE PROBLEM Heavy codeswitching — usually by speakers who are highly multilingual — is often the language norm in daily communication, and this also does not fit the traditional model. There is often also a nativised standard language, which is an adapted western language (most notably English and French) that accommodates local meanings and habits and generally the cultural and linguistic needs of the local community (Kachru 1976). So, all in all, there are several parallel language norms in some non-Western countries, each of which fulfills a different function. While this situation is usually described as deviant, it is highly common and deserves its own place in norm-language theories. However, the only language-norm paradigm that has so far had any theoretical impact is the Western one, which gives other situations an aura of deviancy, and this obstructs efforts towards a broader and revised mainstream theory. Other examples of underlying assumptions that negatively affect the attention paid to non-Western situations can be found in Smakman and Heinrich (2015). This volume demonstrates how Western theoretical models are dominant to such a degree that they reduce non-Western communicative settings to near-oddities, while logically speaking, 11 SOCIOLINGUISTICS Дик Смакман. Вестник РУДН. Серия: ЛИНГВИСТИКА. 2019. Т. 23. № 1. С. 9—22 these setting should be part of a larger theoretical framework. The volume provides a string of examples from countries where Western paradigms do not fit. The importance of dance as a form of intergenerational communication in communities in Alaska is explained, as are the intricate workings of prestige in Saami communities in the far north of Europe. Knowledge of older traditions, values, and language forms are a source of prestige in both the Saami and Alaskan native communities, and thus it is hard to differentiate the knowledge about linguistic and non-linguistic things. Prestige of someone’s language lies not only in the actual language use but in the knowledge, actions, and experiences of the speaker. Politeness and gender roles in Tokyo are also described in this volume to demonstrate how assumptions hereon in mainstream dis- course are in fact incorrect. The global agreement amongst such urban and less urban communities is not part of any mainstream theory. The volume reveals that many of the situations that do not fit into mainstream paradigms at some level actually apply to very large groups of speakers. 2. THE NATURE OF THE PROBLEM Smakman (2015) tried to clarify the mechanisms behind the Western dominance in sociolinguistic theory-making by investigating academic output in the field. He took into consideration issues such as the likely cultural backgrounds of members of edi- torial boards and authors. He correlated these with population size, command of Eng- lish, and economic development of the country where the university of the editors and authors was. Smakman’s data were drawn over several decades and showed no tendency of improvement in the last three or four decades. His data showed that the dominance of Western researchers, and Anglowestern ones in particular, is obvious and easily visible: famous sociolinguists tend to be native speakers of English, journals are mainly run by academics working at Anglophone universities — most of whom will be native speakers of English and all of whom will be acting in line with the local cultural norms of academia —, and by far most internationally renowned introductory books are by Anglophone authors. Being a good speaker of English as a second language seems to correlate with the possibilities of getting research published as well. The correlation between publishing success and human development (place on the Human Development Index) of the country where an author’s university is based is even more striking, with by far most articles coming from affluent countries with western-based demo- cratic systems. Relatively less attention is paid to non-Anglowestern societies in the articles studied, but the contribution of data about these regions is nevertheless considerable (Smakman 2015). The good news is that although the areas with most language variation are understudied, the general attention paid to non-Western languages and cultures is nevertheless positive. However, because non-Anglophone languages and cultures are studied by a relatively high percentage of authors inspired by the Anglowestern models and modes of research, ideologies from this cultural realm will inevitably underlie the analyses and serve as a set of contrastive premises. This will be visible in the choice of research questions, variants of variables, and socio-psychological explanations of research findings. Offering respondents categories such as ‘man’/‘woman’ and ‘femi- nine’/‘masculine’ is problematic in countries where other genders are an established part of society. Western perceptions of age, too, are problematic. In Thai, age poses a chal- lenge if one wants to investigate its effects, because many personal pronouns in that 12 СОЦИОЛИНГВИСТИКА Dick Smakman. 2. THE NATURE OF THE PROBLEM Russian Journal of Linguistics, 2019, 23 (1), 9—22 language depend on relative rather than absolute age of interlocutors (Intachakra 2001), in addition to social and contextual factors, which necessitates determining age relative to others rather than just the speaker’s biological age. Cheshire (2005) explained how some communities attach more importance to rituals than actual age and how age-related categorisation of Xhosa men (southern Africa) is on the basis of initiation rituals. There- fore, applying Western perceptions of age and gender identity when researching these situations is obviously problematic. 3. CRITERIA TOWARDS CULTURAL CATEGORISATION Mainstream Sociolinguistics is mainly modelled on one specific type of socio- linguistic system, namely that which can be called ‘solidly modern societies’. Solidly modern societies in the Western world represent the material from which mainstream sociolinguistic theory was built. They, however, do not represent a ‘natural’ or ‘normal’ or ‘unproblematic’ type, but are also an expression of specific cultural settings. There exist many ways to be solidly modern. Different combinations of a bundle of features define modernity differently for various regions around the world (Eisenstadt 1973), which is why non-Western modernized settings may be very different from those depicted in mainstream Sociolinguistics (Greenberg 2015). Different types of societies must give rise to different types of sociolinguistic study. What gives structure to society needs to be taken into account first before specific sociolinguistic theories are applicable. The culture of societies thus needs to be defined. Many different models exist to define culture (some well-known ones are mentioned below), and they share certain criteria. Below are ten often used criteria to determine or describe culture that such models have yielded. 3.1. Region Regions are not always helpful in explaining why sociolinguistic systems differ. Regions have often been defined in an ad-hoc manner, usually for political rather than cultural reasons. It would be far-fetched to expect, for instance, that concepts such as ‘Southeast Asia’ or ‘the Slavic area’ could be employed to determine patterns of language use. The borders around regions cannot be drawn with any degree of objectivity. Region is nevertheless one of the most convenient and common ways of categorising culture, as it is generally agreed that in very broad terms, ethnicity, religion, and other obvious features correlate with region. Regional categorisations often reflect one of the region’s dominant ideologies, while ignoring other important ideologies that are equally native. Sociolinguistic differences cannot easily be generalised across regions. 3.2. Relationship with uncertainty The Uncertainty Avoidance dimension, as proposed by Hofstede (1980), refers to the way a society deals with uncertainties that the future may bring. Some societies are more controlling than others; they tend towards creating safety and security, while others take a more laissez-faire approach. This criterion will in particular be able to provide insight into power and gender relations, including the language choices of individuals and groups that stem from these. 13 SOCIOLINGUISTICS Дик Смакман. Вестник РУДН. Серия: ЛИНГВИСТИКА. 2019. Т. 23. № 1. С. 9—22 3.3. Invidividualism vs collectivism A distinction is often made between societies that focus more on the individual and their needs, and those in which groups are the main focus in communication. Typically, members of individualist societies focus on the interests of their families, friends, and themselves. In collectivist societies, on the other hand, loyalty within groups, e.g. professional ones, outside the family is more common. The group that one belongs to takes preference in deciding on obligations and responsibilities and loyalties, rather than one’s own or one’s family’s interests. Western societies are considered to be more individualistic in this sense, while Asian societies are stereotypically group-oriented. Morales, López-Sáez, and Vega (1998) critically addressed this cultural distinction, while Hofstede (1980) assumed this distinction to be a feasible criterion to distinguish between cultures. The workings of this criterion will present themselves in spoken, day-to-day discourse, with some speakers addressing the interlocutor’s face as well as a group’s face more than their own, while speakers from another culture give relatively much attention to their own face. 3.4. Economic/human development Halliday (2001) has convincingly argued that a different economic organisation of society must be expected to manifest syntactically, insisting that “major upheavals in human history are also linguistic upheavals” (180). He suggested a distinction between hunter-gatherer societies, settlement-pastoral communities, classic iron-age cultures, and cultures marked by the advancement of learning and modernity. Size, structure, density, composition, etc. of hunter-gatherer societies, agricultural societies, industrial societies, and post-industrial knowledge societies differ considerably, and, in accordance with these differences, society is regulated and maintained differently as well. Besides linguistic effects, this also has sociolinguistic effects. What is missing in Sociolinguistics so far is how cultures create different types of sociolinguistic systems as a result of different economic organisation of societies. The relatively recent economic changes in national economies (due to globalisation, digitisation, and technical advance- ment) will affect communication in various economies differently, and this needs more attention. Human development largely depends on economic structure but takes a broader approach, the idea being that wealth alone does not determine perceptions of happiness and well-being. The Human Development Index, as developed by the United Nations Development Programme (http://hdr.undp.org/en/content/human-development-index-hdi), can be used to this end. People’s worldviews change when they as individuals and the society around them undergo socio-economic development (Inglehart & Welzel 2004, 2005). The HDI combines three factors: length and happiness of one’s life, educational level, and standard of living. The latter largely reflects economic development. Countries can be placed on a cline with categories: ‘developing countries’, ‘less developed countries’, ‘developed countries’, and ‘unstable multilingual communities’. This aspect as a source of communication style has received relatively little attention. 14 СОЦИОЛИНГВИСТИКА Dick Smakman. Russian Journal of Linguistics, 2019, 23 (1), 9—22 3.6. High/low context To categorise cultures across the globe, the British polyglot Richard Lewis (1996) departed from the existing cultural model of High-context cultures and Low-context cultures (Hall 1959, 1976), which roughly refers to the overtness of information in communication. Lewis’ model charts countries triangularly in terms of the tendency towards Reactive, Linear-active, or Multi-active communication. Linear-active commu- nication is associated with an equal contribution of interlocutors to conversations, planned communication patterns, polite directness, goal-orientedness, factuality, re- strained body language, and attaching a strong role to the written word. Multi-active communication, on the other hand, involves relatively much talking, broad topic planning, confrontation, emotion, people-orientedness, placing feelings before facts, relationship-orientedness, importance of the spoken word, and unrestrained body language. Reactive patterns involve much listening, reacting to actions of the inter- locutor, mainly looking at general principles, polite and unconfrontational indirectness, people/harmony-orientedness, the assumption that statements are promises, and subtle body language. Besides addressing linguistic differences based on this phenomenon, Lewis hypothesised in detail about how culture affects communication practices within communities through the overtness of information. 3.7. Femininity/masculinity Cultures can be stereotypically masculine or feminine (Hofstede 1980; Hofstede Hofstede & Minkov 2010). Highly masculine societies are driven by competition, achievement, and success. High femininity refers to concerns about the lives of others and about the quality of life in general. Standing out in a crowd is not admirable in feminine societies. Although the stereotypical assumptions underlying this binary qualification may be perceived as Western and sexist in themselves, this division is often felt to exist and determine language choices across the globe. 3.5. Power relations Individuals and groups in societies are generally not equal, and societies have ways to express the way culture handles this difference (Hofstede 1980). Power distance reflects the acceptance of this difference within a society by its powerful and less powerful members and by all kinds of institutions and organisations. This does not only affect the language (like politeness forms) but it also directly affects the way speakers address each other (structure of use of politeness forms), and even whether they actually do. SOCIOLINGUISTICS 3.9. Tendency towards indulgence Another criterion to define culture is a society’s tendency to give in to indulgence (Hofstede, 1980). The idea is that some societies allow their members to indulge them- selves more (basically, enjoy themselves) while other societies aim more at the control and restraint of such natural urges. It should be clear that this aspect of culture will affect language use, in particular intonation and lexical patterns. 3.8. Conservative versus progressive cultures Some societies more than others maintain links with their own past. While conservative, traditional cultures are hesitant towards change and prefer to rely on existing truths and societal patterning, more progressive societies assume that the truth depends on the situation. Although generalisations should not be made, and cities in particular may often deviate from certain patterns, it could be suggested that North- 15 SOCIOLINGUISTICS Дик Смакман. Вестник РУДН. Серия: ЛИНГВИСТИКА. 2019. Т. 23. № 1. С. 9—22 West Europe is at the more progressive end of the scale, while, for instance, certain Eastern European and African countries may tend more towards more conservative/ traditional patterns of thought. Communicative patterns, especially lexical choices, may reveal this aspect of culture. Progressive manners of communication may involve lexical choices that reveal less sensitivity to social hierarchy. West Europe is at the more progressive end of the scale, while, for instance, certain Eastern European and African countries may tend more towards more conservative/ traditional patterns of thought. Communicative patterns, especially lexical choices, may reveal this aspect of culture. Progressive manners of communication may involve lexical choices that reveal less sensitivity to social hierarchy. 3.10. Cultural values Approaching culture through values entails capturing how people in various cultures value fundamental aspects in life such as religion, happiness, materialism, gender roles, freedom of choice, self-expression, tradition, and authority. The well-known World Values Survey (WVS 2015) has tried to do this. Halman et al. (2008) and Inglehart & Welzel (2004) subdivided cultures in the world into nine categories: (1) Confu- cian, (2) South Asia, (3) African-Islamic, (4) Latin America, (5) English-speaking, (6) Catholic Europe, (7) Protestant Europe, (8) Orthodox, and (9) Baltic. Countries were grouped schematically with other countries within one of these cultural patterns, and at the same time they were plotted on the basis of two value scales, namely ‘Survival—Self-Expression’ and ‘Traditional—Secular-Rational’. Traditional Values emphasize religion, ties between parent and child, respecting authority, and family values. Divorce, abortion, euthanasia, and suicide tend to be rejected in these societies. Nationalism is strong. The opposite of Traditional Values are Secular-Rational Values, which place less emphasis on traditional aspects such as religion and the family, and which are more liberal to euthanasia, divorce, and abortion. Survival Values place emphasis on physical and economic security, and are associated with ethnocentrism and low levels of tolerance and trust. The opposite of Survival Values, namely Self- Expression Values, emphasise liberal values like protection of the environment, tolerance towards foreigners and non-heterosexual people, and gender equality. A relatively high demand of individuals to participate in the decision-making of authorities is also part of this value. Where one’s culture is situated on these scales, affects relationships and, as a result, communication patterns. Several other authors have discussed human values that determine (perceptions of) culture. An important one is Schwartz (2012), who approached values in a less regional manner and presented a theory involving basic human values, including the nature of values, the features common to values, and what distinguishes one value from another. Schwartz distinguished ten basic personal values that are recognized across cultures. Schwartz’ idea was that values form a circular structure that reflects the motivations each value expresses. This circular structure captures the conflicts and compatibility among the ten values and is presented as potentially culturally universal. 16 СОЦИОЛИНГВИСТИКА Dick Smakman. Russian Journal of Linguistics, 2019, 23 (1), 9—22 3.11. Other investigations Other researchers have also made efforts to define and align criteria to determine cultures or, simply, describe cultures. Smith, Fischer, Vignoles, and Bond (2013) took a social semiotic approach, Wodak (2001) developed a gender-centred approach, and Fairclough (2003) revealed culture through critical discourse analysis. The body of research on culture, including considerable empirical evidence through large-scale surveys, as well as research on individuals shows the interest in fathoming the topic of culture, and this interest seems to be increasing in a globalised world. In such a world, cultural categories become more fluid, and this fluidity leads to more categories and more awareness that culture and individual are not connected in straightforward ways and that playing with culture by individuals is increasingly common. 4. THE CHALLENGES OF APPLYING CULTURE CRITERIA Western ideas about religion, authority, divorce, age, gender, or other factors underlie the research (Inglehart & Welzel 2004; Lewis 1996; WVS 2015). The underlying conceptualisation is thus again a Western one, i.e. Western ideas about religion, authority, divorce, age, gender, or other factors underlie the research (Inglehart & Welzel 2004; Lewis 1996; WVS 2015). These criteria tend to confuse culture with country and nation state (Greenberg 2015; Pennycook 2018). Given the fact that Sociolinguistics is ultimately about diversity, the use of models sweeping the existing multilingual and multicultural make-up of all societies around the world under the carpet is questionable. The views are static, placing given cultures in some place in the proposed grid. These cultures have, however, all arrived there at some point of time; they have a trajectory, and they are moving on to other places, too. The idea that society, culture, and language are dynamic should be part of any model. A final issue worth noting is that Westernisation and industrialisation have had their impacts on traditional societies, and many societies have found their own ways of incorporating these inevitable tendencies, making them part of their contemporary culture. Different cultures do this in very different ways. These two tendencies, which inevitably involve a degree of hybridisation, may to a degree hide the original culture, while communicative and other patterns arising from the original culture may be as alive as ever. 4. THE CHALLENGES OF APPLYING CULTURE CRITERIA Hofstede (n.d.) stated that “[w]e can <...> use such country scores based on the law of the big numbers.” However, he starts by saying that “[i]t may well be that the differ- ences among individuals in one country culture are bigger than the differences among all country cultures”. Indeed, there is tension between the idea of generalisability on the one hand, and individual/situational variation and communicational fluidity on the other. To make things even more complex, today most cultures are transforming under the influence of globalisation and self-reflection, giving way to what sociologists call ‘liquid modernity’ (Bauman 2000), as well as ‘late modernity’ or ‘reflexive modernity’ (Giddens 1991). Applying the above criteria (and others) in actual research is also problematic. There is no agreement on the nature and degree of impact of the criteria, and intercul- turally recognised cultural descriptions do not exist yet. To put it simplistically: all the criteria make sense, they all apply in some shape or form, but they tend to gener- alise, and they are often too subjective to be used in empirical research. Morales et al. (1998), for example, found that qualifying individuals as either ‘individualistic’ or ‘collectivist’ could not be used to predict politeness strategies they employ under var- ious circumstances. Those individuals have their own relationship with their cultures, and what is more, each individual is part of a collective, making a distinction between ‘individualistic’ and ‘collectivist’ societies a rather forced one. Lozerand (2015) expanded this argument and deconstructed ideas about ‘collectivist’ non-Western societies as an ‘Orientalist’ discourse of the 19th century, in which Western scholars departed from what they perceived to be physical resemblance of non-Western indivi- duals, projecting on these ‘similar looking people’ a high degree of ‘psychological resemblance’ as well. This then led them to conclude that the non-Western societies lacked ‘individual originality’ and were thus, in a word, ‘collectivist’. This idea was then uncritically reproduced in Western Oriental Studies, from which it found entry into other disciplines. Criteria are also used in culturally biased ways. Models that define cultural patterns tend to ignore the emic/etic differentiation. Indeed, concepts like ‘religion’, ‘authority’, ‘divorce’, ‘age’ or ‘gender’ mean something very different across societies in the world. 17 SOCIOLINGUISTICS Дик Смакман. Вестник РУДН. Серия: ЛИНГВИСТИКА. 2019. Т. 23. № 1. С. 9—22 The underlying conceptualisation is thus again a Western one, i.e. 5. SOLUTIONS The above issues do not merely apply to Linguistics or Sociolinguistics but to a broad range of disciplines. The focus here is on Sociolinguistics. Smakman (2015) and Smakman, Barasa, and Smith-Christmas (submitted) presented a number of practical solutions to the problem at hand. They suggested reconsidering the reviewing process for journals and critically addressing widespread norms of ‘good academic language’; see also Smakman and Duda-Osiewacz (2014). They also suggested that translation can be used as a tool; authors write in their native tongue, and a translator and the author together translate the text into English. Furthermore, authors can be found in different ways from what is currently the case; for instance, by reaching out to specific authors, rather than waiting for articles to be submitted. They furthermore addressed the issue of funding and of intercultural academic cooperation. Finally, they suggested that the selection of editors deserves attention, as well as the availability of articles and their dissemination. Such initiatives could contribute to cultural emancipation in sociolinguistic theorisation and growth of non-Western research. More publications on lesser known sociolinguistic settings obviously help globalise Sociolinguistics to a degree (Meyerhoff & Nagy 2008) and in the process help make lesser known cultures part of mainstream theory. One such effort is the volume by Stanford and Preston (2009), which focuses on under-represented minority communities; on descriptions of specific aspects of lesser-known sociolinguistic systems. Furthermore, the individual chapters in Bolton and Kwok (1992) described a linguistic phenomenon in a specific country or community. The Routledge Handbook of Sociolinguistics around the World (Ball 2010) dealt with larger areas and can thus be considered a good source of background information on sociolinguistic systems across the globe because it focuses on “notable features” (xviii) of regional and smaller-scale cultural settings. Smakman and Heinrich (2015, 2018) also placed lesser known sociolinguistic situations on the map. 18 СОЦИОЛИНГВИСТИКА Dick Smakman. Russian Journal of Linguistics, 2019, 23 (1), 9—22 An effort towards good practice that the author of this article has undertaken is Smakman and Barasa (2016). In this article, the different cultures of the authors (Dutch and Kenyan) led to a publication that has both Anglowestern and non-Anglowestern influences. They described the phenomenon of the ‘norm language’, which is often formalised and expressed through ‘standard’, ‘national’ or ‘official’ languages. 5. SOLUTIONS The approach was one of not taking the western standard language concept as a point of departure but looking at the phenomenon that it is the upshot of, namely the apparent need of peoples to form and maintain some sort of linguistic norm. Smakman and Barasa (2016) explained the relevant factors when defining the language; colonialism, multilingualism of individuals, the existence of parallel language norms (including a nativised standard), and the possible absence of an “exclusive” interpretation of the standard language; see also Smakman (2012). The solutions they offered were to separate the functions of the standard language, to distinguish between the spoken and written norm, to treat the “codeswitched” variety as a possible standard, and to detach the norm language from its automatically assumed reliance on prestige and power. That way, culture is viewed from multiple angles, which leads to a certain nuance and fewer generalisations. The way forward seems to be to consider new methodologies, and in particular be inspired by Linguistic Anthropology and Ethnography. These two disciplines have an edge over Sociolinguistics when it comes to culture-sensitive approaches, as they have a longer history of critically dealing with culture as a factor. This is due to the simple fact that these fields develop insights from within societies and communities rather than insights that are strongly influenced by the researchers’ backgrounds. Methodologically, research interpretation is viewed critically, and participant interpre- tation plays an important role. This requires long and intensive periods of fieldwork, observation and participation. It also requires awareness of one’s own cultural biases when interpreting data; our ‘conceptual lock’ (Gould 2000); “reality does not speak to us objectively, and no science can be free from constraints of psyche and society” (276); one needs to analyse one’s own cultural experiences when analysing those of others. Sociolinguistics compensates for the relative lack of scrutiny of individuals and smaller groups with elaborate and detailed theories and methodologies, including controlled circumstances in lab-like settings. A solution then is to involve sociolinguists more in ethnographic and anthropological research, and in longer-term research. The outcome would be an approach that does not depart from existing descriptions of situa- tions as an explanatory tool or from pre-determined variable-based research questions. Instead, the approach delves into the lowest, local, and most idiosyncratic cultural level, as well as the interpretation thereof by participants. REFERENCES Ball, M. J. (Ed.) (2010). The Routledge Handbook of Sociolinguistics around the World. London: Routledge. Bauman, Z. (2000). Liquid Modernity. Cambridge: Polity Press. 5. SOLUTIONS It subsequently compares observa- tions with research from other field studies and with existing cultural models. © Dick Smakman, 2019 19 SOCIOLINGUISTICS Дик Смакман. Вестник РУДН. Серия: ЛИНГВИСТИКА. 2019. Т. 23. № 1. С. 9—22 Bolton, K., & Kwok, H. (Eds.). (1992). Sociolinguistics Today; International Perspectives. London, New York Routledge. Coulmas, F. (2013). Sociolinguistics. The Study of Speakers’ Choices. Cambridge: Cambridge University Press. Eisenstadt, S. N. (1973). Tradition, Change and Modernity. New York: Wiley. Evans-Pritchard, E. E. (1951). Social Anthropology. London: Cohen & West Ltd. Fairclough, N. (2003). Political Correctness: The Politics of Culture and Language. Discourse and Society, 14(1), 17—28. Giddens, A. (1991). Modernity and self-identity. Self and society in the late modern age. Cambridge: Polity Press. Gould, S. J. (2000). Wonderful Life. The Burgess Shale and the Nature of History. New York: Vintage. Greenberg, M. L. (2015). The Slavic area. Trajectories, borders, centres and peripheries in the Second World. In D. Smakman & P. Heinrich (Eds.), Globalising Sociolinguistics. Challenging and Expanding Theory (pp. 164—177). London: Routledge. Hall, E. T. (1959). The Silent language. New York: Doubleday. Hall, E. T. (1959). The Silent language. New York: Doubleday. Hall, E. T. (1976). Beyond Culture. New York: Doubleday. Hall, E. T. (1976). Beyond Culture. New York: Doubleday. Halliday, M. A. K. (2001). New ways of meaning. The challenge to applied linguistics. In A. Fill & P. Mühlhäusler (Eds.), The Ecolinguistic Reader (pp. 175—202). London: Continuum. Halman, L., Inglehart, R. F., Díez-Medrano, J., Luijkx, R., Moreno, A., & Basáñez, M. (2008). Changing Values and Beliefs in 85 Countries. Trends from the Values Surveys from 1981 to 2004. Leiden: Brill. Hofstede, G. (1980). Culture's Consequences: International Differences in Work-Related Values. Beverly Hills CA: Sage Publications. Hofstede, G. (n.d.). Hofstede insights. Retrieved from https://www.hofstede-insights.com. Hofstede, G., Hofstede, G. J., & Minkov, M. (2010). Cultures and Organizations: Software of the Mind (3 ed.). USA: McGraw-Hill. Inglehart, R. F., & Welzel, C. (2004). What insights can multi-country surveys provide about people and societies? APSA Comparative Politics Newsletter, 15(2), 14—18. Inglehart, R. F., & Welzel, C. (2005). Modernization, Cultural Change, and Democracy: The Human Development Sequence. Cambridge: Cambridge University Press. Jenkins, J. (2009). World Englishes: a Resource Book for Students (2nd ed.). London: Routledge. Kachru, B. B. (1976). Models of English for The Third World: White Man's Linguistic Burden or Language Pragmatics? TESOL Quarterly, 10(2), 221—239. Lewis, R. D. (1996). When Cultures Collide. Leading across Cultures. 5. SOLUTIONS London: Nicholas Brealey International. Lozerand, E. (2015). Il n’y a pas d’individu au Japon. Critique et archeologie d’un stereotype. In C. Galan & J.-P. Giraud (Eds.), Indiviu-s et démocratie au Japon (pp. 19—71). Toulouse: Presses Universitaires du Midi. Meyerhoff, M., & Nagy, N. (2008). Social lives in language. Sociolinguistics and multilingual speech communities celebrating the work of Gillian Sankoff. Amsterdam / Philadelphia: John Benjamins. Morales, J. F., López-Sáez, M., & Vega, L. (1998). Discrimination and beliefs on discrimination in individualists and collectivists. In S. Worchel, J. F. Morales, D. Páez, & J.-C. Dechamps (Eds.), Social identity. International Perspectives (pp. 199—210). London / Thousand Oaks (Cali- fornia) / New Delhi: Sage Publications. 20 СОЦИОЛИНГВИСТИКА Dick Smakman. Russian Journal of Linguistics, 2019, 23 (1), 9—22 Pennycook, A. (2018). Posthumanist Applied Linguistics. Abingdon/New York: Routledge nnycook, A. (2018). Posthumanist Applied Linguistics. Abingdon/New York: Routledge. artz, S. H. (2012). An Overview of the Schwartz Theory of Basic Values. Online Readings. Psychology and Culture, 2(1). Smakman, D. (2012). The definition of the standard language: a survey in seven countries. Inter- national Journal of the Sociology of Language, 218, 25—85. Smakman, D. (2015). The westernising mechanisms in sociolinguistics. In D. Smakman & P. Heinrich (Eds.), Globalising Sociolinguistics. Challenging and Expanding Theory. London: Routledge. Smakman, D., & Barasa, S. N. (2016). Defining ‘Standard’. Towards a cross-cultural definition of the language norm. In I. Tieken-Boon van Ostade & C. Percy (Eds.), Prescription and Tradition in Language. Establishing Standards across Time and Space (pp. 23—38). Bristol / Buffalo / Toronto: Multilingual Matters. Smakman, D., Barasa, S. N., & Smith-Christmas, C. (submitted). Towards cultural diversification in Sociolinguistics. Smakman, D., & Duda-Osiewacz, A. (2014). A contrastive rhetoric analysis of scholarly publications by Polish and Anglophone authors. Journal of Language Teaching and Learning, 4(2), 29—47. Smakman, D., & Heinrich, P. (2015). Globalising Sociolinguistics. Challenging and Expanding Theory. London: Routledge. Smakman, D., & Heinrich, P. (Eds.). (2018). Urban Sociolinguistics. The City as a Linguistic Process and Experience. London: Routledge. Smith, P. B., Fischer, R., Vignoles, V. L., & Bond, M. H. (2013). Understanding social psychology across cultures. Engaging with others in a changing world. London: Sage. Stanford, J., & Preston, D. R. (2009). The Lure of a Distant Horizon. Variation in Indigenous Minority Languages. In J. Stanford & D. Preston (Eds.), Variation in Indigenous Minority Languages (pp. 1—20). Amsterdam: John Benjamins. Wodak, R. (2001). Gender and Language: Cultural Concerns. In N. J. 5. SOLUTIONS Smelser & P. B. Baltes (Eds.), International Encyclopedia of the Social & Behavioral Sciences (pp. 5954—5960). Oxford: Pergamon. WVS, W. V. S. (2015). Findings and Insights. Retrieved from http://www.worldvaluessurvey.org/ WVSContents.jsp. For citation: For citation: Smakman, Dick (2019). Cultural bias and Sociolinguistics. Russian Journal of Linguistics, 23 (1), 9—22. doi: 10.22363/2312-9182-2019-23-1-9-22. Article history: Received: 19 August 2018 Received: 19 August 2018 Revised: 07 October 2018 Revised: 07 October 2018 Accepted: 30 October 2018 Дата поступления в редакцию: 19 августа 2018 Дата поступления в редакцию: 19 августа 2018 Дата поступления в редакцию: 19 августа 2018 Дата принятия к печати: 30 октября 2018 Дата принятия к печати: 30 октября 2018 Для цитирования: Smakman, Dick. Cultural bias and Sociolinguistics // Вестник Российского университета дружбы народов. Серия: Лингвистика = Russian Journal of Linguistics. 2019. Т. 23. No 1. С. 9—22. doi: 10.22363/2312-9182-2019-23-1-9-22. 21 SOCIOLINGUISTICS SOCIOLINGUISTICS Дик Смакман. Вестник РУДН. Серия: ЛИНГВИСТИКА. 2019. Т. 23. № 1. С. 9—22 Bionote: DICK SMAKMAN, Ph.D., Lecturer at Leiden University Centre for Linguistics, Department of English. His research interests embrace sociolinguistics, phonetics, language learning and teaching, Dutch, English. He is a member of the editorial board of the International Journal of the Sociology of Language (IJSL). He publishes on the topics of sociolinguistics and the phonetics of English as a second language. Contact information: e-mail: d.smakman@hum.leidenuniv.nl Сведения об авторе: ДИК СМАКМАН, Центр лингвистики Лейденского университета, г. Лейден, Нидерланды. Сфера научных интересов — социолингвистика, фонетика, изучение и преподавание языков, голландский и английский языки. Член редакционного совета научного журнала International Journal of the Sociology of Language (IJSL). Имеет публикации по проблемам социолингвистики и по преподаванию фонетики английского языка как иностранного. Контактная информация: e-mail: d.smakman@hum.leidenuniv.nl Контактная информация: e-mail: d.smakman@hum.leidenuniv.nl
W2601572606.txt	null	en		Spatial dialectics and the geography of social movements: the case of Occupy London	Transactions of the Institute of British Geographers/Transactions - Institute of British Geographers	2,017	cc-by	11,213	Spatial dialectics and the geography of social movements: the case of Occupy London Sam Halvorsen This paper develops spatial dialectics as an analytical method capable of exposing and explaining the contradictions, dilemmas and tensions that cut through the spatialities of social movements. Despite scholarly recognition of internal divides in movements such as Occupy, there is greater need to conceptualise the inherently contradictory nature of social movements, in particular by reflecting on the role of spatiality. Building on recent work on multiple spatialities of activism, the paper shifts attention to contradiction as a key factor in spatial mobilisation, further arguing that the recent turn to assemblage thought is ill equipped for such a task. Dialectics is introduced via Bertell Ollman’s influential account of its ontological and epistemological bases, before turning to Edward Soja’s reading of Henri Lefebvre to incorporate spatiality. Spatial dialectics disrupts the linearity of thesis–antithesis–synthesis, placing contradictions not only within the historical unfolding of relations but also within co-dependent yet antagonistic moments of space, through Lefebvre’s ‘trialetic’ of perceived, conceived and lived space. Building on ‘militant research’, which combined a seven-month ethnography, 43 in-depth interviews and analyses of representations of space, spatial dialectics is put to work through the analysis of three specific contradictions in Occupy London’s spatial strategies: a global movement that became tied to the physical space of occupation; a prefigurative space engulfed by internal hierarchies; and a grassroots territorial strategy that was subsumed into logics of dominant territorial institutions. In each case, Occupy London’s spatial strategies are explained in the context of unfolding contradictions in conceived, perceived and lived spaces and the subsequent dilemmas and shifts in spatial strategy this led to. In conclusion, the paper highlights broader lessons for social movements’ spatial praxis generated through the analysis of Occupy London. Key words research spatial dialectics; social movements; contradiction; multiple spatialities; Occupy London; militant Department of Geography, University of Cambridge, Cambridge CB2 3EN Email: sth31@cam.ac.uk Revised manuscript received 2 November 2016 Introduction 2011 was a remarkable year for social movements, with ‘Occupy’ setting up protest camps in hundreds of North American cities in September before going global on 15 October, the day it took root in London. Like other movements of 2011, Occupy London was an explosion of rage and hope (Castells 2012) that sought to critique the systemic failures of neoliberal capitalism and develop working alternatives for a more just and democratic society. As the movement took form in the protest camp, however, internal contradictions became clearly visible, contradictions that cut through the multiple spatialities through which the movement mobilised and developed, and which played an important role in the initial demise of Occupy London.1 First, the seemingly global movement that claimed to represent the ‘99 per cent’ became increasingly tied to the physical space of occupation. Second, Occupy sought to prefigure a space for horizontal organisation yet hierarchies developed around class and gender. Third, the attempt to pursue a progressive territorial strategy of occupation was subsumed within the practices of dominant territorial institutions. How can these contradictions be explained and what was their wider significance? I argue that dialectics is a useful conceptual framework for analysing phenomena such as Occupy London and explaining contradictions in their spatial praxis. The dialectical concept of contradiction – a mutually supportive yet undermining relationship – has been relatively underexplored in analyses of social movements compared with studies of capital (e.g. Harvey 2014). Social movement literature often highlights The information, practices and views in this article are those of the author(s) and do not necessarily reflect the opinion of the Royal Geographical Society (with IBG). ISSN 0020-2754 Citation: 2017 doi: 10.1111/tran.12179 © 2017 The Author. Transactions of the Institute of British Geographers published by John Wiley & Sons Ltd on behalf of Royal Geographical Society (with the Institute of British Geographers). 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. Sam Halvorsen 2 internal tensions and dilemmas, including recent work on Occupy (Juris et al. 2012; Rohgalf 2013; Sbicca and Perdue 2014). Yet there has been less attempt to analyse the inherently contradictory nature of social movement mobilisation and the implications this has for their spatial strategy. Recent theorising of social movement spatialities implicitly points in this direction (Miller 2013; Nicholls 2009) but stops short at elaborating an explicitly dialectical approach, and the latest turn to assemblage analysis risks downplaying the antagonistic nature of spatial mobilisation (Davies 2012; McFarlane 2009). While helpful for understanding how people and things come together to allow a social movement to work (or not), assemblage thought is ill equipped for explaining contradiction and abstracting to broader historical and geographical moments. Integrating dialectical and spatial analyses of social movements provides a framework for understanding how and why contradictions inevitably develop in the course of mobilising particular spatial strategies. The paper makes a specific contribution in the burgeoning literature on the geography of social movements by developing spatial dialectics as an analytical method through which scholars can expose and make sense of the contradictions, dilemmas and tensions that cut through the spatialities of social movements, abstracting from the vantage points of both temporality and spatiality. Building on Soja’s (1996) reading of Henri Lefebvre, spatial dialectics provides a means for grappling with the simultaneity of spatial contradiction, disrupting the linearity of thesis–antithesis–synthesis and placing contradictions not only within the historical unfolding of supportive yet undermining relationships (e.g. changing relation between activists and the state) but also within co-dependent yet antagonistic moments of space, through Lefebvre’s ‘trialectic’ of perceived, conceived and lived space. In developing spatial dialectics, this paper takes forward recent attempts to develop frameworks for analysing multiple spatialities (Jessop et al. 2008; Leitner et al. 2008; Miller 2013) by exploring how and why contradictions develop among different spatial moments and the implications this has on social movement strategy. After outlining an understanding of dialectics, drawing in particular on Bertell Ollman, the following section builds on Edward Soja to spatialise dialectics as a method for analysing social movement geography. The remainder of the paper puts spatial dialectics to work in an analysis of three cuts of Occupy London’s spatial praxis, organised around the contradictions described in the opening paragraph. Across these cuts, the paper draws out how contradictions unfold in the course of spatial mobilisation, considering their broader historical and geographical moments. The conclusion pulls out wider lessons on the contradictory nature of social movement spatiality. Dialectics Dialectics is a mode of thinking that seeks to engage with and understand a world in flux that can be traced through Western thought at least to the ancient Greeks (Ollman and Smith 2008). Much contemporary use refers to dialectics in the philosophy by Hegel – who presented an idealist understanding of history as a process of constantly resolving contradictions – and in particular following Marx’s materialist re-reading which sought to expose the ‘contradictions inherent in the movement of capitalist society’ (Marx 1976, 103). Although Marx rarely discussed dialectics in any detail – his 1857 Introduction being the prime example (Marx 1973, 100–9) – there have been ongoing attempts to develop his ‘method’ since then (e.g. Jameson 2009; Lefebvre 2009; Lukacs 1971), including in human geography since the late 1960s (Castree 1996; Harvey 1996). Bertell Ollman (2003) has provided one of the most detailed works on Marx’s method and his account of the ontological and epistemological moments of dialectics provides a useful starting point. Following Ollman (1976 2003), Marx’s dialectical ontology is a ‘philosophy of internal relations’ in which all ‘things’ are conceived of as relations. Unlike the ‘common sense’ view of relations as ‘spatio-temporal ties’ with other things, Ollman (2003, 36) highlights that Marx interiorised relations in things (for example, the class relation in the commodity form). Dialectics dissolves the apparent independence of parts and wholes and instead ‘views the whole as the structured interdependence of its parts’ (Ollman 2003, 140; see also Harvey 1996, 48–55). Examining the internal relation of any part thus brings to light the whole, something often understood as ‘totality’ in which ‘all social phenomena change constantly in the course of their ceaseless dialectical interactions with each other’ (Lukacs 1971, 13). In an unstable world of flux and flow, relations are both mutually supporting and undermining and are thus contradictory: ‘undoubtedly the most important’ aspect of Marx’s relational ontology (Ollman 2003, 17). This is because contradiction allows Marx to avoid a static and one-sided view of capitalism and to understand change as immanent to capitalist development (Harvey 2014; Ollman 2003). Marx’s dialectics provides no synthesis, as some readings of Hegel imply, but demonstrates the immanent ‘power of the negative’ that sets in motion a ‘going beyond’ (Lefebvre 1968, 6), a constant ‘movement of breaking and opening’ (Holloway et al. 2009, 8). If internal relations are key to Marx’s dialectical ontology, then it is the process of abstraction that is central to his epistemology (Ollman 2003). In his 1857 Introduction, Marx describes the ‘method of political economy’ as a movement of abstracting from the ‘real concrete’ – our initial ‘chaotic conception of the ISSN 0020-2754 Citation: 2017 doi: 10.1111/tran.12179 © 2017 The Author. Transactions of the Institute of British Geographers published by John Wiley & Sons Ltd on behalf of Royal Geographical Society (with the Institute of British Geographers). Spatial dialectics and social movements whole’ – towards ever simpler determinations, or ‘thinner abstractions’, in order to finally arrive at a ‘rich totality of many determinations and relations’ (1973, 100–1). Ollman summarises abstraction as an ‘intellectual activity of breaking’ the world as it appears to us into ‘mental units with which we think about it’ (2003, 60). Abstraction takes place at different levels of extension (the spatial and temporal boundaries used in analysis), levels of generality (from the most unique to general characteristics of any entity) and vantage points (the perspective from which to view other components, e.g. the workers or capitalists) (Ollman 2003). This allows dialectics to expose and understand the existence of change and interaction within any form or relation, thus bringing to life the contradictory movement of social relations, at once mutually supporting and undermining. Alongside Marxist understandings of dialectical analysis that operate at the level of generality of the capitalist mode of production (Ollman 2003), focusing on how social relations tend to internalise contradictions of capital (see Harvey 2014), dialectics also provides a broader framework for analysing how and why contradictory social relations arise and develop in the course of human organisation and interaction. The historical work of Mann (1986) is particularly helpful for pointing towards social power, rather than capital, as a basis for dialectical analysis. As he summarises: In pursuit of their goals, humans enter into cooperative, collective power relations with one another. But in implementing collective goals, social organization and a division of labor are set up. (1986, 6–7) These divisions can lead to core contradictions in social organisation, with social groups creating the conditions for their own domination. Through abstraction, dialectics helps make sense of how particular contradictory social relations develop and unfold historically, rather than naturally pre-exist. Yet contradictory social relations do not unfold on the head of a pin (Miller and Martin 2000) and incorporating spatiality into dialectics has significant implications for how the method is put to work. Spatial dialectics and social movements Despite David Harvey’s longstanding effort to insert geography into historical materialism (and vice-versa) (see Harvey 1973; Sheppard 2006), it has been Edward Soja, via his reading of Lefebvre, who has done most in developing a spatialised dialectic. Harvey has long taken the view that dialectics is at the core of Marx’s method (see especially Harvey 1996) and has sought to apply this approach to his analysis of space-time (most clearly achieved in Harvey 2006). Yet Soja (1996) extends this and, building on Lefebvre (1976 1991), 3 argues for the spatialisation of the dialectic itself, inserting a third term into the duality of historicalitysociality. In a first step, Soja (1980) re-iterated Lefebvre’s argument that the production of space and society is a co-constitutive dialectic, thus clarifying concerns of Harvey (1973) and Castells (1977) that Lefebvre was seemingly fetishising space as an autonomous field with a capacity to externally shape human activity. This concern has been carefully revisited by Charnock (2014, 318), who holds that those who follow Lefebvre risk being ‘lost in space’ which, at worst, could lead to an ‘affirmative notion of emancipatory politics’ of the sort that led to the fetishisation of space in many Occupy camps. Yet Soja’s reading of Lefebvre develops a ‘socio-spatial dialectic’ in which space is neither a ‘separate structure with its own autonomous laws’ nor a mere ‘expression of the . . . social (i.e. aspatial) relations of production’, but ‘simultaneously social and spatial’ (1980, 208). In a second step, Soja (1996) develops the notion of trialectics to incorporate spatiality, historicality and sociality as three moments of the dialectic. Incorporating spatiality has consequences for dialectical analysis. Spatial dialectics does not deny that tensions and contradictions unfold historically in society but disrupts a linear view of social change. Space is the realm of simultaneity (Massey 2005, 9) and the spatiality of contradictory relations intersects and interrupts the historical moment of social organisation. Specifically, Soja (1996) opens up the spatiality of trialectics into Lefebvre’s seminal triad of spatial moments: material spatial practice (perceived space); representations of space (conceived space) and spaces of representation (lived space). This provides a clear framework from which to insert spatiality into dialectical analyses, acknowledging how historical social relations unfold through contradictions in moments of space. Lefebvre’s spatial triad has informed much work in human geography (see Harvey 2006) and has started to be applied to social movement analysis (e.g. Garmany 2008; Uitermark 2004). Miller (2013) recently integrated Lefebvre’s triad with discussions on multiple spatialities of activism, providing an excellent starting point for a spatial dialectical analysis of social movements. This paper embraces such an approach but makes the explicitly dialectical move to explore how contradictions arise and unfold within and between different spatial moments of activism, thus drawing out the inherent tensions of spatial strategies. Spatial dialectical analysis acknowledges, and seeks to explain, the contradictions that develop in social movements as they mobilise across space, something that the recent turn to assemblage analyses of social movement spatiality fails to do. Assemblage is a broad term used by geographers to emphasise ‘gathering, coherence and dispersion’ of ‘heterogeneous elements ISSN 0020-2754 Citation: 2017 doi: 10.1111/tran.12179 © 2017 The Author. Transactions of the Institute of British Geographers published by John Wiley & Sons Ltd on behalf of Royal Geographical Society (with the Institute of British Geographers). Sam Halvorsen 4 that may be human or non-human, organic and inorganic, technical and natural’ (Anderson and McFarlane 2011, 124). Like dialectics, assemblage provides a relational approach orientated towards the co-constitution of wholes and parts (Sheppard 2008). Assemblage has been useful to studies of social movements by moving away from tendencies to reify spatialities (e.g. network, territory) and ground them in the specific contexts through which they are produced (Davies 2012). Moreover, assemblage has expanded understandings of agency, theorising it as diffuse and distributed across multiple actants and processes involved in social movements (McFarlane 2011). This has led to richer empirical accounts of social movement spatiality. Yet assemblage thought is much less useful for understanding and explaining contradictions in social movement spatialities and risks downplaying their inherently contradictory nature. Assemblage thinking is oriented towards researching how different components are gathered and assembled (or ‘plugged and ‘unplugged’) into provisional unities that allow for some form of transformation, with little interest in exploring antagonism or abstracting to historical and spatial moments beyond the assemblage (see Anderson et al. 2012; McFarlane 2011). Assemblages focus attention on ‘latent possibilities’ that emerge not from antagonistic relations but through the ‘lines of flight’ that are generated in the process of assembling (McFarlane 2011, 211), and reject any underlying contradictions that guide social processes (Brenner et al. 2011). Moreover, assemblages’ ontology of ‘relations of exteriority’ (Anderson et al. 2012, 177) refuses to abstract beyond the surface appearances of what Marx (1973, 100) would term the ‘chaotic conceptions’ gathered in rich empirical data. Assemblage thought is thus ontologically and epistemologically ill equipped to understand and explain the contradictions of social movement spatiality. Spatial dialectics takes forward research on social movement spatiality by explicitly outlining an approach that explains how and why tensions develop in spatial strategy. There is now a wealth of studies demonstrating the significance of particular spatialities, such as place (Routledge 1993), scale (Miller 2000) and networks (Featherstone 2008), for social movement mobilisation, with recent work examining the coimplication of multiple spatialities to contentious politics (Jessop et al. 2008; Leitner et al. 2008; Nicholls et al. 2013). Across this work are examples of dilemmas appearing in spatial strategies, for example how networking is distorted by the weight of particular nodes (people or places) (Routledge 2008) or how right to the city movements fall into a ‘local trap’ (Purcell 2006). Research on urban social movements has been most effective at drawing out tensions in activists’ spatial praxis, highlighting systemic contradictions at play in the city (Cox 2001; Harvey 2012; Miller and Nicholls 2013; Mitchell 2003; Nicholls 2011). Spatial dialectics takes this forward by exposing the inherently contradictory nature of social movement spatialities. Specifically, it directs attention to how contradictions unfold in the context of both historical and geographical moments of social movements, examining how spatial strategies involve mutually supportive yet undermining relations that require activists to shift priorities between spatialities, in turn creating further dilemmas. This provides critical perspectives on the success and failure of social movements. Researching Occupy London Understanding the dilemmas facing Occupy London was a key concern for my research at both a theoretical and political level. Methodologically, I built on a rich tradition of militant research, which I understand as ‘a committed and intense process of internal reflection from within particular struggle(s) that seeks to map out and discuss underlying antagonisms while pushing the movement forward’ (Halvorsen 2015a, 469). Through my active involvement, I generated a close understanding of Occupy London and the challenges that arose in its spatial strategies, largely tied to the occupation of space in the ‘public’ courtyard of a Cathedral in London’s financial district. Formally, this involved a seven-month period of ‘engaged ethnography’ (Mathers and Novelli 2007) along with 43 in-depth interviews with fellow occupiers (all names have been changed) and an analysis of representations of space (e.g. media, legal) produced by Occupy London. The research spanned August 2011 to June 2012, covering the initial, camp-based phase of Occupy and the immediate posteviction period in which many occupiers began to generate new spatial strategies. My positionality – intimately tied up with internal experiences and debates in the movement – was such that taking an explicitly dialectical approach to my research analysis was a helpful, if challenging, move. There is a risk with militant research that the activistresearcher becomes so committed to the particular ‘situation’ of struggle (Colectivo Situaciones 2003) that they become blind to the broader totality of struggles within which it is located (Halvorsen 2015a). Dialectics forces a confrontation with the inevitably partial and limited context of a struggle (something that assemblage approaches also risk forgetting) and encourages a more critical stance. There remains a challenge in building a relationship between the moments of analysis and praxis, although this is not unique to dialectics and a fuller discussion lies beyond this paper (see Kindon et al. 2007; Shukaitis and Graeber 2007). Spatial dialectics also provides a useful approach for analysing Occupy by moving beyond partial accounts of ISSN 0020-2754 Citation: 2017 doi: 10.1111/tran.12179 © 2017 The Author. Transactions of the Institute of British Geographers published by John Wiley & Sons Ltd on behalf of Royal Geographical Society (with the Institute of British Geographers). Spatial dialectics and social movements internal activist divides. Much research has pointed out the development of internal tensions within Occupy movements, often explained by the limitations of particular forms, such as the tendency of non-hierarchal assemblies to be dominated by participants able to attend hours of meetings (Lupo 2014; Smith and Glidden 2012), or the ways in which particular racial or ethnic power relations manifested themselves in regional contexts (Khatib et al. 2012; Razsa and Kurnik 2012). Spatial dialectics moves a step further to highlight the inherently contradictory nature of social movement mobilisation, pointing to tensions that arise when mobilising across particular spatialities and the contradictory spatial strategies developed in response. Spatial dialectics thus counters a tendency to romanticise forms of resistance (Sparke 2008) and focuses attention on the contradictions of all spatial praxis. In what follows I present a spatial dialectic analysis of Occupy London organised around three cuts. Each cut draws out a core contradiction in Occupy London’s spatial strategy that my research exposed: a global movement tied to a physical space of occupation; a prefigurative space engulfed by internal hierarchies; and a grassroots territorial strategy that was subsumed into logics of dominant territorial institutions. In examining distinct spatial strategies, these cuts speak to recent work on the multiple spatialities of activism (Leitner et al. 2008; Nicholls et al. 2013) incorporating ‘autonomous geographies’ (Pickerill and Chatterton 2006) as an increasingly important spatiality (or set of spatialities) of creative resistance that is often marginalised by a focus on contention in social movements (cf. Clough and Blumberg 2012; Springer et al. 2012). Most importantly, these cuts give the reader an insight into how spatial dialectics might be put to work in social movement analysis. Networks and the protest camp Occupy London was mobilised as part of a global movement seeking to simultaneously challenge capitalist institutions (e.g. the Stock Exchange) and create working alternatives. On 15 October 2011, approximately 3000 people gathered outside St Paul’s Cathedral following a callout to ‘Occupy the London Stock Exchange’, setting up a protest camp that lasted four and a half months, followed by a second, smaller camp and a number of buildings that were occupied during the period leading up to May 2012. During this period, the ‘space of representation’ of Occupy’s networks – a global movement seeking radical structural change – became increasingly tied and trapped to the place of protest (i.e. camp), which acted as a barrier to occupiers’ perceived and conceived spaces of activism, limiting the movement’s capacity to identify and mobilise across more topological connections. 5 Occupy London appeared as part of a global lived space of social change through the desires and imaginations that resonated across hundreds of cities worldwide in 2011. Some occupiers spoke of direct inspiration from what was happening elsewhere. As occupier Jonny said: ‘I saw Occupy Wall Street and that had implanted in me that desire to just be involved’. Putting up camp on 15 October, occupiers produced representations of space that identified with extensive networks – from the universal ‘we are the 99%’ to specific solidarity with Tahrir Square and others – that informed occupiers’ sense of radical possibility.2 Yet just days into the occupation it was clear that Occupy London’s lived space was radically shrinking, getting trapped by the place-based strategy of the protest camp. When I interviewed Juan, one of the few occupiers who maintained a commitment to building relations with social movements from the global south, which he did through a solidarity information tent he constructed on camp, he told me of the lack of support and interest he received on camp: people just weren’t that interested . . . and not many people stopped and looked at [the tent], it was really very difficult, people just had this attitude of like we are already at the occupation, we are already doing what is necessary and what you’re talking about is just slightly unnecessary, it was a surplus requirement . . . Juan’s frustration was not isolated and my own participation in the International working group demonstrated a similar trend. While a small number of occupiers (largely international themselves) attempted to build networks for communication and, potentially, organisation with movements elsewhere, there was little time, energy or enthusiasm for developing this initiative in the camp’s general assemblies and it soon fizzled out. Instead, the priorities of many occupiers were determined by the spatial form they had created, demonstrating a socio-spatial dialectic in which the form of occupation was in turn shaping, and constraining, the social movement itself (Soja 1980). This narrowing of Occupy London’s lived space of contention was produced through its strategic prioritising of place-based spatial practices combined with the subsequent fetishisation of the conceived space of the protest camp. Occupy’s prioritisation of the protest camp, an intensive place-based strategy of resistance and creation (Feigenbaum et al. 2013), can be understood from the vantage points of on-going contradictions in capitalist production and social movement organisation. Capital’s contradiction between circulating and fixed capital not only leads to economic crises (Harvey 1982) but opens up political opportunities for social movements to turn territorial capitalist institutions into ISSN 0020-2754 Citation: 2017 doi: 10.1111/tran.12179 © 2017 The Author. Transactions of the Institute of British Geographers published by John Wiley & Sons Ltd on behalf of Royal Geographical Society (with the Institute of British Geographers). Sam Halvorsen 6 fixed ‘target spaces’ (Sparke 2012). Aside from targeting the Stock Exchange, occupiers used the occupation to expose the City of London Corporation’s (CoLC) role in providing an un-regulated tax haven to multinational corporations (see Shaxson 2012) with a working group created for that purpose. Just weeks since London’s largest riot of a generation, occupiers saw a political opportunity in channelling popular rage onto a fixed target articulated ‘in the face of the financial system’.3 One occupier, Rachel, who lived in the neighbourhood where the riots started, framed this as her motivation for joining Occupy: I found it really dramatic to think that we had created a generation of people that were so disempowered and dispossessed and didn’t even know who the enemy was . . . From an organisational perspective, Occupy sought to resolve a tension in the previous alter-globalisation movements that, by mobilising across extensive, horizontal networks, prevented themselves from growing long-term roots. As alter-globalisation veteran Naomi Klein put it when speaking at Occupy Wall Street: ‘we chose summits as our targets . . . Summits are transient by nature, they only last a week. That made us transient too. We’d appear, grab world headlines, then disappear’ (2011, np). Following the 2008 crisis and subsequent politics of austerity, there was a lack of grassroots institutional basis from which to mobilise (in the UK, but also elsewhere) and Occupy’s open-ended occupations filled this void. Social movements build networks through fixity in place and extensive relations strung across space (Nicholls 2009), and activist networks may be both topological and topographical (Routledge and Cumbers 2009). At any point in time, however, activists may emphasise more fixed or mobile forms and this may produce contradictions that inhibit a movement’s capacity for social change (see also Schrader and Wachsmuth 2012). The protest camp provided a placebased spatial strategy that was appropriate for its historical moment, opening up a new repertoire of contention. Yet, by emphasising fixed, topographical networks, it posed a tension with Occupy’s topologically conceived spaces and limited the capacity of its spatial practices. Thus, the attempt of small numbers of well-resourced ‘imagineers’ (Routledge 2008), many with experience from alter-globalisation movements, to build alliances and networks at multiple scales was hindered by a lack of interest or capacity from the majority of occupiers to follow through on this work and general assemblies were predominantly oriented to producing, representing and defending the protest camp. Many occupiers in London, as elsewhere, were fetishising the protest camp (Halvorsen 2017; Hammond 2013; Marcuse 2011; Miller and Nicholls 2013), embodying their identity and normative ideals in the physical form of the occupation (Juris 2012). Paul, who camped at St Paul’s, expressed this to me: some people got lost, they couldn’t see the woods from the trees, and all they could see was the camp, and didn’t see any wider purpose . . . so that was one of the problems, you get a group of people for who the camp is an end in itself. This narrowing of perspective had a critical effect on the production of occupiers’ lived space that shifted away from the global moment of 15 October 2011 towards an affective commitment to the place of the camp itself as the basis for creating social change. This fetishisation of the camp posed dilemmas for Occupy London, both leading to internal hierarchies as I explore below, but also posing strategic limitations. First, although creating a place-based target space generated some media coverage of financial and political inequality and occupiers were invited to dialogue with some financial institutions based in the City of London, Occupy London had little contact or impact on material spatial practices of power and governance that are nested at more expansive scales and require concerted spatial strategies (cf. Miller 2000; Nicholls 2011). Second, the reluctance to mobilise across topological social movement networks limited the resources and reach of Occupy London’s spatial practices, which, as it became increasingly inward looking, led many to lose touch and feel alienated from the movement’s overarching aims, with visibly dwindling numbers of occupiers over the months. These issues were partially resolved following the eviction at St Paul’s on 28 February 2012, reopening occupiers’ lived space to extensive networks. As one (ex-)occupier, Tania, told me ‘quite shortly after the eviction I started feeling really very relieved that we could finally put our energies into other things other than the camp’. Indeed, the post-camp months saw an explosion of energy into re-building Occupy’s global network, resulting in the project ‘Occupy May’ that saw two global days of action, on 12 and 15 May, to coincide with movements in North America and Europe. Nevertheless, although some sub-groups of occupiers remained active over subsequent months and years, in many cases the strong-tie bonds developed in the protest camp were lost and activists struggled to define their lived space, presenting a new set of strategic dilemmas. Autonomous geographies and internal hierarchies Occupy London, as elsewhere, represented a disillusionment with capitalist institutions and liberal democracy, especially in the wake of the 2008 crisis, and a desire to prefigure non-hierarchical forms of organising ISSN 0020-2754 Citation: 2017 doi: 10.1111/tran.12179 © 2017 The Author. Transactions of the Institute of British Geographers published by John Wiley & Sons Ltd on behalf of Royal Geographical Society (with the Institute of British Geographers). Spatial dialectics and social movements (Graeber 2013; Sitrin and Azzellini 2014; Vasudevan 2015a). As its first public statement read: ‘The current system is unsustainable. It is undemocratic and unjust. We need alternatives; this is where we work towards them’.4 Key to Occupy’s spatial strategy was attempts to forge what Pickerill and Chatterton term ‘autonomous geographies’: relational spaces of resistance and creation ‘where people desire to constitute non-capitalist, egalitarian and solidaristic forms of political, social, and economic organisation’ (2006, 1). These are lived spaces of dreams for better worlds (Pickerill and Chatterton 2006), but depend heavily on perceived (materially practised) and conceived (representations) spaces to sustain them, and it is here that the contradictions arose for Occupy London. Many occupiers were mobilised by an alienation from mainstream politics and its failure to generate a lived space for its strategy of austerity. Martha, who camped at St Paul’s from 15 October, told me: I didn’t know anything about politics to be honest. The only thing I knew, like a lot of people, is like the government is shit basically, they are mugging us over . . . This reflected the government’s decision not to directly address people’s everyday needs, spending £500 billion to bailout the banks while cutting welfare and social spending (e.g. tripling of university tuition fees, capping benefit payments, cuts to disability support). Austerity measures had a particularly negative impact at a local level, with basic services often being withdrawn (e.g. child care, library provision) and neighbourhood communities asked to rebuild their support networks through voluntarism and enterprise under the banner of the Conservative’s ‘Big Society’ (CLES 2014; Featherstone et al. 2012). This crisis of welfare in austerity Britain (Brown et al. 2013) provided a political opportunity for Occupy London to create a counter-space of representation (lived space) for the so-called 99 per cent to develop an inclusive, democratic self-managed community.5 Occupy London’s proposal to forge a ‘real-andimagined’ (Soja 1996) counter-space in central London led to an influx of people seeking refuge in a placebased community, generating tensions in occupiers’ spatial practices. Rocia, an ex-addict who dedicated her time on camp to looking after the vulnerable, told me how many occupiers, including the homeless and people suffering from mental health issues and drug addiction, perceived the camp as a welcoming space of care: ‘they thought Occupy was a more loving environment that didn’t involve so much living around total chaos, it was more positive generally’. Occupy London’s limited resources were quickly stretched by the ongoing labour of social reproduction and care, posing a dilemma for occupiers’ perceived space. Paul, who was involved in the Outreach working group, told me: 7 A lot of the people there really didn’t know why we were there and had no idea, they were just there, and it is fair enough if people want to live, if homeless people want to live there, they’re free to do so, but I think we sort of got distracted a lot by, we weren’t focusing on the movement . . . Paul hints at a division between the spatial practices of living and social reproduction and those of doing activism in ‘the movement’. This division soon led to clear hierarchies, as Jane, who camped at St Paul’s, described: There was such a division between physical and organizational, you know theoretical labour at the camp, and you know there was a real lack of respect for people who didn’t express themselves in an intellectual language and a real readiness to allow the people who were professionals and professional talkers . . . to determine what the message of Occupy was going to be, and so that was a really deep problem. The ‘professional’ group of Occupiers drew on extensive networks beyond the camp (e.g. with journalists and academics), and were able to create representations of space that had wide reach. Moreover, many of them lived off site and did not rely on the material space of the camp (although their representations of space remained tied to the spaces of occupation). In contrast, those occupiers maintaining Occupy’s material space of living had little reach beyond the place of the camp and were often marginalised in meetings, lacking both time and skills to participate. Moreover, most of them lived and relied on the camp’s infrastructure. This division became spatially constituted in the camp and generated a visible class divide. Simon, who did shifts in the Info Tent but lived off site, described this to me: There were two camps in a way, there was a class divide I always felt, there was the middle-class at the Info Tent and the working-class were at the kitchen end, which was quite an interesting thing to see and I hardly went up that top end . . . I would describe them as upper and lower LSX . . . one was more the intellectual space and one more the living space maybe . . . The ‘upper’ camp was located on the main road near the Cathedral’s entrance, providing the visible, public face to Occupy, where most visitors would gather, while the ‘lower’ camp was tucked away behind the Cathedral, hidden from immediate public view, thus posing additional tensions in occupiers’ differing capacities to create spaces of representation.6 In response, several weeks into occupation, the ‘lower’ camp began to confront dominant spaces of representation, intervening, at times violently, in general assemblies, rejecting the often complex, intellectualised process of consensus decision-making, demanding greater access to resources (e.g. media, finance) and criticising the small group of occupiers creating the space of representation. ISSN 0020-2754 Citation: 2017 doi: 10.1111/tran.12179 © 2017 The Author. Transactions of the Institute of British Geographers published by John Wiley & Sons Ltd on behalf of Royal Geographical Society (with the Institute of British Geographers). Sam Halvorsen 8 This eventually led key working groups from the ‘upper’ camp, such as media and finance, to meet off camp, detaching themselves from the material production of Occupy’s space. Despite the same desire to create a lived space of autonomy, Occupy was being pulled apart and becoming an unpleasant space. Simon summarised the transition in his relation to Occupy’s lived space: ‘I used be proud of being part of Occupy London and that slowly dissipated such that now I am embarrassed’. The tensions in the spatiality of conceived and perceived spaces also took on a gendered dimension, with a small group of women assuming key tasks of welfare and domestic labour. Maria, from the welfare working group, told me: welfare is the hardest work, it’s the work that nobody wanted to do . . . they were just busy, they had other stuff to do, everyone was fighting for a better world, and you know that’s how these things get missed. Similarly, Miranda, from recycling and waste, told me she was often brought to tears due to exhaustion and lack of support for work that many ‘didn’t find sexy enough or revolutionary enough to actually care about’. The burden of spatial practices of social reproduction fell on a small group of women who also suffered the insecurity of looking after vulnerable people in an open space in central London. In response, female occupiers sought to construct their own spaces of representation and mobilise resources through extra-local networks. For example, a callout was made for professional welfare support on camp, providing much needed resources, and a women’s network of occupiers developed across the different camps in the UK, tapping into the skills and experiences of established feminist networks (e.g. Global Women’s Strike). Although these strategies enhanced the resources of gendered spatial practices, they did little to address the underlying tensions in material spatial practices. Occupy London relied on a spatial division of labour in its perceived and conceived space, posing a core dilemma in its aim to prefigure an alternative lived space of community. Miller and Nicholls (2013, 467) highlight this ‘flaw’ in protest camp strategy, and point to the unrealistic requirement that occupiers drastically alter their everyday spatial activity. In Occupy London the burden of material spatial practice took on class and gendered dimensions due to the differing capacity of occupiers to mobilise resources and representations of space beyond the place of occupation. In the post-camp phase, Occupy London sought to address this contradiction, in part by working with neighbourhood-based organisations that have had greater success at building lasting networks of support, integrated into the spatial practices of a community’s everyday life (e.g. child care sharing), a spatial strategy that also developed in movements such as Occupy Wall Street and the indignados (Miller and Nicholls 2013; Moreno-Caballud and Sitrin 2011). In part, this shift is a question of scale and the need for social movements to address the disjuncture between what Cox (1998) terms ‘spaces of dependence’ and ‘spaces of engagement’ (see also Miller 2000). It also involves a transformation in occupiers’ lived spaces, away from the prefigurative place of the protest camp and towards spatialities more rooted in everyday life. This highlights the constraints on ‘real-and-imagined’ thirdspaces that, although they may appear as strategic priorities (Soja 1996), rely on tensions in perceived and conceived spaces. Shifts in spatial strategy towards neighbourhood organising are unlikely to resolve underlying contradictions, however, and present new dilemmas, such as how much of the state’s crisis of social reproduction should be absorbed by community activists. Spatial dialectics points towards no synthesis, only unfolding strategic dilemmas in spatial praxis. Territoriality and power Occupation is a popular territorial strategy of social movements, controlling demarcated space to both resist (e.g. privatisation) and create (e.g. new subjectivities and values). Although Anglophone human geographers have largely examined territoriality as the top-down exertion of power over space, following Sack’s (1986) seminal definition, there is growing acknowledgement that territoriality is also produced from below by social movements (Routledge 2015; Vasudevan 2015b), presenting overlapping (Agnew and Oslender 2013) and clashing (Zibechi 2012) territorial claims to that of the state and dominant institutions. How the relationship between these two territorial logics unfolds is decisive in determining the outcomes of social movement occupations. This relation also poses a tension in the lived space of occupation, torn between the legitimacy of bottom-up autonomous claims on space and the state’s sovereign rights. The territorial strategies of occupiers and dominant institutions will vary depending on particular circumstances of legitimacy. In the first night of Occupy London, when eviction looked possible, support from the (soon to be ex-) canon of St Paul’s Cathedral, which owned part of the land being occupied, was crucial in delegitimising the use of state violence. Instead, occupiers entered a lengthy legal battle with the City of London Corporation (CoLC), who also owned part of the occupied land, and this became the central battleground in the lived space of Occupy London’s territorial strategy. In turn, however, this opened up contradictions in the perceived and conceived moments of Occupy’s territoriality, posing dilemmas for the movement. Occupy London’s territorial strategy involved both a legal defence of the occupation and also the broader ISSN 0020-2754 Citation: 2017 doi: 10.1111/tran.12179 © 2017 The Author. Transactions of the Institute of British Geographers published by John Wiley & Sons Ltd on behalf of Royal Geographical Society (with the Institute of British Geographers). Spatial dialectics and social movements contestation of the territoriality of private property, asserting the right to produce overlapping material practices and representations of territory that challenge exclusive ideas of sovereignty and develop self-managed forms of governance. On 17 November 2011, the CoLC initiated proceedings for possession and injunctions through the High Court, pinning notices on tents around camp. Although a few occupiers ignored the notices, refusing to enter into any negotiation, the majority of occupiers realised that negotiation was essential, both in terms of defending the camp and also the potential to set legal precedents for future protest camps. As occupier Rachel told me: there was always a possibility that the law could be stretched and changed and in that case I don’t think that we could have walked away because it would have been ignoring the possibility of changing something. Over the coming days, the legal working group developed its strategy, bringing together expert lawyers with experienced activists. Camp meetings were increasingly taken up by discussions of legalities, and from late November until the end of occupation groups of occupiers went to court each day, watching the dock proceedings or standing in solidarity outside, often gaining media attention. In this way Occupy London’s production of territoriality became gradually entangled with the state-legal apparatus and the CoLC’s territorial claims. Although there was a sense by occupiers that this relationship had a potential for social change – both in terms of the legal precedents and allowing the protest camp to continue – there was less reflection on how power relations were unfolding on camp through the production of territoriality. In order to produce territoriality from below – opening up space to diverse encounters and new social relations, including experiments with consensus decision-making and popular education (see Halvorsen 2015b) – Occupy London was obliged to internalise dominant territorial material practices and representations. Although mobilised in opposition to hierarchical decision-making, occupiers had to develop a territorial organisation capable of representing itself in court. The format of the high court meant that one named defendant had to be nominated (although others could participate), providing a challenge to Occupy London’s claim to leaderless organisation.7 Martha, who became the sole defendant, told me it was basically a situation where had we not have had a named defendant we wouldn’t even get a chance to say a bloody word in our defence . . . and in which case they would have just written the order and we would have been gone. Despite doing her utmost to represent discussions from general assemblies in court, this was inevitably an impossible task, given the diversity of opinions on 9 camp. There was thus a clash between Occupy’s desire to represent its territoriality through consensus decision-making and the need for a clear and coherent legal argument to be pursued in court. This tension was confounded by the heavy reliance of occupiers on the experience and knowledge of a small group of lawyers and activists. The technical-legal language of court was difficult to translate on camp and participating in the legal battle required a level of commitment that most occupiers did not have time for. It soon became clear that Occupy London’s territorial strategy was dominated by the material practices and representation of space of a small group of well-resourced and experienced individuals with limited means for broader participation. As the legal case gained momentum, some occupiers became concerned that the occupation was being managed through ‘control points’. Simon reflected on this to me: suddenly these control points started to appear . . . the fact that we fought the court case without any actual real discussion about those things, these kind of clandestine decisions made on the behalf of people who hadn’t been part of it . . . The legal battle presented a particular contradiction for Occupy because, no matter how transparent and open the legal working group attempted to be, there was a daily need (in the court room) to operate within the dominant institutional apparatus and ‘political technologies’ of state territoriality (Elden 2013). Similar tensions have been highlighted in research on squatters movements, noting both a tendency for activists to become institutionalised within hierarchical territorial structures (Martınez 2013) and dominant groups to form around those with what Kadir (2010) terms ‘squatters’ capital’: skills and experience of building occupations. In Occupy, the seemingly external logic of top-down territoriality was gradually internalised in camp through mundane material practices (e.g. occupiers were required to remove several tents to create passages in case of fire emergencies) and conditions imposed on its representations of space (e.g. horizontal decision-making embedded in a vertical, scalar structure). This led to a noticeable shift in the lived space being produced through Occupy’s territorial strategy, from prefigurative claims of self-governance and the counter-territoriality of occupation towards a space of negotiation and lobbying, using the court case to push for new legal precedents on public space occupation (which were largely unsuccessful). This led to a rift within occupiers, with some leaving the movement around the time the court case got underway and others staying involved but increasingly concerned about the direction the movement was headed. Ultimately, an eviction notice was served and the St Paul’s camp was cleared, by which stage the occupation had come to ISSN 0020-2754 Citation: 2017 doi: 10.1111/tran.12179 © 2017 The Author. Transactions of the Institute of British Geographers published by John Wiley & Sons Ltd on behalf of Royal Geographical Society (with the Institute of British Geographers). 10 signify quite different things to different occupiers, making on-going organisation increasingly challenging post-camp. Occupy London’s production of territoriality was an inherently contradictory process, torn between two logics that can be understood within Lefebvre’s (1991, 165) dialectic of appropriation-domination, two ‘opposite and inseparable’ processes. This is not the same as what Allen terms an ‘oppositional rhetoric’ in which ‘power is something that can be identified, pushed back, interrupted’ (2003, 186). Dialectical thinkers such as Lefebvre never think of domination as ‘from above and “over there”’ (Allen 2003, 186), but something generated internally through spatial practice and representation. Territorial strategies typically involve building a relation with overlapping and clashing territorialities and this leads to dilemmas and contradictions. Inevitably, activist territorialities become engaged in relations with the state and activists have to think carefully about how to organise their material practices and representations of space in that context, taking into account how this may alter the lived space of the movement. Post 2011, several occupation-based movements have gradually moved towards a politics of territorial institutional engagement and, in some cases, active participation. This is most clearly the case in the Spanish radical municipal movements and the Podemos party, which developed in part from the indignados, while the grassroots mobilisation within the Labour party indicates a possible move in this direction in the UK, although the links to Occupy are less direct. Such shifts in territorial strategy are unlikely to remove the contradictions of activist territoriality, but will pose a different set of dilemmas and strategic challenges, as the contemporary experiences of Latin American social movements indicate (see Rossi and B€ ulow 2015). Conclusion This paper has argued that spatial dialectics provides an analytical approach for understanding how and why contradictions develop within social movements’ spatial praxis. By abstracting to Occupy London’s historical and geographical moments it has demonstrated how in pursuing particular spatial strategies occupiers created tensions that undermined their original aims and goals, posing contradictions that led to new spatial strategies. As Harvey recently summarised, contradictions are not inherently ‘bad’ and can be crucial for progressive change, yet they do ‘have the nasty habit of not being resolved but merely moved around’ (2014, 4). Spatial dialectics demonstrates that contradictions do not only unfold historically but also geographically, thus moving away from a teleological understanding of social movements that has long dominated sociological approaches (Tarrow 1998; Tilly 2004; cf. Miller 2000; Sam Halvorsen Nicholls 2011). Building on recent attempts to operationalise multiple spatialities as an analytical approach (Miller 2013), this paper shifts attention to contradiction as a key factor in spatial mobilisation. It argued that the recent turn to assemblage thought is illequipped for such a task and there is greater need to confront the inherently antagonistic nature of social movement spatiality. I end by reiterating and learning from the key contradictions of Occupy London. First, movements must make strategic decisions between mobilising networks across extensive topological spaces and intensive topographic places. Although territorial and relational networks are not mutually exclusive (Nicholls 2009; Routledge and Cumbers 2009), activists must nevertheless make decisions about how to invest the limited resources in their spatial practices and what representations of space to prioritise. These framings will be influenced by and in turn shape the lived space of networks. As Nicholls (2011) argues, the mobilisation of social movement networks often requires intensive placebased relations, which in turn generates a spatial unevenness between powerful cores and weaker peripheries, leading to antagonism and fragmentation among activists. A key concern of Occupy was the tendency for occupiers to fetishise the protest camp, holding its global networks hostage to place and causing a rift in occupiers’ lived experience. It is noticeable that most 2011 camp-based movements have not reproduced protest camps in subsequent years. Yet spatial dialectics highlights that contradictions will not be resolved teleologically. Constant attention must be given to the ways in which spatial forms of activism co-constitute wider networks, the ‘unevenness’ of social movement space (Nicholls 2011) and the subsequent tensions in the conceived, perceived and lived spaces of mobilisation. Second, strategies to produce ‘autonomous geographies’ require divisions in material spatial practices that may pose contradictions in the lived space being produced. In particular, place-based projects to build autonomous community require a difficult balance between spatial practices of social reproduction and intellectual work of ‘activism’. This tension has long plagued autonomous spaces such as squats (Kadir 2010; Vasudevan 2015a) and protest camps (Feigenbaum et al. 2013), which often struggle to resolve it. A key consideration is how the spatial form of protest coincides, or not, with everyday spatialities of social reproduction (Miller and Nicholls 2013) and the differing capacity of social groups (e.g. class, gender) to negotiate and represent such divides. Social movements may respond with spatial strategies that scale-up or network their perceived and conceived spaces of prefigurative organisation, but this is likely to shift contradictions to new spatialities rather than resolve them. ISSN 0020-2754 Citation: 2017 doi: 10.1111/tran.12179 © 2017 The Author. Transactions of the Institute of British Geographers published by John Wiley & Sons Ltd on behalf of Royal Geographical Society (with the Institute of British Geographers). Spatial dialectics and social movements Third, the production of territoriality by social movements has to develop a strategy for relating to dominant territorial claims, tied to the state, which poses dilemmas in the lived space of occupation, torn between the legitimacy of two opposed yet entangled logics. The spatial practices and representations of activist territoriality are obliged to develop an organisational form capable of negotiating power with dominant institutions. Occupy London’s territorial strategy relied on a subgroup of experts. While this allowed other occupiers to continue their territorial activism, it led to a clear hierarchy and concerns over ‘control points’, gradually subsuming dominant territorial claims into Occupy’s lived space. The territorial strategies of social movements may involve more or less direct confrontation or involvement in dominant institutional practices, but the spatial dialectics of appropriation-domination presents an on-going contradiction in grassroots struggles for counter-spaces (Lefebvre 1991). Spatial dialectics directs attention to the unfolding of social movements’ contradictory spatial practices and the case study presented here only gives a snapshot of particular dilemmas that developed in the protestcamp phase of mobilisation. Despite recognition of the many divides and tensions that cut through Occupy (e.g. Juris et al. 2012), there is a greater need to appreciate the inherently contradictory nature of social movements, in particular by reflecting on the role that spatiality plays in their mobilisation. The growing interest in the multiple spatialities of contention (Nicholls et al. 2013) can be further developed by explicitly acknowledging how the spatial strategies of social movements more often than not plant the seeds of their own demise, at minimum requiring a change in spatial priority while at times leading to de-mobilisation. Contradictions are not confined to the abstract space of capitalist production but are inevitable to any grassroots production of space (Lefebvre 1991). In the quest to change the world, social movements appropriate space and this creates tensions between how space is practised, imagined and directly lived, placing them in what Lefebvre terms ‘the dialectical relationship between “possible and “impossible”’ (1991, 60). The radical potential implied in movements such as Occupy can only be fully appreciated once these tensions are brought to the surface and analysed. Acknowledgements Many thanks to Andrew Davies, Jason Dittmer, Alan Ingram, Tariq Jazeel and Judith Ryser for comments on earlier drafts and in particular to Pooya Ghoddousi for many stimulating conversations. I owe a debt of gratitude to four anonymous reviewers for their detailed comments that greatly improved the final version and to the editorial guidance of Gavin Bridge, 11 Robyn Dowling and Fiona Nash. Any errors are mine alone. This work was supported by the Economic and Social Research Council (grant: S/J500185/). Notes 1 Activists continue mobilising under the banner of Occupy London at the time of writing (http://occupylondon.org.uk/ Accessed 1 November 2016). 2 See initial statement and global democracy statement (http://occupylondon.org.uk/about-2/ Accessed 1 November 2016). 3 http://occupylondon.org.uk/occupylsx-callout-lets-do-this/ Accessed 1 November 2016. 4 http://occupylondon.org.uk/about-2/ Accessed 1 November 2016. 5 See initial statement and other public declarations by Occupy London (http://occupylondon.org.uk/about-2/ Accessed 1 November 2016). 6 Similar spatial divisions of labour were also noted in other camps, see Franck and Huang (2012). 7 http://occupylondon.org.uk/about/statements/statementof-autonomy/ Accessed 1 November 2016. 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https://openalex.org/W2900994094	https://www.matec-conferences.org/articles/matecconf/pdf/2018/86/matecconf_icct2018_02007.pdf	English	null	Application of SVM Optimized by IPSO in Rolling Bearing Fault Diagnosis	MATEC web of conferences	2,018	cc-by	5,058	1 Introduction can be obtained by nonlinear mapping.From the computational geometry, the hyperplane is understood, and the maximum geometric interval is equivalent to the minimization . Rolling bearing is an important part of rotating machinery. Its running state directly affects whether the whole equipment can work normally. Once failure occurs, it will cause a chain of destruction and bring unnecessary economic loss[1~3]. When the sample set is linear and inseparable, the idea of "soft spacing" is introduced[9], that is, the existence of a small number of outlines is allowed, relaxation variables 0  i are often added, and a penalty factor C is required, which represents the degree of tolerance of outliers. It is one of the important parameters that affect the classification performance of SVM.as shown in the following: However, the selection of SVM parameters (such as penalty parameter C and kernel parameter g) has a great influence on the classification effect. Therefore, some scholars apply intelligent search algorithm to find the optimal parameters of support vector machine[4~7]. To sum up, this paper proposes a new classifier based on improved particle swarm optimization to optimize SVM parameters, making it play an advantage in rolling bearing fault diagnosis          N i b x w y t s C w i T N i i , ,2,1 , 1 ) ( . 2 1 min 1 2    (1) Application of SVM Optimized by IPSO in Rolling Bearing Fault Diagnosis Mingzhu Lv 1,2 , Xiaoming Su1 and Shixun Liu3 Mingzhu Lv 1,2 , Xiaoming Su1 and Shixun Liu3 1Mechanical Engineering Department, Shenyang University of Technology, Shenyang, China 2Automatic Control Department, Liaoning Equipment Manufacturing Professional Technology Institute, Shenyang, China 3CQC North Laboratory, Shenyang, China 1Mechanical Engineering Department, Shenyang University of Technology, Shenyang, China 2Automatic Control Department, Liaoning Equipment Manufacturing Professional Technology Institute, Shenyang, Chin 3CQC North Laboratory, Shenyang, China Abstract. Aiming at the problem that the classification effect of support vector machine (SVM) is not satisfactory due to improper selection of penalty factor C and kernel parameter g, this paper proposes a new modified classifier that uses the improved particle swarm optimization (IPSO) to optimize the parameter of SVM (IPSO-SVM) by introducing the dynamic inertia weight, global neighborhood search, population shrinkage factor and particle mutation probability. The classification result is tested by Common data sets named BreastTissue、Heart and Wine from the Libsvm toolbox, the results show that IPSO-SVM classifier is obviously superior to SVM and PSO-SVM classifier in terms of prediction accuracy and classification time．Then it is applied to the fault diagnosis in two classification problems and multiple classification problems of rolling bearings. The simulation results show that the IPSO-SVM classifier has stronger global convergence ability and faster convergence speed, and the ideal classification results can be obtained. Finally, the IPSO-SVM classifier is used to diagnose the fault of the actual bearing. The results show that the classifier has a better classification stability and is worthy of further promotion in engineering field. 2 Optimization of SVM parameters using improved Particle Swarm Optimization algorithm (1) The common method to solve the linear inseparability problem is to map a low-dimensional sample set to a high-dimensional sample set, which is called kernel function. The commonly used kernel functions include polynomial kernel function, radial basis kernel function, multilayer perceptual neuron kernel function and so on. By introducing kernel ) ( x x k  MATEC Web of Conferences 227, 02007 (2018) ICCT 2018 MATEC Web of Conferences 227, 02007 (2018) ICCT 2018 https://doi.org/10.1051/matecconf/201822702007 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). 2.2 IPSO algorithm The paper proposes a IPSO algorithm to optimize the parameters of SVM. The algorithm is based on the traditional basic particle swarm optimization (PSO) algorithm, which introduces dynamic inertia weight, global neighborhood search, population shrinkage factor and particle mutation factor to accelerate the convergence speed and improve the search accuracy. It effectively solves the problem that PSO algorithm is easy to fall into local optimum. The velocity and the position of the particles at the next iteration (k) are calculated in terms of the values at current iteration (k-1) as follows: 2.Considering the existence of better particles near the neighborhood of global and local optimal particles, a small perturbation is added to the global and local optimal solutions by using the method of global domain search, as shown by formula (8).In this way, the algorithm can be avoided from falling into local optimum, and its ability to develop the solution space can be improved. ) ) 1( ( ] ) 1( [ ) ( ) 1 ( 2 2 )1 ( 1 1 ) 1 ( ) (             k ij gj k ij ij k ij k ij x rand p r c x rand p r c v k v    (8) (8) ) ( ) ( ) 1 ( 2 2 ) 1 ( 1 1 ) 1 ( ) (         k ij gj k ij ij k ij k ij x p r c x p r c v v  (4) k ij k ij k ij v x x   ) 1 ( (5) ) ( ) ( ) 1 ( 2 2 ) 1 ( 1 1 ) 1 ( ) (         k ij gj k ij ij k ij k ij x p r c x p r c v v  (4) where is called a disturbance.a large number of experiments show that if 01 .0   can effectively improve the performance of individual optimization and global optimization. (4) k ij k ij k ij v x x   ) 1 ( (5) 3.The population contraction factor is introduced. 2.1 SVM basic theory In the initial stage of search, it is possible for inertia weigh to take a larger value in order to enhance the global search ability, and in the later stage of search, it is very possible for inertia weigh to take a smaller value in order to improve the ability of searching for local space solution. where ) ( rand is the random numbers in the interval [0.0,1.0],k is the current number of iterations, K is the maximum number of iterations (set at the beginning), 9.0 max   4.0 min   Reference [11] has confirmed the (3) where ) ( rand is the random numbers in the interval [0.0,1.0],k is the current number of iterations, K is the maximum number of iterations (set at the beginning), 9.0 max   , 4.0 min   .Reference [11] has confirmed the law of variation with the number of iterations. In the initial stage of search, it is possible for inertia weigh to take a larger value in order to enhance the global search ability, and in the later stage of search, it is very possible for inertia weigh to take a smaller value in order to improve the ability of searching for local space solution. where g is the kernel parameter, which represents the width of the kernel function, is another important parameter affecting the classification performance of SVM. Therefore, in order to ensure the good performance of SVM classifier, appropriate penalty factors C and kernel parameters g must be selected. 2.1 SVM basic theory The core idea of SVM is to maximize the spacing between categories, giving them greater credibility and generalization, and which data points near the border are called support vectors[8].SVM is usually designed as a two-classifier to find a positive class and a negative class to segment the sample. The optimal hyperplane functions ) , ( j i x x k and Lagrange multipliers i , we write the formula (1) as a dual form: MATEC Web of Conferences 227, 02007 (2018) ICCT 2018 MATEC Web of Conferences 227, 02007 (2018) https://doi.org/10.1051/matecconf/201822702007      x x k y y N i j i j i N j i ) , ( 2 1 min 1 1    exponential function and a random number distributed in the interval [0.0,1.0].such as formula (6) and (7).                  N i C y t s x x k y y i N i j i N i i j i j i N i j i , ,2,1 , 0,0 . ) , ( 2 1 min 1 1 1       exponential function and a random number distributed in the interval [0.0,1.0].such as formula (6) and (7). rand k m         max min max ) (     (6) 95 .0 95 .0 2 2   K k m (7) rand k m         max min max ) (     (6) (2) (6) It has been proved in reference [10] that the radial basis function has better universality, and its expression is: 95 .0 95 .0 2 2   K k m (7) (7)   j i x x g j i e x x k    , (3)   j i x x g j i e x x k    , (3) where ) ( rand is the random numbers in the interval [0.0,1.0],k is the current number of iterations, K is the maximum number of iterations (set at the beginning), 9.0 max   , 4.0 min   .Reference [11] has confirmed the law of variation with the number of iterations. 2.3 Proof of algorithm 4.Inspired by the idea of genetic algorithm mutation, the particle is initialized with a probability of 10% after each update of the speed and position, which expands the search space, which not only keeps the particle activity but also ensures the diversity of the population. It effectively avoids the precocity of the algorithm. Three groups of data sets, Breast Tissue, Heart and Wine, are selected from the Libsvm common data sets as simulation samples to verify the classification effect of the improved classifier IPSO-SVM in MATLAB R2013a. The Breast Tissue data set is the result of an analysis of breast cancer diagnosis. Is one of the data sets commonly used in machine learning literature. The Heart data set is a comparison of two different heart disease patterns. The Wine data set is the result of chemical analysis of three different varieties of wine in the same region of Italy. In summary, the steps of the IPSO-SVM algorithm are summarized as follows:  Parameter initialization.Suppose the population size, learning factors 1c and 2c , iteration times K , maximum and minimum of inertia weight, the disturbance size, The population contraction factor, parameter C and g . The range of parameter C is [0.1 100], and the range of parameter g is [0.011 1000].In the particle swarm optimization algorithm, the population size of PSO- SVM and IPSO-SVM is 20, the evolutionary algebra is  The position and velocity at which the initial particle is randomly generated. The fitness of each particle is calculated, the individual extreme value and the global extreme value are sorted out, and the average fitness of each generation population is calculated. 200 times, the learning factors 1c and 2c are 1.5 and 1.7 respectively, the maximum inertia weight is 0.9, the minimum is 0.4, the shrinkage factor is 0.7298, and the probability of individual variation is 10.The classification accuracy and time of these three classifiers for Breast Tissue, Heart and Wine data sets are shown in Table 1.  Update particle velocity and position information. The velocity of each particle is updated according to formula (6)~(8), and the position of particle is updated according to formula (9) ~ (10), and then the adaptive mutation operation of particle is performed with a probability of 10%.  Test termination conditions. Determine whether to reach the maximum number of iterations (or reach Table 1. 2.2 IPSO algorithm Thus 7298 .0   , the following population location update formula is available: where l is the equilibrium factors of "diffusion" and "attraction" operations . The experimental results show that the population diversity can be maintained properly  Input the optimal parameters C and g into the SVM classifier. The training data set has the best feature, and then applies to the test data set.  Input the optimal parameters C and g into the SVM classifier. The training data set has the best feature, and then applies to the test data set. and effectively when 1.4  l . Thus 7298 .0   , the following population location update formula is available:  The test results of fault classification are obtained, and the performance index of the results is analyzed. ) ( ) 1 ( ) ( k ij k ij k ij v x x     (10) (10) 2.2 IPSO algorithm In the process of operation of the algorithm, if the diversity of the population decreases gradually, the population does not approach the global optimal position any more, but moves away from the location one after another, which is equivalent to performing the "diffusion" operation, and when the diversity of the population increases gradually, The population began to move closer to the overall optimal position, that is, the "attraction" operation was performed [12].Therefore, it is necessary to introduce a contraction factor to ensure the convergence of the algorithm. The calculation expression of the shrinkage factor is shown in the formula(9). where i is the index of particle, j is the index of position in particle, ω is called the “inertia weight” that controls the impact of the previous velocity of the particle on its current one.is the velocity of the i th particle in swarm on j th index of position in p k ijv article, and k ijx is the position. 1r and 2r are the random numbers uniformly distributed in the interval [0.0,1.0]. 1c and 2c are self-learning factors and social learning factors, respectively, which are called “acceleration coefficients”. In this paper, the basic particle swarm optimization (PSO) algorithm is improved in the following four aspects, and a new algorithm(IPSO algorithm) is formed. l l l 4 2 2 2      (9) (9) 1.By changing the original fixed inertia weight and introducing a dynamic velocity inertia weight, It is defined as the form of the product between an 2 MATEC Web of Conferences 227, 02007 (2018) ICCT 2018 MATEC Web of Conferences 227, 02007 (2018) https://doi.org/10.1051/matecconf/201822702007 the precision requirements), if so, output the optimal parameters C and g , otherwise return to step b.  Input the optimal parameters C and g into the SVM classifier. The training data set has the best feature, and then applies to the test data set. the precision requirements), if so, output the optimal parameters C and g , otherwise return to step b. the precision requirements), if so, output the optimal parameters C and g , otherwise return to step b. where l is the equilibrium factors of "diffusion" and "attraction" operations . The experimental results show that the population diversity can be maintained properly and effectively when 1.4  l . 2.3 Proof of algorithm Classifying results of 3 classifiers on Breast Tissue, Heart and Wine data sets Classifier Type Breast Tissue Heart Wine Prediction Accuracy（%） Classification Time（s) Prediction Accuracy（%） Classification Time（s) Prediction Accuracy（%） Classification Time（s) SVM 61.5385 91.60 74.2857 170.70 96.6292 47.12 PSO-SVM 57.6923 20.13 78.5714 47.60 97.7528 15.44 IPSO-SVM 73.0769 14.01 85.7143 47.59 100 13.49 As can be seen from Table 1, IPSO-SVM classifiers have superior classification performance for three data sets with different sample dimensions and different sample totals. Compared with the other two classifiers, the IPSO-SVM classifier has the highest prediction accuracy and the shortest calculation time. 3. Application of IPSO-SVM classerfier in fault diangosis of rolling bearing As shown in Figure 1 , the flow chart of the IPSO-SVM classifier in the fault diagnosis of rolling bearing mainly includes data acquisition , feature extraction , division data , parameter optimization and category prediction . Table 1. Classifying results of 3 classifiers on Breast Tissue, Heart and Wine data sets Classifier Type Breast Tissue Heart Wine Prediction Accuracy（%） Classification Time（s) Prediction Accuracy（%） Classification Time（s) Prediction Accuracy（%） Classification Time（s) SVM 61.5385 91.60 74.2857 170.70 96.6292 47.12 PSO-SVM 57.6923 20.13 78.5714 47.60 97.7528 15.44 IPSO-SVM 73.0769 14.01 85.7143 47.59 100 13.49 Table 1. Classifying results of 3 classifiers on Breast Tissue, Heart and Wine data sets As can be seen from Table 1, IPSO-SVM classifiers have superior classification performance for three data sets with different sample dimensions and different sample totals. Compared with the other two classifiers, the IPSO-SVM classifier has the highest prediction accuracy and the shortest calculation time. 3. Application of IPSO-SVM classerfier in fault diangosis of rolling bearing 4 Fault Diagnosis Results of Rolling Bearings Classifier Type Prediction Accuracy （%） Classification Time（s) Best C Best g SVM 55 118.21 1.6713 0.1000 PSO-SVM 95 24.80 1.6752 0.1000 IPSO-SVM 97.5 16.75 6.7207 0.1000 0 20 40 60 80 100 120 1 1.5 2 2.5 3 3.5 4 Test Set Sample Number Test set Sample Class Comparison of SVM prediction results of test set with RBF kernel function accuracy = 55% True Category Forecast Category Figure 2. Prediction accuracy of SVM classifier 3 3.5 4 e Class Comparison of PSO-SVM prediction results of test set with RBF kernel function accuracy = 95% True Category Forecast Category Table 2. 4 Fault Diagnosis Results of Rolling Bearings Classifier Type Prediction Accuracy （%） Classification Time（s) Best C Best g SVM 55 118.21 1.6713 0.1000 PSO-SVM 95 24.80 1.6752 0.1000 IPSO-SVM 97.5 16.75 6.7207 0.1000 Table 2. 4 Fault Diagnosis Results of Rolling Bearings Figure 1. Flow chart of rolling bearing fault diagnosis based on IPSO-SVM classifier Vibration data used in this paper have been obtained from the bearing test data set of the Western Reserve University Bearing Data Center website. These bearing fault signals have been widely used to validate the effectiveness of new algorithms for bearing fault diagnosis.The test bearings support the motor shaft. Single point faults were introduced to the test bearings using electro-discharge machining.Vibration data was collected using accelerometers, multiple groups of data are obtained under different conditions. The bearing monitored is a deep groove ball bearing manufactured by SKF, rotational speed of 1797rpm and sampling frequency of 12KHz are selected. 0 20 40 60 80 100 120 1 1.5 2 2.5 3 3.5 4 Test Set Sample Number Test set Sample Class Comparison of SVM prediction results of test set with RBF kernel function accuracy = 55% True Category Forecast Category The validity of the proposed method is verified by the data of four states. at normal condition, the inner raceway fault, the outer raceway fault, and rolling element fault. In addition, the defective diameters of slight fault and serious fault are 0.007in.and 0.021in respectively. 3.1 Multi-classification fault data acquisition and feature extraction The performance of IPSO-SVM classifier is verified by taking four classification problems as an example. Four cases of slight fault grade (pitting diameter 0.007 in) are selected: bearing normal, inner raceway fault, outer raceway fault and the rolling body fault, which are set up as label 1˛2 ˛ 3 and 4 respectively. 4096 data points are collected in each state. The 13 dimensional fault eigenvector is obtained by EMD decomposition and Hilbert-Huang envelope spectrum analysis. 60 samples are selected in each state, a total of 240 samples are selected. It was randomly divided into two parts, 120 as training samples and 120 as test samples. Figure 2. Prediction accuracy of SVM classifier Comparison of PSO-SVM prediction results of test set with RBF kernel function accuracy = 95% Figure 2. Prediction accuracy of SVM classifier Comparison of PSO-SVM prediction results of test set with RBF kernel function accuracy = 95% Figure 2. Prediction accuracy of SVM classifier 4 Comparison of PSO-SVM prediction results of test set with RBF kernel function accuracy = 95% 0 20 40 60 80 100 120 1 1.5 2 2.5 3 3.5 4 Test Set Sample Number Test set Sample Class y True Category Forecast Category 3. Application of IPSO-SVM classerfier in fault diangosis of rolling bearing As shown in Figure 1 , the flow chart of the IPSO-SVM classifier in the fault diagnosis of rolling bearing mainly includes data acquisition , feature extraction , division data , parameter optimization and category prediction . As shown in Figure 1 , the flow chart of the IPSO-SVM classifier in the fault diagnosis of rolling bearing mainly includes data acquisition , feature extraction , division data , parameter optimization and category prediction . 3 MATEC Web of Conferences 227, 02007 (2018) https://doi.org/10.1051/matecconf/201822702007 MATEC Web of Conferences 227, 02007 (2018) ICCT 2018 Figure 1. Flow chart of rolling bearing fault diagnosis based on IPSO-SVM classifier reach its optimal fitness, it not only converges slowly, but also tends to fall into local optimum, while IPSO- SVM has converged in the second generation and quickly jumped out of the local optimal solution. Not only the global optimum fitness can be achieved rapidly, but also the fastest convergence rate can be obtained, which improves the efficiency of fault classification. SVM has converged in the second generation and quickly jumped out of the local optimal solution. Not only the global optimum fitness can be achieved rapidly, but also the fastest convergence rate can be obtained, which improves the efficiency of fault classification. Table 2. 4 Fault Diagnosis Results of Rolling Bearings Classifier Type Prediction Accuracy （%） Classification Time（s) Best C Best g SVM 55 118.21 1.6713 0.1000 PSO-SVM 95 24.80 1.6752 0.1000 IPSO-SVM 97.5 16.75 6.7207 0.1000 0 20 40 60 80 100 120 1 1.5 2 2.5 3 3.5 4 Test Set Sample Number Test set Sample Class Comparison of SVM prediction results of test set with RBF kernel function accuracy = 55% True Category Forecast Category Figure 2. Prediction accuracy of SVM classifier 0 20 40 60 80 100 120 1 1.5 2 2.5 3 3.5 4 Test Set Sample Number Test set Sample Class Comparison of PSO-SVM prediction results of test set with RBF kernel function accuracy = 95% True Category Forecast Category Figure 3. Prediction accuracy of PSO-SVM classifier Table 2. 3.2 Fault diagnosis result analysis The fault diagnosis of rolling bearings for 4 classification problems is carried out. The performance comparison and classification results of each classifier are shown in Table 2 and Figure2~4.From the comparison of the prediction accuracy and classification time of each classifier in Table 2, we can see that the classification effect of IPSO-SVM classifier is the best, and from Figure2~4, we can clearly see that the IPSO- SVM classifier has the least number of misclassified samples. Figure 3. Prediction accuracy of PSO-SVM classifier Figure5~6 shows the fitness curve of two particle swarm optimization algorithms, PSO-SVM does not 4 https://doi.org/10.1051/matecconf/201822702007 MATEC Web of Conferences 227, 02007 (2018) MATEC Web of Conferences 227, 02007 (2018) ICCT 2018 0 20 40 60 80 100 120 1 1.5 2 2.5 3 3.5 4 Test Set Sample Number Test set Sample Class Comparison of IPSO-SVM prediction results of test set with RBF kernel function accuracy = 97.5% True Category Forecast Category Figure 4. Prediction accuracy of IPSO-SVM classifier 0 20 40 60 80 100 120 140 160 180 200 40 50 60 70 80 90 100 Evolutionary algebra Fitness Fitness Curve Accuracy[PSOmethod] (Parameter c1=1.5,c2=1.7,Termination algebra=200,Population quantity=20) Optimum fitness Mean fitness Figure 5. Fitness Curve of PSO algorithm 70 75 80 85 90 95 Fitness Fitness Curve Accuracy[IPSOmethod] (Parameter c1=1.5,c2=1.7,Termination algebra=200,Population quantity=20) Optimum fitness Mean fitness 0 20 40 60 80 100 120 1 1.5 2 2.5 3 3.5 4 Test Set Sample Number Test set Sample Class Comparison of IPSO-SVM prediction results of test set with RBF kernel function accuracy = 97.5% True Category Forecast Category Figure 4. Prediction accuracy of IPSO-SVM classifier 0 20 40 60 80 100 120 1 1.5 2 2.5 3 3.5 4 Test Set Sample Number Test set Sample Class Comparison of IPSO-SVM prediction results of test set with RBF kernel function accuracy = 97.5% True Category Forecast Category 4 Comparison of IPSO-SVM prediction results of test set with RBF kernel function accuracy = 97.5% accuracy optimized by the parameters can be improved significantly, which shows the importance of parameter selection in the fault diagnosis of rolling bearings.  The classification performance of IPSO-SVM classifier is more stable. Unlike PSO-SVM classifier, it is not easy to fall into the local optimal solution, resulting in the decrease of classification accuracy and the slow convergence time. 4 Conclusion 0 20 40 60 80 100 120 140 160 180 200 40 50 60 70 80 90 100 Evolutionary algebra Fitness Fitness Curve Accuracy[PSOmethod] (Parameter c1=1.5,c2=1.7,Termination algebra=200,Population quantity=2 Optimum fitness Mean fitness Fitness Curve Accuracy[PSOmethod] SVM is a commonly used fault diagnosis classifier. The selection and optimization of its parameters is very important to the classification effect of the classifier. In this paper, an improved classifier based on IPSO parameter optimization is proposed. Three Libsvm data sets, two-classification problems of rolling bearing fault diagnosis and multiple- classification problems are simulated and analyzed. The superior performance of IPSO-SVM classifier is verified by comparing with SVM and PSO-SVM classifiers. It not only improves the accuracy of classification prediction, but also shortens the time of classification. It is found that fault samples with the same amplitude and similar frequency spectrum are prone to misclassification. The method to solve this problem is to use a variety of signal processing techniques to extract more effective fault feature vectors. Fitness Evolutionary algebra Figure 5. Fitness Curve of PSO algorithm 0 20 40 60 80 100 120 140 160 180 200 60 65 70 75 80 85 90 95 Evolutionary algebra Fitness Fitness Curve Accuracy[IPSOmethod] (Parameter c1=1.5,c2=1.7,Termination algebra=200,Population quantity=2 Optimum fitness Mean fitness Figure 6. Fitness Curve of IPSO algorithm 0 20 40 60 80 100 120 140 160 180 200 60 65 70 75 80 85 90 95 Evolutionary algebra Fitness Fitness Curve Accuracy[IPSOmethod] (Parameter c1=1.5,c2=1.7,Termination algebra=200,Population quantity=2 Optimum fitness Mean fitness Figure 6. Fitness Curve of IPSO algorithm Fitness Curve Accuracy[IPSOmethod] Fitness Curve Accuracy[IPSOmethod] Acknowledgements This work was completed in CQC(Shenyang) North Laboratory. The authors would like to thank Scientific Research Project No.51675350. 3.2 Fault diagnosis result analysis 0 20 40 60 80 100 120 1 1.5 2 2.5 3 Test Set Sample Number Test set Sample Class Figure 4. Prediction accuracy of IPSO-SVM classifier 0 20 40 60 80 100 120 140 160 180 200 40 50 60 70 80 90 100 Evolutionary algebra Fitness Fitness Curve Accuracy[PSOmethod] (Parameter c1=1.5,c2=1.7,Termination algebra=200,Population quantity=20 Optimum fitness Mean fitness Figure 5. Fitness Curve of PSO algorithm 0 20 40 60 80 100 120 140 160 180 200 60 65 70 75 80 85 90 95 Evolutionary algebra Fitness Fitness Curve Accuracy[IPSOmethod] (Parameter c1=1.5,c2=1.7,Termination algebra=200,Population quantity=20 Optimum fitness Mean fitness Figure 6. Fitness Curve of IPSO algorithm Combined with the above analysis, the following results can be obtained:  The parameters C and g directly affect the classification accuracy. The SVM classification  The SVM classifier is more suitable for solving the two-classification problem. When dealing with the multi-classification problem, the prediction accuracy drops sharply, while the IPSO-SVM classifier can obtain the highest prediction accuracy and the shortest classification time for both the two- classification problem and the multi -classification problem. Therefore, it is more suitable for fault diagnosis of rolling bearings.  The SVM classifier is more suitable for solving the two-classification problem. When dealing with the multi-classification problem, the prediction accuracy drops sharply, while the IPSO-SVM classifier can obtain the highest prediction accuracy and the shortest classification time for both the two- classification problem and the multi -classification problem. Therefore, it is more suitable for fault diagnosis of rolling bearings. Figure 4. Prediction accuracy of IPSO-SVM classifier References 1. Wang F, Chen X, Liu C, et al. Reliability Evaluation and Life Prediction of Rolling bearing based on KPCA and WPHM [J]. Vibration. Testing and Diagnostics, vol. 37,no. 3, pp.476-483, Jun. 2017. , pp , 2. Wang X, Yan W. Fault diagnosis of Rolling bearing based on variational Mode decomposition and SVM [J]. Vibration and Shock, vol. 36, no. 18, pp.252-256, Sep. 2017. Figure 6. Fitness Curve of IPSO algorithm Combined with the above analysis, the following results can be obtained: 3. Qin B, Sun G, Ge J, et al. Research on Rolling bearing Fault diagnosis based on Hilbert envelope singular value and IPSO-SVM [J].  The parameters C and g directly affect the classification accuracy. The SVM classification 5 5 MATEC Web of Conferences 227, 02007 (2018) MATEC Web of Conferences 227, 02007 (2018) ICCT 2018 https://doi.org/10.1051/matecconf/201822702007 Mechanical Transmission, vol. 47, no. 3, pp.166-181, Mar. 2017. Mechanical Transmission, vol. 47, no. 3, pp.166-181, Mar. 2017. Measurement and Instrumentation,vol. 31,no. 8, pp.1239-1246, Aug. 2017. 4. Zhou Si, Liao J, Shi X. RBF-SVM kernel parameter selection method and its application in fault diagnosis[J]. Journal of Electronic Measurement and Instruments, vol. 28, no. 3, pp.240-246, Mar. 2014. 8. Fu D, Zhai Y, Yu Q. Research on Rolling bearing Fault diagnosis based on EMD and support Vector Machine [J]. Machine Tools and Hydraulic Pressure, vol. 45,no. 11, pp.184-187, Jun. 2017. 9. Bian B. Rolling bearing Fault diagnosis method based on LCD noise reduction and LS-SVM [J]. Modular Machine Tools and Automatic Machining Technology, vol. 2, pp.:119-122, Feb. 2017. pp , 5. Samanta, B, Nataraj, C. Use of particle swarm optimization for machinery fault detection[J]. Engineering Applications of Artificial Intelligence, vol. 22, pp.308-316, Oct.. 2009. 6. Li, R, Sopon, P, He, D. Fault features extraction for bearing prognostics. Journal of Intelligent Manufacturing[J]. vol. 23, pp.313- 321, Apr.. 2012. 10. Song G, Wang H, Liu H, et al.Analog circuit fault diagnosis using lifting wavelet transform and SVM[J].Journal of Electronic Measurement and Instrument，vol. 24, no. 1, pp.:17-22, Jan. 2010 7. Mei H, Yin L, Liu D, et al. Improved Particle Swarm Optimization for Fault diagnosis of SVM Analog Circuits [J]. Journal of Electronic 6 6
https://openalex.org/W2990991941	https://timreview.ca/sites/default/files/article_PDF/TIMReview_November2019%20-%20F%20-%20Final.pdf	English	null	Uncovering Research Streams in the Data Economy Using Text Mining Algorithms	Technology innovation management review	2,019	cc-by	5,198	Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Data is only as valuable as the decision it enables. Ion Stoica Computer scientist cision it enables. Ion Stoica Computer scientist Data-driven business models arise in different social and industrial sectors, while new sensors and devices are breaking down the barriers for disruptive ideas and digitally transforming established solutions. This paper aims at providing insights about emerging topics in the data economy that are related to companies’ innovation potential. The paper uses text mining supported by systematic literature review to automatize the extraction and analysis of beneficial insights for both scientists and practitioners that would not be possible by a manual literature review. By doing so, we were able to analyze 860 scientific publications resulting in an overview of the research field of data economy and innovation. Nine clusters and their key topics are identified, analyzed as well as visualized, as we uncover research streams in the paper. Introduction could be of future interest for scientists on the one side as well as practitioners on the other side. Given the volume of publications, this paper uses a literature review and text mining approach to analyze keywords and abstracts of scientific publications in the context of the data economy in terms of their relevance, relation, and potential for automated innovation. In this paper, we provide the following results: on the one hand, we show what a text mining supported systematic literature review could look like. This approach can be easily adapted to analyze other research fields and topics. On the other hand, we provide content-related insights in the field of data economy and innovation. Due to rapid technological and organizational progress in digitalization, a diverse ecosystem of innovative technologies, platforms, and digital market players has emerged, leading to what we now call the “data economy”. One characteristic of this data economy is the huge amount of available data, which is often referred to as the “big data” paradigm. There are many sources available in scientific and practitioner communities, which need to be analyzed in order to stay informed, and thus capable of acting. The volume of sources means that a complete manual analysis is time consuming. Information overload leads to challenges for scientists as well as practitioners to identify and track the main topics in which innovation might take place. The challenge, however, is a prerequisite for achieving sustainable competitive advantage, due to volatile market changes and disruptive innovation approaches. Innovation bring to value creation. Business models in the digital economy (Otto, 2015; Zimmerman, 2000) are characterized by developing products into hybrid or purely digital services. The close integration of digital and physical products in combination with a vast amount of internally and externally available data enables data-driven service offerings for traditional products, as well as innovations to add more value to tangible products (Yoo et al., 2010). However, what does the term “data economy” mean exactly? Despite, or perhaps even because of the high attention given to it, a common understanding of the term “data economy” is still missing. Schumpeter's (1912) work on economic development theory, in which he describes an innovation as a "new combination" that asserts itself on the market and establishes a "creative redesign", is regarded as fundamental for introducing the concept of innovation. Numerous authors and scientists have taken up and interpreted innovation differently (Schumpeter, 1912). The following definitions reveal diverse understandings of the concept of innovation. Barnett argues that innovation is a qualitative differentiation from existing ideas or objects. The distance or the extent of novelty is the decisive factor to distinguish between "non-innovation" and innovation (Barnett, 1953). Nevertheless, a number of definitions have formed in practice, which are presented as follows: According to the German Association for the Digital Economy (BVDW), data economy deals with the monetization of information based on acquired data, which is transformed into valuable information using an algorithm, and then made accessible on the basis of business management functions. A data economy can be operated as its own business model or it can support, modify, or replace existing value creation models by increasing digitalization (German Association for the Digital Economy, 2018). Many authors take up the characteristic of novelty in their definition of innovation, while nevertheless interpreting novelty in decisively different ways. Thus, Vedin sees innovation in the first application of the new idea, method, or use of a novel object (Vedin, 1980). In his work on innovation diffusion theory, Rogers also takes up this approach, but adds a perspective that defines the concept more clearly. He thus interprets that something new only leads to an innovation if the adapting user perceives it in the same way (Rogers, 1983). This definition implies that (early) users adapt an innovation, which is to be understood as a first step in the later diffusion process. Data Economy Organizations invest a lot in digitalization programs and projects aiming to benefit from data economics. The discussion around digital business as “a business model whose underlying business logic deliberately acknowledges one or more characteristics of digitalization and aims to take advantage of them” (Otto et al., 2015), shows the growing importance of data within enterprises business (Moody & Walsh, 1999). Digitalization and advancing an organization’s business model in this direction requires considering the opportunities and challenges that data and information This paper aims at facing this challenge and enables an automatized, repeatable way to identify topics of interest and track the fields of innovation as discussed in published research literature. By systematically reviewing scientific publications, major research streams and their (sub-)topics are revealed. This will help scientists and practitioners to identify potentials for innovation and give guidance regarding which topics Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke organization does. These changes always lead to something new that creates some kind of value (Bessant & Tidd, 2007). Van der Kooij finds a generic definition for innovation and highlights the aspect of change as well. Here, an innovation is a change in the function of a system (product, process, organization, or society) that has a stepwise character. In short, it is the result of a process of human activity. The steps could be small, incremental, or large, and hence result in discontinuities (Van der Kooij, 2017). and cleaning as a second step, and data extraction as a final step. The third and last phase, “Reporting the review”, concludes with results that answer the predefined research questions (Kitchenham & Charters, 2007). To obtain valid results it is important to follow a systematic search strategy while doing a literature review. This can be done by defining the objectives and formulating specific research questions to be answered by carrying out the review. The research questions addressed by this article are derived from the objectives mentioned in the introduction. Our research answers the following research question (RQ): An innovation is thus created by combining the two characteristics of novelty and use, as defined by Ahmed and Shepherd (2010). This paper follows the Ahmed and Shepherd’s definition and consider innovation as a combination of something new that using or applying brings a change to the status quo. Which subject areas are relevant in the context of data economy innovation and what are the major research streams and (sub-) topics? Innovation In addition to novelty, the concept of innovation is here linked with adaptation, that is, the application of a novel idea, method, or use of a new product by users. Following this definition, an innovation can be understood as a novel idea or invention that eventually finds commercial application. Zawislak et al. also define innovation as the application of knowledge to generate technical or organizational changes capable of offering advantages to the firm that accomplishes them (Zawislak et al., 2008). By the European Commission’s definition, data economy measures the overall impacts of the data market on the economy as a whole. It involves the generation, collection, storage, processing, distribution, analysis elaboration, delivery, and exploitation of data enabled by digital technologies (European Commission, 2019). A study by Digital Reality, a worldwide leader in building data centers, defines data economy as the financial and economic value created by the storage, retrieval and analysis –using sophisticated software and other tools – of large volumes of business and organizational data at very high speeds (so-called ´big data´). This can involve, for example, realizing improved operational efficiency or implementing improved strategic decisions (Digital Reality, 2018). Francis and Bessant view innovation from the perspective of the change that comes with innovation (Francis & Bessant, 2005). Regarding this view, Bessant and Tidd distinguish four categories of innovation. “Product innovation” refers to changes in the things (product/services) an organization offers. “Process innovation” implies changes in the way in which things are created and delivered. “Position innovation” refers to changes in the context in which things are introduced, while “paradigm innovation” describes changes in the underlying mental models that frame what the This paper therefore defines “data economy” as an umbrella term, which includes digital business models independent of a particular industry, for example, data products and services, digital technologies, data value chains, and their technical implications for data creation, processing, provision, and use to gain benefits for an organization. Figure 2. Search Query from Scopus The final limitation on stage five was performed outside the Scopus search engine. We used Scopus’ export functionality to export a BibTex formatted file of the search results, including the fields: Author (a), Document title (b), Year (c), Source title (d), volume, issue, pages (e), Citation count (f), Source & document type (g), DOI (h), Affiliations (i), Language (j), Abstract (k), Author keywords (l), and Index keywords (m). A python script was implemented to extract the information, which was exported in BibTex format within a relational database system in order to have a better structure for further analyses of relations. The script systematically loops through the BibTex file and stores article information, as listed above, for each entry. as or-conditions, set the basis for retrieving appropriate publications for our review. Furthermore, we needed to ensure a connection to innovation. For this reason, we added the keyword “innovation” as a mandatory condition in the title, abstract, or keyword of the publication, and chained this as a prerequisite regardless of all the other keywords, that is, where that particular term has to be matched. With this combination of keywords, we ensured a focus on publications in the area of data economy and innovation. Following this approach, we were able to retrieve 1,163 publications as the foundational data set. For the second step, we had to ensure the quality of our data set, and therefore combined the results with different filters and inclusion criteria, in order to gain a higher level of quality. In the second stage, 908 publications were returned, after limiting the result set to journal articles and conference papers. The third stage included only articles published in English, which returned 863 articles. Stage four excluded another three articles due to missing author names. We also consciously decided not to exclude subject areas in Scopus in order to cover a wide range of research. The final search string, as the result of combining our keyword search together with the limitation criteria, is depicted in Figure 2. In order to focus our analysis on innovation topics within the data economy, we limited our dataset by searching for specific words within the articles’ abstracts, and excluded all articles that did not include these terms. For filtering, we choose the words (a) problem, (b) challenge, (c) demand, (d) requirement, (e) obstacle, (f) limit, (g) barrier, and (h) necessity. Research Design The first step to conduct a phase of the SLR is the study selection. As a first step, we focused on Elsevier’s Scopus database as a source for exploring peer-reviewed publications. Scopus offers easy access for meta-data on publications and has one of the largest databases for scientific publications with over 70 million publications (https://www.elsevier.com/solutions/scopus). For the second step, we defined suitable keywords to meet the objectives of our review and to answer the above research questions. We used the keywords "digital economy", "data economy", "digital business model", "data driven business model", "digital business", "digital platforms", "data technologies", "digital disruption", and "digital transformation". These keywords, chained This paper adopts the methodology of systematic literature review (SLR) (Kitchenham, 2004, Figure 1). The SLR consists of three phases: planning, conducting, and reporting. Within the first phase, “planning the review”, the goal is to create a basic framework and design the content arrangement. This involves identifying the need for a review, specifying the addressed research questions, and developing a review protocol for controlling the review. “Conducting the review” in the second phase means executing the review protocol designed in the planning phase, which includes the creation of a dataset. This begins with the selection of suitable publications as a first step, quality assessment Figure 1. SLR process phases according to Kitchenham and Charters (2007) Figure 1. SLR process phases according to Kitchenham and Charters (2007) reams in the Data Economy Using Text Mining Algorithms nn, Henry Goecke Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke TITLE-ABS-KEY ("digital economy" OR "data economy" OR "digital business model" OR "data driven business model" OR "digital business" OR "digital platforms" OR "data technologies" OR "digital disruption" OR "digital transformation") AND TITLE-ABS-KEY ("Innovation") AND (LIMIT-TO (DOCTYPE, "cp") OR LIMIT-TO (DOCTYPE, "ar")) AND (LIMIT-TO (LANGUAGE, "English" )) Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke First, information density is highest in the abstract (Scheumie et al., 2004). Second, access to scientific full- text content for text mining is difficult due copyright and licensing reasons (O’Mara-Eves et al., 2015). Based on our approach, the final number of articles, as result of stage five, returned 334 documents. The full study selection and cleaning process is depicted in Figure 3. To answer the specified research questions, we analyzed the relations between different keywords and formed clusters of different topics and sub-topics. The assigned relations between keywords were done by creating a correlation matrix. We looped through different keywords and selected all publications containing a specific keyword. After that we mapped all other keywords assigned to these publications and linked these relations within our database. In order to answer the specified research questions, we analyzed the abstracts and topic for data extraction. To get first insights into the data set, we used available general meta information. For example, we considered the year of publication in the area of data economy and innovation. In addition, in co-authored papers, author affiliations were analyzed, as well as country of work, in order to depict where research on the focus topic is done. From a content perspective, we took subject areas from different sources within our data set to get a distribution overview. For our main research, we focused on annotated keywords , since they can serve to help articulate a highly concise summary of a document (Siddiqi and Sharan, 2015). Within the available datasets, the keywords exported from Scopus database differed between indexed and author keywords. While author keywords are exempt from semantics rules and annotated directly by the authors, indexed keywords are assigned by Scopus using a taxonomy to form a semantic system and organize the platforms entries. Using this system enables a more consistent analysis through better comparability between different keywords. For further analyses, we used indexed keywords only. Figure 2. Search Query from Scopus We argue that these terms, related to challenges and obstacles, within the abstracts enables through filtering the identification of novel approaches and applications to a specific problem. It was deliberately decided not to do a full-text analysis of the publications for two reasons: Figure 3. Study selection on the Scopus Engine Figure 3. Study selection on the Scopus Engine Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Findings Summary and analysis of results y y f It should be noted as a general result that the number of scientific publications in this field has increased more than 2,800  over the last 10 years (Figure 4). Although the publication date was not considered as a filter criterion in the search process, the following graph starts at 1998, because before 1998 only one article (in 1985) was published. From a geographical point of view, most of the publications we studied where published within the United States of America, Germany, United Kingdom, China, and the Russian Federation, as seen in the following Figure 5. Figure 6 shows the main subject areas: computer science (28 ), business, management & accounting (16 ), and engineering (14 ). Surprisingly, the social science sector is also strongly represented with 11  of all scientific Figure 4. Number of scientific publications by year Figure 4. Number of scientific publications by year Figure 4. Number of scientific publications by year Figure 5. Number of scientific publications by country Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Figure 5. Number of scientific publications by country nodes and 114,414 edges. By using force-directed algorithms, where nodes repulse and edges attract each other, we identified nine relevant clusters. publications on our topic. Figure 7 shows the leading research institutions in this field. Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Figure 7. Number of scientific publications by affiliation ng Research Streams in the Data Economy Using Text Mining Algorithms Markus Spiekermann, Henry Goecke Figure 7. Number of scientific publications by affiliation artificial intelligence), which are based on article abstracts. As can be clearly seen, many scientific publications address challenges within their abstracts, and therefore are rated as likely to provide insights about innovation potential. The topics of innovation management (76 ), machine learning (71 ), decision making (63 ), and knowledge management (31 ) are overrepresented in the publications dealing with challenges compared to all publications. However, contrary to expectations, we also found artificial intelligence having only a small increase (20 ) in the number of mentions by comparison. While the ratio of artificial intelligence (AI) is relatively similar, the sub- class of machine learning reveals a considerable difference. This dominance of ML shows that authors prefer ML vs. AI terminology. communities in the network (Blondel et al., 2014). As shown in Figure 8, the network graph has nine clusters. These were resized according to their degree of their interconnectedness to give a better presentation of the most relevant nodes. Table 1 sums up the identified clusters, including the top keywords from each cluster. The name of the cluster is based on the node with the most incoming and outgoing edges. The number of all edges in total are given according to the keyword within the table. Based on the keywords related to the presented clusters, we derived a proposal for interpretation. This explanation was used to form a common understanding of the clusters in communicating preliminary results. Keyword relations in a network graph By using filter techniques, such as a giant component (see Fulton et al., 2001) as used in the network theory, and a degree range setting of 65, only 658 nodes and 23,706 edges were left. In order to spatialize the network graph, the Forceatlas2 algorithm was used. Forceatlas2 is a force-directed layout where nodes repulse and edges attract (Jacomy et al., 2014). Furthermore, a modularity class filter was applied to examine the resulting Gephi (http://gephi.org) software was used in order to identify and visualize subject areas and relations between keywords from the scientific literature. This software enabled the creation of a network graph, which illustrates the relations between keywords, as shown in Figure 8. In this graph, one can see so-called keyword nodes, as well as the edges that establish connections between nodes. The unfiltered graph includes 5,231 Figure 6. Portion of scientific publications by subject area Figure 6. Portion of scientific publications by subject area Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Uncovering Research Streams in the Data Economy Using Tex Can Azkan, Markus Spiekermann, Henry Goecke vering Research Streams in the Data Economy Using Text Mining Algorithm an, Markus Spiekermann, Henry Goecke Node analysis l f h We argue that the nine clusters are to be regarded as categories for potential innovation within the overall data transformation towards a data economy. Organizations should pay attention to these topics, while transforming their business and developing digital services and business models. As well, they should track ongoing research to identify novel approaches and applications to different areas and topics. Analysis of the results shows that authors on this topic seem to assume that challenges are associated with data handling, innovation management, decision-making, and machine learning. We assume that more research in specific areas will lead also to higher innovation potential, especially in combination with other topics and technologies. In order to obtain more precise evaluation, we carried out a keyword comparison. For this purpose, we compared the number of keywords between the articles reviewed in stages 4 and 5. This was done to identify the ratio of keywords within all articles in order to discover possible articles for innovation topics. The following graph is presented in more detail for two concrete examples, first, for "Big Data" (Figure 10). The clearly visible connection to "Digital Transformation" and “Technological innovation” supports our argumentation about automatic identification of innovation potential. Figure 9 shows a comparison between the top four article subject areas in data economy, as well as two selected subject areas (knowledge management and Secondly, the node "machine learning" shows a very strong connection with the 5th cluster "Decision making" (Figure 11). Along with machine learning, one Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Figure 8. Network graph to visualize the relations between keywords n Azkan, Markus Spiekermann, Henry Goecke Figure 8. Network graph to visualize the relations between keywords Figure 8. Network graph to visualize the relations between keywords Table 1. Cluster results with related terms (below) Table 1. Cluster results with related terms (below) Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Figure 9. Keywords vs. keywords in context of challenges Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Figure 11. Gephi analysis of the node "machine learning" p y Figure 11. Gephi analysis of the node "machine learning" Figure 11. Gephi analysis of the node "machine learning" can identify also strong connections to applications within the medical sector, shown by the nodes "medical informatics", "digital pathology", and "clinical decision making". according to geography as well as affiliation. In addition, we identified major research streams by performing a network analysis and forming clusters based on the number of interconnections between different topics and their sub-topics. This provided an overview about relevant topics within the data economy that can help researchers derive topics where future research will probably emerge. Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Figure 9. Keywords vs. keywords in context of challenges Figure 9. Keywords vs. keywords in context of challenges Figure 10. Gephi analysis of the node "Big Data" Figure 10. Gephi analysis of the node "Big Data" Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke oÉÑÉêÉåÅÉ Ahmed, Pervaiz K., Shepherd, Charlie. 2010. Innovation Management. Context, strategies, systems and processes (1st ed.). Harlow: Financial Times Prentice Hall. Schuemie, Martijn J., Weeber, Marc, Schijvenaars, Bob JA, Mulligen, Rik M. Van, van der Eijk, C. Cristiaan, Jelier, C. Cristiaan, Mons, Barend, and Kors, Jan A. 2004. Distribution of information in biomedical abstracts and full-text publications. Bioinformatics, 20: 2597–2604. Barnett, Homer G. 1953. Innovation: The basis of cultural change. New York: McGraw- Hill. Blondel, Vincent D., Guillaume, Jean-Loup, Lambiotte, Renaud, and Lefebvre, Etienne. 2008. Fast unfolding of communities in large networks. J. Stat. Mech. Theory Exp. Schumpeter, Joseph. 1912. Theorie der wirtschaftlichen Entwicklung. Eine Untersuchung über Unternehmergewinn, Kapital, Kredit, Zins und Konjunkturzyklus (9. Aufl.). Berlin: Duncker und Humbolt. Federal Association of the Digital Economy. 2018. Data Economy. Datenwertschöpfung und Qualität von Daten. Siddiqi, Sifatullah, and Sharan, Aditi. 2015. Keyword and Keyphrase Extraction Techniques: A Literature Review. Int. J. Comput. Appl. 109: 18–23. Coupland, Rob. 2018. Digital Realty. Data Economy Report. Tidd, Jie, and Bessant, John R. 2015. Innovation and Entrepreneurship (3rd ed.). Wiley. Ding, Ying, Chowdhury, Gobinda, Foo, Schubert. 2001. Bibliometric cartography of information retrieval research by using co-words analysis. Inf. Process. van der Kooij, Bouke. 2017. Search for a Common Ground in the Fogs of Innovation Definitions. SSRN Electronic Journal. European Commission 2019. Building a European data economy. www.ec.europa.eu/digital-single- market/en/policies/building-european-data- economy. Vedin, Bengt-Arne. 1980. Management and organization for innovation: radical product renewal in large corporations. Göteborg: Chalmers TH. Yoo, Youngjin, Henfridsson, Ola, and Lyytinen, Ola. 2010. The new organizing logic of digital innovation: An agenda for information systems research. Information Systems Research. Vol. 21, No. 4, December: 724–735. Francis, Dave L. and Bessant, John. 2005. Targeting Innovation and Implications for Capability Development. Technovation, 25, (3): 171-183. Bollobas, Bela. 2001. Random Graphs. Cambridge Studies in Advanced Mathematics, 73. Zimmerman, Hans-Dieter. 2000. Understanding the Digital Economy: Challenges for New Business Models. AMCIS 2000 Proceedings. Jacomy, Mathieu, Venturini, Tommaso, Heymann, Sebastien, Bastian, Mathieu. 2014. ForceAtlas2, a continuous graph layout algorithm for handy network visualization designed for the Gephi software. PLoS One. 9: 1–12. Zawislak, Paulo Antônio, Borges, Mauro, Wegner, Douglas, Santos, Andre and Cristina, Castro-Lucas. 2008. Towards the Innovation Function. Journal of Technology Management & Innovation. Kitchenham, Barbara. 2004. Procedures for Undertaking Systematic Reviews. Joint Technical Report, Computer Science Department, Keele University (TR/SE-0401) and National ICT Australia Ltd. Kitchenham, Barbara, and Charters, Stuart. 2007. Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Rogers, Everett M. 1983. Diffusion of Innovations (3rd ed.). New York: Free Press. Conclusion This paper presents an automatized way to derive areas for innovation in the field of data economy. By conducting a systematic literature review in combination with basic text-mining methods, we identified 1,163 publications in the Scopus database. We analyzed them to identify a suitable dataset of publications containing terms related to challenges and requirements, as a way to answer our predefined research questions. We focused on these publications because abstracts dealing with challenges and related terms also refer to innovation topics. With pattern recognition based on text mining, we identified 334 articles based on abstracts that included specified terms for our analysis. Researchers and practitioners are welcome to test the usefulness and applicability of our approach, especially evaluating our argumentation that derives innovation potential from challenges and requirement-related publications. Further research in the field of data economy may challenge our results with a more detailed view of specific clusters to gain even more insights. We then illustrated the development of topics and sub- topics related to data economy and innovation over the time, and depicted the main contributors in this area Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke oÉÑÉêÉåÅÉ Guidelines for performing Systematic Literature reviews in Software Engineering Version 2.3. Engineering, 45. Moody, Daniel, and Walsh, Peter. 1999. Measuring the Value Of Information: An Asset Valuation Approach. Seventh Eur. Conf. Inf. Syst.: 1–17. O’Mara-Eves, Alison, Thomas, James, McNaught, John, Miwa, Makoto, and Ananiadou,Sophia. 2015. Using text mining for study identification in systematic reviews: A systematic review of current approaches. Syst. Rev. 4: 1–22. Otto, Boris. 2015. Quality and Value of the Data Resource in Large Enterprises. Inf. Syst. Manag. 32: 234–235. Otto, Boris, Bärenfänger, Rieke, Steinbuß, Sebastian. 2015. Digital Business Engineering: Methodological Foundations and First Experiences from the Field. Proc. 28th Bled eConference: 58–76. Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Uncovering Research Streams in the Data Economy Using Text Mining Algorithms Can Azkan, Markus Spiekermann, Henry Goecke Keywords: Data Ecosystem, Data Economy, Digital Economy, Data Ecosystem, Digital Transformation, Data Market, Big Data, Literature Review, Network Graph, Text Mining. Citation: Azkan, C., Spiekermann, M., Goecke, H.. 2019. Uncovering Research Streams in the Data Economy Using Text Mining Algorithms. Technology Innovation Management Review, 9(10): 62-74. http://doi.org/10.22215/timreview/1283 ^ÄçìííÜÉ ^ìíÜçêë Can Azkan is a scientist and PhD candidate at the Fraunhofer Institute for Software and Systems Engineering ISST in Germany. He studied Mechanical Engineering at the Technical University of Dortmund and the San Diego State University, while he gained practical experience in the field of industrial engineering and digital business models in machine und plant engineering. His research at Fraunhofer ISST focuses on value co-creation in emerging data ecosystems and the management of data as a corporate asset. Markus Spiekermann currently works as Head of Department "Data Business" at the Fraunhofer Institute for Software and Systems Engineering in Dortmund, Germany. He leads research projects and is active in several related advisory boards. His main research focuses on the topics of data engineering and data management, alongside on the valuation of data assets especially within data ecosystems. Before his time at Fraunhofer, he worked as IT-Professional and Software Engineer from 2008 to 2016. He obtained his Bachelor and Master of Science degree in the field of information systems with a focus on IT Management at the FOM University of Applied Sciences in Essen. Since 2017 Dr. Henry Goecke has been head of the Research Group "Big Data Analytics" at the German Economic Institute. Previously he worked at the German Economic Institute as scientific assistant of the Director, at the IW Consult as Senior Economist, at the TU Dortmund University as research and teaching assistant as well as lecturer at the University of Cologne and the Hochschule Fresenius. He studied Economics at the TU Dortmund University, Strathclyde University of Glasgow, and the Leuphana University of Lüneburg. His research interests are on the impact of social media, artificial intelligence, big data, and data economy. Citation: Azkan, C., Spiekermann, M., Goecke, H.. 2019. Uncovering Research Streams in the Data Economy Using Text Mining Algorithms. Technology Innovation Management Review, 9(10): 62-74. http://doi.org/10.22215/timreview/1283 Keywords: Data Ecosystem, Data Economy, Digital Economy, Data Ecosystem, Digital Transformation, Data Market, Big Data, Literature Review, Network Graph, Text Mining.
https://openalex.org/W4390767862	https://www.revistaagraria.com/index.php/agraria/article/download/346/308	es		Evaluacion de colecciones nativas de zacaton alcalino Sporobolus airoides Torr. en la formacion de variedades bajo sequia	Agraria	1,985	cc-by-sa	0
https://openalex.org/W2091118421	https://europepmc.org/articles/pmc4459543?pdf=render	English	null	Metalearning: a survey of trends and technologies	Artificial intelligence review	2,013	cc-by	8,604	Artif Intell Rev (2015) 44:117–130 DOI 10.1007/s10462-013-9406-y Metalearning: a survey of trends and technologies Christiane Lemke · Marcin Budka · Bogdan Gabrys Published online: 20 July 2013 Published online: 20 July 2013 © The Author(s) 2013. This article is published with open access at Springerlink.com y © The Author(s) 2013. This article is published with open access at Springerlink.com Abstract Metalearning attracted considerable interest in the machine learning community in the last years. Yet, some disagreement remains on what does or what does not constitute a metalearning problem and in which contexts the term is used in. This survey aims at giving an all-encompassing overview of the research directions pursued under the umbrella of metalearning, reconciling different deﬁnitions given in scientiﬁc literature, listing the choices involved when designing a metalearning system and identifying some of the future research challenges in this domain. Keywords Metalearning · Metaknowledge extraction · Life-long learning M. Budka (B)· B. Gabrys Bournemouth University, Poole House, Talbot Campus, Fern Barrow, BH12 5BB Poole, UK e-mail: mbudka@gmail.com M. Budka (B)· B. Gabrys Bournemouth University, Poole House, Talbot Campus, Fern Barrow, BH12 5BB Poole, UK e-mail: mbudka@gmail.com B. Gabrys e-mail: bgabrys@bournemouth.ac.uk C. Lemke Unister GmbH, Leipzig, Germany e-mail: christiane.lemke@gmail.com 1 Introduction The term metalearning ﬁrst occurred in the area of educational psychology. One of the most cited researchers in this ﬁeld, John Biggs, described metalearning as being aware of and taking control of one’s own learning (Biggs 1985). Hence, metalearning is viewed as an understanding and adaptation of learning itself on a higher level than merely acquiring subject knowledge. In that way, a person aware and capable of metalearning is able to assess his or her learning approach and adjust it according to the requirements of a speciﬁc task. Metalearning as used in a machine learning context has many similarities to this descrip- tion. Subject knowledge translates into base-learning, where experience is accumulated for one speciﬁc learning task. Metalearning starts at a higher level and is concerned with accu- B. Gabrys e-mail: bgabrys@bournemouth.ac.uk C. Lemke Unister GmbH, Leipzig, Germany e-mail: christiane.lemke@gmail.com 123 118 C. Lemke et al. mulating experience over several applications of a learning system according to Brazdil et al. (2009). lating experience over several applications of a learning system according to Brazdil et (2009). In the last 20 years, machine learning research was faced with an increasing number of availablealgorithmsincludingamultitudeofparametrisation,preprocessingandpostprocess- ing approaches as well as a substantially extended range of applications due to increasing computing power and wider availability of computer-readable data sets. By promoting a better understanding of machine learning itself, metalearning can provide an invaluable help avoiding extensive trial and error procedures for algorithm selection, and brute force searches for suitable parametrisation. Looking at how to proﬁt from past experience of a predictive model on certain tasks can enhance the performance of a learning algorithm and allow to better understand what makes a given algorithm perform well on a given problem. The idea of metalearning is not new, one of the ﬁrst and seminal contributions having been provided by Rice (1976). However, the literal term only started appearing in machine learning literature in the 1990s, yet still many publications deal with problems related to metalearning without using the actual word. This contribution tries to grasp every point of view metalearning has been investigated from, citing books, research and review papers of the last decade. We hope this survey will provide a useful resource for the data mining and machine learning community. The remainder of this paper is organized as follows. In Sect. 1 Introduction 2 we review deﬁnitions of metalearning given in scientiﬁc literature, focusing on common themes occurring in all of them. Section 3 describes different notions of metalearning, linking them to the deﬁnitions given in Sect. 2. In Sect. 4 practical considerations arising when designing a metalearning system are discussed, while open research directions are listed in Sect. 5. 2 Deﬁnition In the 1990s, the term metalearning started to appear in machine learning research, although the concept itself dates back to the mid-1970s (Rice 1976). A number of deﬁnitions of metalearning have been given, the following list cites the main review papers and books from the last decade: 1. Metalearning studies how learning systems can increase in efﬁciency through experience; the goal is to understand how learning itself can become ﬂexible according to the domain or task under study (Vilalta and Drissi 2002a). 2. The primary goal of metalearning is the understanding of the interaction between the mechanism of learning and the concrete contexts in which that mechanism is applicable (Giraud-Carrier 2008). 3. Metalearning is the study of principled methods that exploit metaknowledge to obtain efﬁcient models and solutions by adapting machine learning and data mining processes (Brazdil et al. 2009). 4. Metalearning monitors the automatic learning process itself, in the context of the learning problems it encounters, and tries to adapt its behaviour to perform better (Vanschoren 2010). Learning systems that adapt and improve by experience are a key concept of deﬁnitions 1, 3 and 4. This in itself however does not sufﬁce as a description, as it basically applies to all machine learning algorithms. Metalearning becomes metalearning by looking at different problems, domains, tasks or contexts or simply past experience. This aspect is inherent in all of the deﬁnitions, although somewhat disguised in deﬁnition 3 using the term metaknowledge Learning systems that adapt and improve by experience are a key concept of deﬁnitions 1, 3 and 4. This in itself however does not sufﬁce as a description, as it basically applies to all machine learning algorithms. Metalearning becomes metalearning by looking at different problems, domains, tasks or contexts or simply past experience. This aspect is inherent in all of the deﬁnitions, although somewhat disguised in deﬁnition 3 using the term metaknowledge 123 Metalearning: a survey of trends and technologies 119 instead. Metaknowledge as described by the authors stands for knowledge to be exploited from past learning tasks, which may both mean past learning tasks on the same data or using data of another problem domain. Deﬁnition 2 differs in emphasising a better comprehension of the interaction between domains and learning mechanisms, which does not necessarily imply the goal of improved learning systems, but the pursuit of a better understanding of for which tasks individual learners succeed or fail. instead. 2 Deﬁnition Metaknowledge as described by the authors stands for knowledge to be exploited from past learning tasks, which may both mean past learning tasks on the same data or using data of another problem domain. Deﬁnition 2 differs in emphasising a better comprehension of the interaction between domains and learning mechanisms, which does not necessarily imply the goal of improved learning systems, but the pursuit of a better understanding of for which tasks individual learners succeed or fail. Rephrasing, the common ground the above deﬁnitions share, we propose to deﬁne a metalearning system as follows: Deﬁnition 1 1. A metalearning system must include a learning subsystem, which adapts with experience. Deﬁnition 1 1. A metalearning system must include a learning subsystem, which adapts with experience. 2. Experience is gained by exploiting metaknowledge extracted 2. Experience is gained by exploiting metaknowledge extracted (a) …in a previous learning episode on a single dataset, and/or (b) …from different domains or problems. Furthermore, a concept often used in metalearning is that of a bias, which, in this context, refers to a set of assumptions inﬂuencing the choice of hypotheses for explaining the data. Brazdil et al. (2009) distinguishes declarative bias specifying the representation of the space of hypotheses (for example representing hypotheses using neural networks only) and proce- dural bias, which affects the ordering of the hypotheses (for example preferring hypothesis with smaller runtime). The bias in base-learning according to this theory is ﬁxed, whereas metalearning tries to choose the right bias dynamically. 3 Notions of metalearning The dataset is subsequently used for training of a base model and the collection of models obtained in this way forms an ensemble with individual models’ decisions combined typically using voting (in classiﬁcation problems) or averaging (in regression problems). – BoostingproposedinFreundandSchapire(1997),whichmanipulatestheprobabilitywith which samples are drawn from the original training data, to sequentially train classiﬁers focusing on the ‘difﬁcult’ parts of the training set. Hence each consecutive ensemble member focuses on the training examples that cannot be successfully handled by the ensemble developed up to that point. The ensemble is usually built until a speciﬁed number of ensemble members is generated (although other stopping criteria are possible) and their decisions are combined using a weighted voting mechanism. Although the ensemble members can be ‘weak’ learners (i.e. models only slightly better than chance), this property must hold in the context of an increasingly difﬁcult resampled dataset. As a result at some stage the ‘weak’ learner may in fact need to be quite complex and powerful. – BoostingproposedinFreundandSchapire(1997),whichmanipulatestheprobabilitywith which samples are drawn from the original training data, to sequentially train classiﬁers focusing on the ‘difﬁcult’ parts of the training set. Hence each consecutive ensemble member focuses on the training examples that cannot be successfully handled by the ensemble developed up to that point. The ensemble is usually built until a speciﬁed number of ensemble members is generated (although other stopping criteria are possible) and their decisions are combined using a weighted voting mechanism. Although the ensemble members can be ‘weak’ learners (i.e. models only slightly better than chance), this property must hold in the context of an increasingly difﬁcult resampled dataset. As a result at some stage the ‘weak’ learner may in fact need to be quite complex and powerful. The above approaches exploit variation in the data and are referred to as metalearning methods in Brazdil et al. (2009) and Vanschoren (2010). Bagging however does not satisfy point 2 of Deﬁnition 1, as consecutive random samples from the original dataset are independent from each other, so there is no experience from previous learning episodes involved. In the case of boosting however, the ensemble is built sequentially and it is the performance of previous ensemble members (i.e. experience gained while trying to solve the problem) that inﬂuences the sampling process. 3 Notions of metalearning Metalearning can be employed in a variety of settings, with a certain disagreement in literature about what exactly constitutes a metalearning problem. Different notions will be presented in thissectionwhilekeepinganeyeonthequestioniftheycanbecalledmetalearningapproaches according to Deﬁnition 1. Figure 1 groups general machine and metalearning approaches in relation to Deﬁnition 1. Each of the three circles presents a cornerstone of the deﬁnition (1: adapt with experience, 2a: meta-knowledge on same data set, 2b: meta-knowledge from different domains), the approaches are arranged into the circles and their overlapping sections depending on which parts of the deﬁnition applies to them. As an example, ensemble methods do generally work with experience gained with the same data set (deﬁnition 2a) and adapt with experience (deﬁnition 1), however, the only approach potentially applying all three parts of the deﬁnition is algorithm selection, which appears where all three circles overlap. 3.1 Ensemble methods and combinations of base-learners Model combination is often used when several applicable algorithms for a problem are available. Instead of selecting a single algorithm for a problem, the risk of choosing the wrong one can be reduced by combining all or a subset of the available outcomes. In machine learning, advanced model combination can be facilitated by ensemble learning according to Dietterich (2000) and Yao and Islam (2008), which comprises strategies for training and combining outputs of a number of machine learning algorithms. One often used approach of this type is resampling, leading to a number of ensemble generation techniques. Two very popular resampling-based ensemble building methods are: 123 123 120 C. Lemke et al. Fig. 1 Notions of metalearning versus components of a metalearning system Fig. 1 Notions of metalearning versus components of a metalearning system – Bagging introduced in Breiman (1996), which denotes repeated random sampling with replacement to produce a dataset of the same size as the original training set. The dataset is subsequently used for training of a base model and the collection of models obtained in this way forms an ensemble with individual models’ decisions combined typically using voting (in classiﬁcation problems) or averaging (in regression problems). – Bagging introduced in Breiman (1996), which denotes repeated random sampling with replacement to produce a dataset of the same size as the original training set. 3.2 Algorithm recommendation A considerable amount of metalearning research has been devoted to the area of algorithm recommendation. In this special case of metalearning, the aspect of interest is the relationship between data characteristics1 and algorithm performance, with the ﬁnal goal of predicting an algorithm or a set of algorithms suitable for a speciﬁc problem under study. As a motivation, the fact that it is infeasible to examine all possible alternatives of algorithms in a trial and error procedure is often given along with the experts necessary if pre-selection of algorithms is to take place. This application of metalearning can thus be both useful for providing a recommendation to an end-user or automatically selecting or weighting algorithms that are most promising. Vanschoren (2010) points out another aspect: it is not only the algorithms themselves, but different parameter settings that will naturally let performance of the same algorithm vary on different datasets. It would be possible to regard versions of the same algorithm with different parameter settings as different learning algorithms altogether, but the author advocates treating the subject and studying its effects differently. Such an approach has for example been taken in Gomes et al. (2012) and Miranda et al. (2012), where the authors discuss a hybrid metalearning and search based technique to facilitate the choice of opti- mal parameter values of a Support Vector Machine (SVM). In this approach, the candidate parameter settings recommended by a metalearning algorithm are used a starting point for further optimization using Tabu Search or Particle Swarm Optimization techniques, with great success. Reif et al. (2012b) investigate increasing the accuracy and decreasing runtime of a genetic algorithm for selecting learning parameters for a Support Vector Machine and a Random Forests classiﬁer. Based on past experience on other datasets and corresponding dataset characteristics, metalearning is used to select a promising initial population for the genetic algorithm, reducing the number of iterations needed to ﬁnd accurate solutions. An interesting treatment of the above problem can also be found in Jankowski and Grabczewski (2009), where the authors propose to take into account not only the expected performance of the algorithm but also its estimated training time. In this way the algorithms can be ordered according to the estimated training complexity, which allows to produce rela- tively well-performing models very quickly and then look for better solutions, while the ones already trained are producing predictions. 3 Notions of metalearning More often, the following two approaches are considered as metalearning techniques: – Stacked generalisation (or stacking) as introduced in Wolpert (1992), where a number of base learners is trained on the same dataset. Their outputs are subsequently being used – Stacked generalisation (or stacking) as introduced in Wolpert (1992), where a number of base learners is trained on the same dataset. Their outputs are subsequently being used 123 121 Metalearning: a survey of trends and technologies for a higher level learning problem, building a model linking the outcomes of the base learners to the target value. The meta-model then produces the ﬁnal target outcome. for a higher level learning problem, building a model linking the outcomes of the base learners to the target value. The meta-model then produces the ﬁnal target outcome. for a higher level learning problem, building a model linking the outcomes of the base learners to the target value. The meta-model then produces the ﬁnal target outcome. – Cascadegeneralisation(GamaandBrazdil2000),whichworkssequentially.Whenbuild- ing a model, the output of the ﬁrst base learner is appended to the original feature set and passed on to the next learner with the original target values. This process can then be repeated. – Cascadegeneralisation(GamaandBrazdil2000),whichworkssequentially.Whenbuild- ing a model, the output of the ﬁrst base learner is appended to the original feature set and passed on to the next learner with the original target values. This process can then be repeated. Although in these cases the information about base-learning is drawn in the sense of point 2a of Deﬁnition 1, these algorithms are limited to a single problem domain with a bias that is ﬁxed a priori, so that they, using the deﬁnition above, do not undoubtedly qualify as metalearning methods. 1 In a more advanced form it is the relationship between data and algorithm characteristics, and algorithm performance. 2 http://www-ai.cs.uni-dortmund.de/MMWEB/downloads. 3.2 Algorithm recommendation These ideas are further extended in Jankowski (2011), where some modiﬁcations of the complexity measures used are introduced. The classic application area of algorithm selection in machine learning is classiﬁcation. Smith-Miles (2008) however tries to generalise the concepts to other areas including regres- 123 123 122 C. Lemke et al. sion, sorting, constraint satisfaction and optimisation. Metalearning for algorithm selection has also been investigated in the area of time series forecasting, where the term was ﬁrst used in Prudencio and Ludermir (2004b). A comprehensive and recent treatment of the subject can be found in Wang et al. (2009) and Lemke and Gabrys (2010), where time series are clustered according to their characteristics and recommendation rules or combination weights derived with machine learning algorithms. In the area of data mining, algorithm recommendation was identiﬁed as an important research issue at the 2001 KDD conference and the 2006 KDD workshops according to Brazdil et al. (2009). Several systems for algorithm recommendation have been implemented. Following a previous successful European commission funded project, a project named ‘meta-learning assistant for providing user support in machine learning and data mining’ (METAL 2002), investigated model selection and combination approaches. A tool resulting from this project is thedataminingadvisor(DMA),aweb-basedsystemprovidingrankingsofclassiﬁcationalgo- rithms for users as described in Giraud-Carrier (2005), which however is no longer accessible. The last updates on the METAL project webpage date back to 2004/2005, and the webpage is currently ofﬂine. There is however another project focussing on preprocessing for data min- ing with a metalearning approach, called MiningMart (Morik and Scholz 2004). Although the software tool is still available for download,2 its most recent version is dated October 2006. 3.3 Dynamic bias selection In classic algorithm recommendation, the bias depends on the available learning algorithm chosen and is not modiﬁed dynamically. Dynamic bias selection is often mentioned in relation to a continuous ﬂow of training examples (data streams), where metalearning algorithms can be used for bias management and detecting concept drifts, as argued in Brazdil et al. (2009). In their metalearning review, Vilalta and Drissi (2002a) cite Bruha and Famili (2000) as the only example of dynamic bias selection apart from a few projects before 1992. In this contribution, a rule-based system which includes quality information about each rule, inﬂuencing the prediction process is used. The quality of a rule is updated continuously, which makes selection of the bias dynamic. In classic algorithm recommendation, the bias depends on the available learning algorithm chosen and is not modiﬁed dynamically. Dynamic bias selection is often mentioned in relation to a continuous ﬂow of training examples (data streams), where metalearning algorithms can be used for bias management and detecting concept drifts, as argued in Brazdil et al. (2009). In their metalearning review, Vilalta and Drissi (2002a) cite Bruha and Famili (2000) as the only example of dynamic bias selection apart from a few projects before 1992. In this contribution, a rule-based system which includes quality information about each rule, inﬂuencing the prediction process is used. The quality of a rule is updated continuously, which makes selection of the bias dynamic. Very fast decision trees (VFDT) according to Domingos and Hulten (2000) dynamically adjust their biases with new incoming examples, with bias in this case referring to splitting tests in the tree nodes. Another work qualifying as dynamic bias selection is the Learnt Topology Gating Artiﬁcial Neural Networks (LTGANN) by Kadlec and Gabrys (2008). In this contribution, neural networks are used for three different purposes: as base learners, as ‘local experts’ predicting the performance of one assigned base learner and as gating networks. The gating networks are used to discover relationships between different network topologies and their performance, inﬂuencing the topology of new networks added to the system. 3.4 Inductive transfer A different ﬂavour of metalearning runs under the name of ‘inductive transfer’, or ‘learning to learn’. The emphasis here is not on investigating a preferably extensive set of problems as in algorithm selection; it is mostly used for a smaller selection of multiple related learning tasks. However, in the spirit of metalearning, knowledge from one problem domain is transferred across other domains or tasks. A learning mechanism thus accumulates experience that is supposed to improve performance through time. This notion puts a bigger focus on aspect 2 123 Metalearning: a survey of trends and technologies 123 of Deﬁnition 1. Although aspect 1 still applies, most research in inductive transfer looks at a single learning mechanism and not a whole learning system. of Deﬁnition 1. Although aspect 1 still applies, most research in inductive transfer looks at a single learning mechanism and not a whole learning system. Brazdiletal.(2009)distinguishtwomainformsofknowledgetransferusingtheexampleof neural networks. One is representational transfer, where training of the base learner is carried outsequentially. Functionaltransfer refers to training severalneuralnetworks in parallelwhile sharing all or a part of their internal structure. A special case of functional transfer is multitask learning, where a neural network uses output nodes to accommodate for more than one task. Evgeniou et al. (2005) discusses inductive transfer in kernel methods using the example of Support Vector Machines and regularisation networks. Single-task kernel learning algorithms are extended to multi-task learning by forcing all hypotheses to share a common component along with the problem-speciﬁc deviations. Kernel learning from a different perspective is presented in Aiolli (2012): a suitable kernel matrix for a linear Hebbian classiﬁer is learnt with improving a basic kernel by learning chains of kernel transforms. Based on the results on an initial problem set, the kernel can then be applied to related problems. In a special issue on metalearning, Silver and Bennett (2008) present several contributions in the ﬁeld of inductive transfer. Zhang et al. (2008) approaches the subject from a Bayesian perspective, using a framework including latent variables for modelling a shared structure among different learning scenarios. Silver et al. (2008) uses a neural network with extra contextual inputs for different tasks. The task hence shifts to ﬁnding a single learner for the domains, only distinguished (‘indexed’) by the additional inputs. 3 http://www.e-lico.eu/. 3.4 Inductive transfer In a recent survey article Pan and Yang (2010), inductive transfer is presented as a special case of a wider category of ‘transfer learning’, alongside transductive and unsupervised transfer learning. Transfer learning has been deﬁned as a mechanism ‘which aims to help improve the learning of the target predictive function in a new domain, using the knowledge gained in other domains’. Although this does not strictly adhere to Deﬁnition 1, the authors discuss relevant research issues of (1) what knowledge to transfer, (2) how to transfer it, and (3) in which situations to transfer the knowledge. This last point seems especially interesting as it can be equally well cast as in which situations not to transfer the knowledge, since it may happen that the source and destination domains are not related. In such a case the performance of the new model rather than beneﬁting, could suffer from the knowledge transfer (so called ‘negative transfer’) (Pan and Yang 2010). 3.5 Metalearning systems The major usual steps of a modelling process consist of data analysis, data preprocessing, model building and a phase of interpretation and evaluation. Rather than applying metalearn- ing in only a part of this process, latest research started taking a more holistic view by investigating metalearning frameworks and architectures. While not yet mentioned in the seminal review of Vilalta and Drissi (2002a), Brazdil et al. (2009) give an overview of (semi-)automatic metalearning systems for data mining, only two of which target more than one aspect of the knowledge discovery process: Project CITRUS according to Wirth et al. (1997) seems to have been discontinued after a few publi- cations in 1996 and 1997, however, it did target the complete modelling process. A bit more recently, Bernstein et al. (2005) propose the Intelligent Discovery Assistant (IDA), providing a template for ontology-driven assistants for knowledge discovery dealing with preprocess- ing, model building and post-processing. A realization of this idea has been materialized within the e-LICO project,3 which investigated the concept of ‘meta-mining’—ontology and 123 124 C. Lemke et al. metaknowledge driven recommendation of data mining workﬂows as proposed in Nguyen et al. (2011) and evaluated in Nguyen et al. (2012). A somewhat related term, ‘domain- driven data mining’, also gained some recognition in the last years. The idea promotes a ubiquitous intelligence to be incorporated into the data mining process from a more method- ological point of view, increasing reusability and understanding of algorithms. An overview of research done in this area and a discussion of future challenges and issues can be found in Cao (2010). metaknowledge driven recommendation of data mining workﬂows as proposed in Nguyen et al. (2011) and evaluated in Nguyen et al. (2012). A somewhat related term, ‘domain- driven data mining’, also gained some recognition in the last years. The idea promotes a ubiquitous intelligence to be incorporated into the data mining process from a more method- ological point of view, increasing reusability and understanding of algorithms. An overview of research done in this area and a discussion of future challenges and issues can be found in Cao (2010). For continuous streams of data and the example of softsensors in process industry, Kadlec and Gabrys (2009) develop an architecture promoting life-long learning, where base-learners and pre- and post-processing methods can be plugged in and dynamically combined. A metalearning component optimises the system with regard to a global performance function. 3.5 Metalearning systems JankowskiandGrabczewski(2009)describetheimplementationofanextensivemetalearning architecture in detail, dealing with aspects like algorithm selection and parameter tuning, with the latter topic also addressed in Molina et al. (2012) for automatic tuning of parameters of decision trees. METALA is an agent-based architecture with a metalearning component, which has been implemented as a J2EE based framework with details given in Hernansaez et al. (2004). It supports an arbitrary number of algorithms, which are managed by agents. Using metalearn- ing on statistical and information-theoretic task properties, the best algorithm is dynamically selected, if the pool of algorithms and tasks is updated. A distinct concept of lazy metalearning has been extensively studied in Bonissone (2012). The motivation for this work is the apparent lack of automation in model creation, which leads to bottlenecks in the models lifecycle and scalability (two other attempts at automa- tion of model development methodology can also be found in Budka and Gabrys 2010; Budka et al. 2010). The main premise in lazy metalearning is that there is access to a large library of both local and global, pre-computed models together with their meta-information, which are perceived as commodity and which have been enabled by the recent expansion of cloud computing and crowdsourcing. The idea is to build dynamic on-demand ensem- bles using the models from the library as the building blocks, rather than optimizing and tuning of pre-computed models (Bonissone 2012). Since exhaustive enumeration of all possible combinations of models in a library would be intractable, a multi-step selection process based on metaknowledge, query information and correlation of errors of the ensem- ble members is employed. According to Bonissone (2012), a multi-criteria decision making process is followed in order to (1) create the model by pre-selecting the initial building blocks for the assembly and compiling their meta-information, which is an off-line phase, and (2) perform dynamic model assembly, where the best subset of models for a given query is selected on-line, i.e. during runtime. The process is also able to determine the weights of the ensemble models in the fusion schema, based on their local performance around the query. Some other recent approaches to development of metalearning frame- works can be found in Matijaš et al. (2013) for the problem of electricity load forecasting, Abbasi et al. (2012) for ﬁnancial fraud detection or Tsai and Hsu (2013) for bankruptcy prediction. 4 Considerations for using metalearning Before applying metalearning to any problem, certain practical choices have to be made. This includes the choice of a metalearning algorithm, which can even constitute a meta- metalearning problem itself. Selection of appropriate metaknowledge and the problem of 123 125 Metalearning: a survey of trends and technologies setting up and maintaining metadatabases have to be tackled, research efforts of which will be summarised in this section. setting up and maintaining metadatabases have to be tackled, research efforts of which will be summarised in this section. 4.2 Metalearning algorithms 4.2 Metalearning algorithms Vanschoren (2010) gives a survey on efforts to describe properties of algorithms. The author distinguishes qualitative properties (for example type of data that can be handled, learning strategy, incrementality) and quantitative properties (bias-variance proﬁle, runtime properties like scalability and resilience). In an effort to ﬁnd an implementation and vendor-independent method for representing machine learning models, the XML-based standard PMML has been developed and gained some recognition in the last years. A detailed description of PMML can be found in Guazzelli et al. (2009). The choice of a metalearning algorithm naturally depends on the problem and the task to be solved. Generally, traditional classiﬁcation algorithms are very successful in metalearn- ing algorithm selection and can include meta-decision trees (Todorovski and Dzeroski 2003), neural networks, Support Vector Machines or any other classiﬁcation algorithms, with the k-Nearest Neighbours being another popular choice (Brazdil et al. 2009). Applying regres- sion algorithms is less popular, even smaller is the number of available algorithms to learn rankings. One of the simplest ranking method involves dividing the problem space using clustering of available datasets according to a distance measure (usually k-Nearest Neigh- bour) of the metafeatures and using average performance ranks of the cluster into which a new problem falls (Brazdil et al. 2003). Brazdil and Soares (2000) also look at the magnitude and signiﬁcance of the differences in performance. The NOEMON approach introduced by Kalousis and Theoharis (1999) builds classiﬁers for each pair of base forecasting methods with a ranking being generated using the classiﬁers’ outputs. Todorovski et al. (2002) build decision trees using the positions in a ranking as target values. 4.3 Extracting metaknowledge 4.1 Prerequisites As also elaborated on in Brazdil et al. (2009), metalearning can not be seen as a magic cure to machine learning problems for a variety of reasons. First of all, the extracted metafeatures need to be representative of their problem domain, otherwise, an algorithm will fail to identify similar domains. On the same note, if a problem has not been seen before, metalearning will be unable to exploit past knowledge to improve prediction performance. Performance estimation may be unreliable because of the natural limitations of estimating the true performance of the dataset. Different metafeatures might be applicable to each dataset. These issues emphasise the importance of being critical when designing a metalearning system. 4.3 Extracting metaknowledge According to Brazdil et al. (2009), metaknowledge is derived in the course of employing a learning system. A very common form of metaknowledge is the performance of algorithms in certain problem domains, which is to be linked with characteristics of the task. Several possibilities for characterising a problem domain exist. The most straightforward form of metaknowledge extracted from the data include sta- tistical or information-theoretic features. For classiﬁcation problems, Brazdil et al. (2009) mention the number of classes and features, ratio of examples to features, degree of corre- lation between features and target concept and average class entropy. For other application areas, features can look completely different, as for example summarised in Lemke et al. 123 123 126 C. Lemke et al. (2009) for the area of time series forecasting, where features can include, for example, length, seasonality, autocorrelation, standard deviation and trends of the series. (2009) for the area of time series forecasting, where features can include, for example, length, seasonality, autocorrelation, standard deviation and trends of the series. Vilalta and Drissi (2002b) propose measures for the difﬁculty of a classiﬁcation problem that can be used as an input for metalearning. They include class variation, denoting the probability that, by means of a distance measure, any two neighbouring data records have a different class value and example cohesiveness, measuring the density of the example distribution in the training set. In a similar approach, Köpf and Iglezakis (2002) also suggest comparing observations with each other and extract ‘case base properties’, which assess the quality of a dataset using measures such as redundancy, for example induced by data records that are exactly the same, or incoherency, which, for example occurs if data records have the same features but different class labels. Alternatively to looking at the data only, information of individual algorithms and how they solved the problem can be considered, for example their predicted conﬁdence inter- vals. This can be achieved by using a model that is fast to build and train and investigating its properties. In this spirit, Bensusan et al. (2000) suggest building a decision tree for a classiﬁcation problem and using properties of the tree such as nodes per feature, tree depth or shape to characterise it. Another approach is landmarking as proposed in Pfahringer et al. 4.3 Extracting metaknowledge (2000), using the performance of simple algorithms to describe a problem and corre- lating this information with the performance of more advanced learning algorithms. A list of landmarking algorithms can be found in Vanschoren (2010). Landmarking algorithms can also be run on only a small sample of the data available, reducing the training time required. Performance information of different algorithms and learning curves generated when more data is added to the training set can then be used to select an algorithm according to Fürnkranz et al. (2002). Empirical evaluation of different categories of metafeatures in the context of their suitabil- ity for predicting classiﬁcation accuracies of a number of standard classiﬁers can be found in Reif et al. (2012c). The authors distinguish 5 such categories of features i.e. simple, statisti- cal, information-theoretic, landmarking and model-based, which corresponds to the general categorization evident from the literature. As with any learning problem, metalearning is subject to the ‘curse of dimensionality’ (Bishop 1995) and other issues, which can traditionally be solved by selecting a subset of relevant features. Although to the best of our knowledge, in the context of metalearning this issue has only been addressed in relatively few publications (e.g. Kalousis and Hilario 2001; Reif et al. 2012c; Todorovski et al. 2000), we assume that the reason for this is quite simple—meta-feature selection does not differ from feature selection at the base-level, and the machine learning literature is very rich in this regard (a comprehensive review of various feature selection techniques can be found in Guyon and Elisseeff 2003). 4.4 Metadatabases As metalearning proﬁts from knowledge obtained while looking at data from other problem domains, having sufﬁcient datasets at one’s disposal is important. Soares (2009) propose transforming existing datasets (‘datasetoids’) to obtain a larger number of them and show success of the approach on a metalearning post-processing problem. Vanschoren (2010) states that there is no lack of experiments being done, but datasets and information obtained often remain in ‘people’s heads and labs’. He proposes a framework to export experiments to speciﬁcally designed experiment databases based on an ontology for experimentation in machine learning. The resulting database can then, for example, give information on rankings of learning algorithms, the behaviour of ensemble methods, learning curve analyses and the Metalearning: a survey of trends and technologies 127 bias-variance behaviour of algorithms. One example of such database can be The Open Experiment Database.4 An analysis of this database together with a critical review can be found in Driessens et al. (2012). bias-variance behaviour of algorithms. One example of such database can be The Open Experiment Database.4 An analysis of this database together with a critical review can be found in Driessens et al. (2012). An alternative approach to the problem of scarcity metadatabases has been presented in Reif et al. (2012a), where the authors describe a dataset generator able to produce synthetic datasets with speciﬁed values of some metafeatures (like kurtosis and skewness). Although the proposed generator appears to be at a very early stage of development, the idea is deﬁnitely very promising, also from the point of view of performing controlled experiments on datasets with speciﬁed properties. Similarly to feature selection, synthetic data generation has received a considerable attention in the recent generic machine learning and data mining literature, especially in the context of data streams and concept drift (please see Bifet et al. 2011 and references therein). 4 http://expdb.cs.kuleuven.be/. 5 Conclusions and research challenges Research in the area of metalearning is continuing in several directions. One area is the identiﬁcation of metafeatures. As mentioned before, the vast majority of publica- tions investigates extracting features from the dataset, mostly in the form of statisti- cal or information theoretic measures. Landmarking is a different approach using sim- ple base learning algorithms and their performance to describe the dataset at hand. How- ever, Brazdil et al. (2009) argue that characteristics of learning algorithms and gaining a better understanding of their behaviour would be a valuable research avenue with very few publications, for example Vanschoren and Blockeel (2006), that exist in this area to date. A lot of publications on metalearning focus on selecting the base-learning method that is most likely to perform well for a speciﬁc problem. Fewer publications like Brazdil et al. (2003) and Prudencio and Ludermir (2004a) consider ranking algorithms, which can be used to guide combination weights and to increase robustness of a metalearning sys- tem. Regarding adaptivity and continuous monitoring, many approaches go further than the static traditional metalearning approaches, for example by using architectures that support life-long learning such as in Kadlec and Gabrys (2009). However, research in this area can still go a long way further investigating continuous adjustment, rebuilding or discarding of base-learners with the help of metalearning approaches. Users of predictive systems are faced with a difﬁcult choice of an ever increasing number of models and techniques. Metalearning can help to reduce the amount of experimentation by providing dynamic advice in form of assistants, decrease the time that has to be spent on introducing, tuning and maintaining models and help to promote machine learning outside of an academic environment. In this context, architectures and frameworks using metalearning have been named in Sect. 3.2. 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https://openalex.org/W2794183448	https://scholarlypublications.universiteitleiden.nl/access/item%3A2953263/view	English	null	Current Understanding of the Neural Mechanisms of Dissociation in Borderline Personality Disorder	Current behavioral neuroscience reports	2,018	cc-by	9,318	Current Behavioral Neuroscience Reports (2018) 5:113–123 https://doi.org/10.1007/s40473-018-0146-9 Current Behavioral Neuroscience Reports (2018) 5:113–123 https://doi.org/10.1007/s40473-018-0146-9 PERSONALITY AND IMPULSE CONTROL DISORDERS (R LEE, SECTION EDITOR) PERSONALITY AND IMPULSE CONTROL DISORDERS (R LEE, SECTION EDITOR) PERSONALITY AND IMPULSE CONTROL DISORDERS (R LEE, SECTION EDITOR) Abstract Purpose of Review In this article, we aim to give an overview over recent neuroimaging research on dissociation in borderline personality disorder (BPD). Stress-related dissociation is highly prevalent in BPD, while so far only little is known about its neural underpinnings. Recent Findings Based on research in depersonalization and the dissociative subtype of posttraumatic stress disorder, it has been proposed that dissociation involves alterations in a cortico-limbic network. In BPD, neuroimaging research explicitly focusing on dissociation is still scarce. Summary Functional neuroimaging studies have provided preliminary evidence for an altered recruitment and interplay of fronto-limbic regions (amygdala, anterior cingulate, inferior frontal gyrus, medial and dorsolateral prefrontal cortices) and temporoparietal areas (superior temporal gyrus, inferior parietal lobule, fusiform gyrus), which may underlie disrupted affective-cognitive processing during dissociation in BPD. More neuroimaging research with larger samples, clinical control groups, and repeated measurements is needed to deepen the understanding of dissociation in BPD. Keywords Borderline personality disorder . Depersonalization . Dissociation . Traumatic stress . Neuroimaging ds Borderline personality disorder . Depersonalization . Dissociation . Traumatic stress . Neuroimaging Current Understanding of the Neural Mechanisms of Dissociation in Borderline Personality Disorder Annegret Krause-Utz1,2 & Bernet Elzinga1,2 Published online: 12 February 2018 # The Author(s) 2018. This article is an open access publication Published online: 12 February 2018 # The Author(s) 2018. This article is an open access publication 2 Leiden Institute for Brain and Cognition (LIBC), Leiden, The Netherlands 1 Institute of Psychology, Leiden University, Leiden, The Netherlands Definitions and Etiological Models of Dissociation 51]. The traumatic event may be perceived as a film-like scene from a wider distance. Salient sensory information may be processed in a distorted way and perception of time may be substantially altered. Emotional numbing and depersonaliza- tion may help create an inner distance from overwhelming and horrifying experiences that cannot be integrated into existing views of the world, self, and others. Such distortions in time, thought, body (e.g., out of body experiences, such as the sense of floating above one’s body), and emotions may later re- occur as distinctive “trauma-related states of consciousness” (TRASC) [52]. It has been shown that individuals who expe- rienced peritraumatic dissociation are more likely to dissociate in other stressful life situations later on in life [34, 52–54]. Thereby, dissociation can interfere with the development of identity [24, 38, 55] and lead to a compartmentalization (fragmentation) of memories [56]: Salient characteristics of the stressful event are encoded and stored as separate ele- ments, which may later re-occur as unwanted implicit flash- back memories [51, 57–60], increasing the risk of posttrau- matic stress symptoms [34, 54, 61, 62]. Several studies pro- vided evidence for a disruptive effect of dissociation on neu- ropsychological functioning [13, 51, 57, 63], although this relationship is not yet completely understood and cannot be generalized across different (clinical) groups [64–66]. Dissociation has been defined as “disruption of and/or discon- tinuity in the normal, subjective integration of one or more aspects of psychological functioning, including – but not lim- ited to – memory, identity, consciousness, perception, and motor control” [24] (p. 826). The broad variety of symptoms can be classified into the following three categories: (1) a loss of continuity in subjective experience, accompanied by invol- untary and unwanted intrusions into awareness or behavior (e.g., dissociative flashbacks); (2) an inability to access infor- mation or control mental functions that are normally amenable to such control or access (e.g., dissociative amnesia); and (3) a sense of experiential disconnectedness, including distorted perceptions about the self or the environment (e.g., deperson- alization, derealization) [26]. Acute dissociative states (state dissociation) are usually distinguished from a more general tendency to experience dissociative symptoms (trait dissociation), while both may go hand in hand. Of note, dis- sociative experiences are not necessarily pathological but, to a lesser extent, are also present in non-clinical healthy popula- tions [27, 28]. Etiological Models While the precise neural mechanisms of dissociation remain elusive, neuroimaging research in depersonalization disorder, dissociative subtype (D-PTSD), and dissociative identity dis- order has provided important insights into brain networks pos- sibly implicated in dissociation. In the following, current neu- robiological conceptualizations stemming from this research are briefly introduced. Interacting with genetic, neurobiological, and cognitive dispo- sitions, traumatic stress can play an important role in the de- velopment of pathological dissociation [32–44]. A history of trauma is neither necessary nor sufficient for the development of dissociation and/or BPD. Yet, dissociation in BPD has been closely linked to chronic interpersonal trauma, such as severe childhood abuse and neglect [6, 25, 45, 46]. Peritraumatic dissociation immediately during the traumatic event [34] has been partly conceptualized analogous to freezing responses in animals. The proximity of threat may first elicit an orienting response (fight or flight response; [47]), associated with an increase in sympathetic nervous system function (e.g., heart rate, blood pressure, release of stress hormones), to prepare the organism for an active defense mechanism. If the organism cannot escape, it may more likely engage in a passive defense mode, accompanied by tonic immobility (freezing), an in- crease in parasympathic activity and a shutting-down of the arousal system [33, 38, 48, 49]. If chances of winning a fight or escaping are low or not given, passive defense responses such as tonic immobility may reduce the risk of being detected or more severely injured [48, 50]. In general, direct transla- tions from animal studies to humans are not possible due to conceptual and methodological differences [49]. Yet, dissocia- tive responses, such as tonic immobility, in humans might be an evolutionary-based regulatory process, which may enhance survival or the coping with extreme threat [22, 25, 26, 38, 44, Introduction to linearly increase with the level of subjective distress [11]. Furthermore, there is evidence for a detrimental effect of dis- sociation on emotional learning and memory [12–15], which may partly explain why dissociative symptoms can contribute to poor therapy outcome in patients with BPD [16–19]. So far, very little is known about the neural mechanisms of disturbed information processing during dissociation: Only few neuro- imaging studies in BPD explicitly focused on dissociation, compared to the growing body of research in other trauma- related and dissociative disorders (e.g., dissociative subtype of posttraumatic stress disorder). Stress-related dissociative states, including depersonalization, derealization, analgesia, and emotional numbing, are a clinical hallmark of borderline personality disorder (BPD), occurring in about 75–80% of patients [1–6]. In the current version of the Diagnostic and Statistical Manual of Mental Disorders (DSM5), transient “dissociative states under stress” are listed among the diagnostic features of BPD [7]. Such states usually last for minutes or hours [8–10] and are often linked to stress- driven rapid shifts in self-image [10]. The intensity, frequency, and strength of self-reported dissociative states in BPD seem In the following, we introduce current conceptualizations of dissociation, based on more substantial neuroimaging re- search that has been conducted in patients with depersonali- zation, the dissociative subtype of posttraumatic stress disor- der, and dissociative identity disorder (a comprehensive re- view of this literature is beyond the scope of this article; for further reviews see, e.g., [20–25]). Based on this literature, we then describe and discuss recent neuroimaging research on dissociation in BPD (focusing on functional neuroimaging studies) together with its implications, methodological limita- tions, and suggestions for future research. This article is part of the Topical Collection on Personality and Impulse Control Disorders * Annegret Krause-Utz a.d.krause@fsw.leidenuniv.nl 1 Institute of Psychology, Leiden University, Leiden, The Netherlands 2 Leiden Institute for Brain and Cognition (LIBC), Leiden, The Netherlands This article is part of the Topical Collection on Personality and Impulse Control Disorders * Annegret Krause-Utz a.d.krause@fsw.leidenuniv.nl 1 Institute of Psychology, Leiden University, Leiden, The Netherlands 2 Leiden Institute for Brain and Cognition (LIBC), Leiden, The Netherlands This article is part of the Topical Collection on Personality and Impulse Control Disorders * Annegret Krause-Utz a.d.krause@fsw.leidenuniv.nl * Annegret Krause-Utz a.d.krause@fsw.leidenuniv.nl 114 Curr Behav Neurosci Rep (2018) 5:113–123 Definitions and Etiological Models of Dissociation The present article focuses on pathological dissociation, as highly prevalent in BPD, PTSD, and dissocia- tive disorders [9, 10, 29–31]. Cortico-Limbic Disconnection Model: Research in Depersonalization Disorder Already in 1998, Sierra and Berrios proposed that a “discon- nection” of cortico-limbic brain regions underlies symptoms of depersonalization, such as numbing, analgesia, hypervigi- lance, and emptiness of thoughts [67]. In their cortico-limbic disconnection model, the authors propose that increased activ- ity in medial and dorsolateral prefrontal cortices (areas impli- cated in cognitive control and arousal modulation) both direct- ly and indirectly via the anterior cingulate cortex (ACC) [68–70] dampens activity in the amygdala, which is crucial to the initiation of stress and fear responses [71, 72]. In addi- tion to the aforementioned cortico-limbic regions, the posteri- or cingulate, hippocampus, superior temporal gyrus, precuneus, temporoparietal junction, inferior parietal lobe, an- gular gyrus, frontopolar cortex, and medial prefrontal cortex (mPFC) have been implicated in states of depersonalization 115 Curr Behav Neurosci Rep (2018) 5:113–123 [73–75]. These areas are parts of the default mode network, which has been implicated in inward-directed processes, such as autobiographical memory retrieval, daydreaming, rumina- tion, pain processing, and mentalizing [76–81]. Alterations in the abovementioned regions may underlie changes in self-per- ception, such as “feeling unreal” [67, 82] and altered intero- ceptive awareness during depersonalization [68, 74, 75, 83] [84–86]. conscious level—but not when threatening stimuli were pre- sented non-consciously. In the latter condition, these patients showed increased activity in the amygdala, insula, and thala- mus, suggesting that regulatory processes during dissociation may be partly explained by conscious top-down processes, which might not work on a non-conscious level [91]. Further evidence for altered fronto-limbic interactions, in terms of a stronger coupling of the amygdala with fronto- parietal regions, stems from resting-state functional magnetic resonance imaging (RS-fMRI) studies investigating connec- tivity patterns in the absence of experimental stimulation [93]. Nicholson and colleagues (2017) used dynamic causal model- ing (DCM) to more directly investigate directed (effective) connectivity between functional nodes of fear and emotion regulation circuits [94]. In PTSD patients with the dissociative subtype, a predominate pattern of top-down connectivity from the ventromedial PFC (vmPFC) to the amygdala and periaqueductal gray (PAG) and from the amygdala to the PAG was found. In contrast, PTSD patients without the dis- sociative subtype predominately showed bottom-up connec- tivity from the amygdala to the vmPFC and from the PAG to the vmPFC and amygdala. Emotion Overmodulation: Research in the Dissociative Subtype of PTSD and in DID Further evidence for alterations in cortico-limbic and default mode network regions stems from research in PTSD patients with the D-PTSD, which has recently been introduced in DSM5 [7]. As proposed by Lanius and colleagues [22] (p. 640), dissociation may involve an increase in self-control and arousal modulation (“emotion overmodulation”), associ- ated with increased activation in frontal regions (dorsal/rostral ACC, mPFC) and dampened limbic activity in the amygdala and insula. Patients predominantly showing hyperarousal and re-experiencing symptoms (emotion under-modulation) are thought to exhibit the reverse pattern: limbic hyperactivity and diminished recruitment of the ACC and mPFC. Central to this model were observations from a script-driven imagery study, in which PTSD patients were exposed to autobiograph- ical narratives of traumatic events [87, 88]. The majority of patients reported pronounced re-experiencing symptoms (traumatic flashbacks, intense feelings of shame, disgust, guilt, hopelessness, etc.), associated with an increase in heart rate. In contrast, patients with the dissociative subtype dem- onstrated increased activity in areas implicated in emotion regulation, arousal modulation, and sensory filtering (e.g., me- dial frontal gyrus, anterior cingulate, middle temporal gyri, precuneus, occipital areas, and inferior frontal gyrus) [87]. Patients with the dissociative subtype exhibited a stronger coupling of the ventrolateral thalamus with right insula, mid- dle frontal gyrus, superior temporal gyrus, cuneus, and left parietal lobe (regions implicated in emotion regulation), while connectivity between the thalamus and right parahippocampal gyrus and superior occipital gyrus (areas implicated in sensory and emotion processing) was diminished [88]. The thalamus functions as a sensory gate or filter, receiving direct input from both subcortical limbic regions and frontal areas, and may therefore play an important role in dissociative-like states of altered consciousness [21, 89, 90]. More specifically, the thal- amus may either facilitate or impede the flow of information to cortico-limbic structures [21] and thereby contribute to de- fensive regulatory processes [91, 92]. i l i i h h di i i b hib Higher depersonalization/derealization severity further pre- dicted decreased connectivity between the perigenual ACC and ventromedial PFC as well as altered synchrony between the default mode network and the central executive network [98]. Extending the abovementioned finding, these results point to an altered synchrony, i.e., switching between large- scale networks implicated in rest and executive control in the dissociative subtype of PTSD. Cortico-Limbic Disconnection Model: Research in Depersonalization Disorder These differences in resting-state connectivity in brain circuits associated with fear processing [94] may translate into symptoms of emotion over—versus under—modulation in PTSD patients with versus without the dissociative subtype [95, 96]. In this context, the PAG may play an important role in passive defense responses dur- ing dissociation [97]. Emotion Overmodulation: Research in the Dissociative Subtype of PTSD and in DID Interestingly, patients with dissociative identity disorder (DID) demonstrated distinct neural response patterns, depend- ing on the presence or absence of dissociation, which highly resemble observations in PTSD: While being in a “hyper- aroused state” or “traumatic identity state” with voluntary ac- cess to traumatic memories, DID patients showed elevated cardiovascular responses (heart rate, blood pressure) and stronger amygdala and insula activity, along with lower activ- ity in cingulate gyrus, parietal cortex, and para-hippocampus. The opposite response pattern was observed when patients were in their “normal dissociative identity state,” character- ized by dissociative amnesia [99, 100]. Studies including clin- ical control groups are needed to clarify whether altered activ- ity patterns in fronto-limbic areas described above are specific to the dissociative subtype of PTSD or a trans-diagnostic phe- nomenon of emotion overmodulation, extending to other stress-related disorders including DID and BPD. Interestingly, patients with the dissociative subtype exhib- ited enhanced activity in the ventral PFC, suggesting in- creased emotion downregulation, when threatening (fearful versus neutral) facial expressions were presented on a Curr Behav Neurosci Rep (2018) 5:113–123 116 Resting-State Neuroimaging Studies Several studies in BPD examined links between self-reported dissociation and findings of altered baseline glucose metabo- lism and cerebral blood flow during rest. Several studies using positron emission tomography (PET) [104], single-photon emission computed tomography (SPECT) [105], or continu- ous arterial spin labeling [106] did not report significant links between altered baseline metabolism/activity and dissocia- tion, while self-reported dissociation predicted changes in resting-state functional connectivity (RSFC) in two fMRI studies [107, 108]. Wolf and colleagues (2011) found positive correlations between scores on the Dissociation Stress Scale (DSS) and RSFC in the insula and precuneus. Krause-Utz and colleagues (2014) investigated whether scores on the Dissociative Experiences Scale (DES) [109] predicted RSFC of the bilateral amygdala and ACC in BPD. Higher trait dis- sociation positively predicted amygdala RSFC with the dor- solateral prefrontal cortex (dlPFC) and negatively predicted left amygdala RSFC with a cluster in the occipital lobe (cuneus, intracalcarine cortex, and fusiform gyrus). More resting-state fMRI studies with larger sample sizes and clinical control groups are needed to understand the precise mecha- nisms underlying the abovementioned RSFC patterns in BPD. Future studies in BPD may acquire resting-state scans before and after dissociation induction or therapeutic interventions aimed at a reduction of dissociation to gain more insight into the impact of dissociation on RSFC in large-scale brain networks. A re-analysis of this data set showed that self-reported state dissociation further positively predicted functional connectiv- ity of the amygdala with right thalamus, right ACC, left insula, and left precentral gyrus during negative distractors [112]. Altered interaction between these regions may underlie altered processing of disturbing emotional information in the context of a cognitive task in BPD. In another study, higher trait dissociation (scores on the DES) predicted a stronger signal decrease of the default mode network in response to painful heat stimulation in BPD pa- tients with current self-injurious behavior [113]. As previously mentioned, the default mode network plays an important role in self-referential processing and pain processing. Alterations in this network might underlie altered processing of pain, e.g., as being less self-relevant or aversive, in patients with frequent dissociative experiences. Since in other studies no significant correlations between dissociation and limbic reactivity during painful heat stimulation [114, 115] or emotional words [116, 117] were found, future research is needed to investigate whether different kinds of experimental material and subsam- ples may have contributed to these discrepancies. Neuroimaging Research on Dissociation in BPD unpleasant pictures in BPD patients and patients with schizotypal personality disorder. Overall, BPD patients showed increased amygdala reactivity to repeatedly presented (versus novel) emotional pictures and a prolonged return to baseline of amygdala compared to healthy controls. In line with this, Krause-Utz and colleagues (2012) found significant associations between self-reported dissociation and amygdala reactivity to negative pictures, presented as distractors in the context of a working memory task, in BPD. The Emotional Working Memory Task (EWMT) used in this study is a mod- ified version of a Sternberg item-recognition task, in which participants are instructed to memorize task-relevant informa- tion (a set of letters) over a short delay interval. Afterwards, a probe (another set of letters) is presented and participants are instructed to indicate whether one of these letters was part of the previous set (memoranda) or not by pressing a “yes” or “no” button. In half of the trials, a target is present, while in the other trials, the target is absent. During the delay interval, either no distractors (only a fixation cross) or distracting neu- tral versus negative pictures of interpersonal scenes are pre- sented. Negative pictures contain scenes of interpersonal vio- lence, such as sexual or physical assault, a beaten child, or a physically mutilated body. Participants are instructed to ignore these distractors and to respond as fast and accurately as pos- sible to the probes, i.e., to voluntarily inhibit emotion process- ing in favor of cognitive processing. During presentation of neutral and negative interpersonal scenes, BPD patients showed significantly longer reaction times and significantly increased activity in the amygdala (among other regions), sug- gesting higher emotional distractibility than healthy controls. At the same time, self-reported dissociation was negatively correlated to amygdala activity in BPD. Compared to research in D-PTSD and dissociative disorders, studies explicitly focusing on dissociation in BPD are still relatively scarce. Most evidence stems from studies that ex- amined correlations between neuroimaging findings and clin- ical measures of dissociation, without explicitly and/or exclu- sively focusing on dissociation or inducing this symptom [101–103] [104–116]. To our knowledge, so far, only three studies used script-driven imagery to more directly investigate the effect of experimentally induced dissociation on neural processing in BPD. Findings of functional neuroimaging stud- ies are described in more detail below. Script-Driven Imagery Studies in the left inferior frontal gyrus and dlPFC for negative versus neutral words and overall lower activity in the fusiform gyrus and inferior parietal and temporal cortices. Altered activity in brain regions implicated in interference inhibition [120] may underlie the disruptive effect of dissociation on cognitive per- formance in BPD. To our knowledge, so far, three fMRI studies used script- driven imagery to experimentally induce dissociation in indi- viduals with BPD [117–119]. Script-driven imagery is a well- established paradigm, aimed at provoking specific experi- ences through a recollection of autobiographical memories. Together with the experimenter, each participant creates a per- sonalized narrative of an autobiographical situation, e.g., a situation involving marked dissociative experiences (versus an emotionally neutral script). The script is usually recorded on an audio tape by the experimenter and later re-presented in an experimental setting, e.g., during fMRI. Participants are instructed to recall the specific situation, described in the script, as vividly as possible. Consistent across different stud- ies, script-driven imagery successfully induced dissociation, in terms of self-reported dissociative symptoms (on the DSS scale) in previous research [117–119]. In a pilot study by Ludaescher and colleagues (2010), self-reported state dissoci- ation, pain sensitivity, and changes in BOLD responses were assessed in 15 BPD patients exposed to either a dissociation script or a neutral script. While being exposed to the dissoci- ation script, patients showed increased activation in the left inferior frontal gyrus. Higher self-reported dissociative symp- toms predicted higher activation in the left superior frontal gyrus and lower activation in the middle and inferior temporal gyrus. BPD patients with comorbid PTSD (n = 10) addition- ally showed increased activation in the left cingulate gyrus. In this subgroup, self-reported dissociation positively predicted activity in the bilateral insula, while negatively predicting ac- tivity in the right parahippocampal gyrus. Findings of this study provided first evidence for increased frontal activity and diminished temporo-limbic activity during acute dissoci- ation in BPD, especially in a subgroup of patients with comor- bid PTSD. However, the sample size in this study was rela- tively small and no control group was included. Krause-Utz and colleagues (2017) combined script-driven imagery with the EWMT, described above, to investigate the effect of dissociation on working memory performance during emotional distraction. As mentioned above, a previous study using this task revealed negative associations between state dissociation and amygdala activity during emotional distrac- tion in BPD [111]. Task-Related fMRI Studies Some studies in BPD reported significant correlations be- tween clinical measures of dissociation and changes in blood-oxygen-level-dependent (BOLD) responses during the presentation of aversive images [110–112]. Hazlett and col- leagues (2012) found negative correlations between self- reported dissociation and amygdala reactivity during a habit- uation task with repeatedly presented (versus novel) 117 Curr Behav Neurosci Rep (2018) 5:113–123 Script-Driven Imagery Studies When performing the EWMT after disso- ciation induction, BPD patients showed significantly more errors and misses across all conditions compared to healthy controls and BPD patients exposed to a neutral script. BPD patients who underwent dissociation induction further showed an overall deactivation in the bilateral amygdala and lower activity in left cuneus, lingual gyrus, and posterior cingulate compared to patients with a neutral script. During emotional distraction, a stronger positive coupling of the amygdala with right middle/superior temporal gyrus and left inferior parietal lobule along with negative functional connectivity between amygdala and fusiform gyrus was found in patients after dis- sociation induction. These findings seem to provide further evidence for a disruptive effect of dissociation on affective- cognitive processing in BPD. Overall Discussion On a neural level, patients who underwent dissociation induction showed increased activity Curr Behav Neurosci Rep (2018) 5:113–123 118 regions implicated in arousal modulation, body movements, and the filtration of sensory information (ACC, insula, dlPFC, precentral gyrus, fusiform gyrus, inferior parietal lobule, and thalamus) were observed in BPD patients with acute dissoci- ation. A reduced coupling of the amygdala with occipital areas (fusiform gyrus), observed both during resting-state [108] and emotional distraction [112], may translate into altered gating and reduced processing of sensory input. Increased positive amygdala functional connectivity with the dlPFC during rest [108] and with the ACC, insula, and inferior parietal lobule during emotional distraction [112] may reflect increased at- tempts of arousal modulation, as suggested by research in the dissociative subtype of PTSD [22]. Stress-related dissociation in BPD may be a form of emotion modulation, especially in patients with severe childhood trauma, while so far there is not enough known about neural mechanisms which may underlie these processes [25, 29]. Studying the modulating effect of dissociation on amygdala reactivity and connectivity in BPD may have important implications for neurobiological concep- tualizations of the disorder, as amygdala hyperreactivity is currently considered a general key feature of the disorder, despite conflicting findings [122, 123]. causal modeling (DCM), may help gain more insight into these processes [94]. Moreover, longitudinal studies may help to clarify whether altered neural response patterns, observed in previous research, are a predisposition for or the result of frequent dissociative experiences. Cross-sectional correlation- al designs do not allow causal conclusions, e.g., it is also conceivable that patients who are more prone to dissociation show reduced awareness of emotions (alexithymia), which contributes to the observed correlations. A combination of different neuroimaging techniques, such as functional and structural MRI, in future research may help to obtain a more global picture of neural alterations associated with dissocia- tion [124]. The diversity of neuroimaging techniques (e.g., region of interest analyses and seed-based correlations versus data-driven clustering methods) makes it difficult to compare findings across studies, as each technique entails certain ad- vantages and limitations (see [125]). Given the general con- cerns about robustness and reproducibility of neuroimaging findings, large-scale meta-analyses, including original data sets and software, are needed to tease apart robust results from false-positive findings, especially for findings which are not corrected for multiple comparisons [125]. Overall Discussion The aim of this article was to give an overview over neuroim- aging studies on dissociation in BPD. Research in dissociative disorders and D-PTSD provided evidence for alterations in (pre)frontal, subcortical limbic, and tempo-parietal areas (parts of the default mode network). Extending previous ob- servations in depersonalization and D-PTSD [82, 88, 98], dis- sociative states in BPD have been associated with reduced activation in the inferior and superior temporal gyrus [117, 121] and a stronger positive coupling of this area with the amygdala during emotional distraction [118]. In addition, larger gray matter volumes in the middle and superior tempo- ral gyrus were linked to higher trait dissociation in these pa- tients [103]. Findings in BPD further point to a diminished responsiveness or deactivation of the amygdala during the presentation of aversive (trauma-related) pictures [110, 111, 118], while it remains unclear whether this observation is re- lated to certain tasks or sample characteristics (e.g., specific experimental stimuli, trauma history, medication). The amyg- dala is crucial to emotion process and the initiation of stress responses [71] and has been critically implicated in neurobio- logical models, conceptualizing dissociation as self-regulatory strategy to cope with overwhelming emotions [22, 67]. In line with this, altered interactions of the amygdala with brain Winter and colleagues (2015) combined script-driven im- agery with the Emotional Stroop Task (EST) and subsequent memory tasks to investigate the impact of dissociation induc- tion on the cognitive inhibition of emotional material in BPD. In the EST, either neutral or emotional words are presented in different colors. Participants are instructed to name the color of each word as fast and accurately as possible while ignoring its content. Before they performed the EST, 19 female BPD patients were exposed to a dissociation script (BPDd), while 18 BPD patients and 19 healthy controls were exposed to neutral scripts (BPDn). Patients who underwent dissociation induction showed more errors and slower reaction times than the other groups, together with longer reaction times in re- sponse to negative versus neutral words and impaired recall and recognition of EST words during a subsequent memory task compared to BPDn. Implications Pathological dissociation is a complex heterogeneous phe- nomenon comprising various symptoms [26–28, 42, 52]. A more precise differentiation between these symptoms in future fMRI research may help to clarify whether neural alterations are specifically related to distinct dissociative features, such as distortion in time, thought, body, and emotion [52]. Likewise, an extended and more precise neuropsychological assessment may help to better understand which neurocognitive processes are affected by dissociation and how this may translate into altered affective-cognitive functioning. It has been proposed that dissociation specifically involves diminished recollection of trauma-related information [126], while the existing litera- ture in the field is still heterogeneous [65]. To elucidate this relationship, future neuroimaging studies in BPD may inves- tigate the effect of dissociation on processing (e.g., autobio- graphical recall) of trauma-related material compared to gen- erally negative information. The combination of dissociation induction and affective-cognitive neuropsychological tasks in neuroimaging research may contribute to a better understand- ing of this relationship. Future studies may combine neuroim- aging techniques with advanced methods of ecological mo- mentary assessment in daily life to elucidate how specific dissociative symptoms (e.g., derealization versus dissociative amnesia) may relate to certain neural response patterns. A broader assessment of dissociative experiences in everyday Overall Discussion Finally, sample sizes of most studies are relatively small and studies with larger data sets are needed to replicate or extend existing findings. Three fMRI studies in BPD consistently observed in- creased activity in the left inferior frontal gyrus after dissoci- ation induction [117–119], possibly underlying altered sup- pression of impulses during emotional interference [120]. In the study by Winter and colleagues (2015), BPD patients performing an EST after dissociation induction further showed increased activity in the dlPFC, which plays an im- portant role in emotion downregulation [87, 88]. Alterations in the abovementioned areas may underlie altered information processing during dissociation in BPD. However, the precise neural mechanisms of dissociation need yet to be elucidated and findings need to be interpreted in the light of certain lim- itations, as discussed below. Compliance with Ethical Standards life situations might also help to enhance our understanding of the ecological validity of experimental paradigms, which are used to experimentally induce dissociation, such as the script- driven imagery paradigm. More specifically, future studies may address how well naturally occurring dissociative symp- toms correspond to dissociative symptoms provoked in the laboratory, e.g., by dissociation scripts. Conflict of Interest The authors declare that there are no conflicts of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors. Neuroimaging research on dissociation may also help to identify neural processes relevant for psychotherapy. Dissociative symptoms were found to hinder treatment out- come in BPD [16–19] and other psychiatric groups [127, 128], possibly by interfering with emotional learning and memory, e.g., habituation processes during exposure therapy [12–14, 96]. In a recent intervention study, a computerized (continuous) monitoring of dissociative states and suggestions for anti-dissociative skills had a beneficial effect on exposure exercises during trauma therapy [129]. Combining such inter- ventions with repeated neuroimaging assessment (before and after treatment) could help to identify neural pathways asso- ciated with a reduction of dissociative symptoms [55]. Moreover, there is preliminary evidence that dissociative symptoms decrease along with a neurofeedback training targeted on amygdala activity in response to aversive images [95, 96], while it remains unclear which processes (e.g., en- hanced top-down control versus bottom-up processes of emo- tion processing, such as increased emotional awareness, emo- tional clarity, and acceptance of emotions) may be involved. Neurofeedback training (real-time fMRI) may be a promising add-on intervention to help individuals gain more control over dissociative processes, i.e., reduce them at moments when these processes are disruptive and therefore maladaptive [130]. So far, very little is known about neural mechanisms that may be key modulators in this relationship and more studies are needed to identify brain areas that may be possible targets for such neurofeedback interventions. 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https://openalex.org/W3122239502	https://pure.eur.nl/files/48449032/Repub_134282_O-A.pdf	English	null	First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in BRAFV600-mutant advanced melanoma patients: a propensity-matched survival analysis	British journal of cancer	2,021	cc-by	9,570	1Dutch Institute for Clinical Auditing, Rijnsburgerweg 10, Leiden 2333AA, The Netherlands; 2Department of Medical Oncology, Amsterdam UMC, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1118, Amsterdam 1081HZ, The Netherlands; 3Department of Surgical Oncology, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066CX, The Netherlands; 4Department of Pharmacy, Rode Kruis Ziekenhuis, Vondellaan 13, Beverwijk 1942LE, The Netherlands; 5Department of Medical Oncology and Immunology, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066CX, The Netherlands; 6Department of Molecular Oncology & Immunology, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066CX, The Netherlands; 7Department of Medical Oncology, Maastricht University Medical Centre, P. Debyelaan 25, Maastricht 6229 HX, The Netherlands; 8Department of Medical Oncology, Zuyderland Medical Centre Sittard, Dr. H. van der Hoffplein 1, Sittard-Geleen 6162BG, The Netherlands; 9Isala Oncology Center, Isala, Dokter van Heesweg 2, Zwolle 8025AB, The Netherlands; 10Department of Medical Oncology, University Medical Centre Groningen, Hanzeplein 1, Groningen 9713GZ, The Netherlands; 11Department of Medical Oncology, Leiden University Medical Centre, Albinusdreef 2, Leiden 2333ZA, The Netherlands; 12Department of Internal Medicine, Medisch Spectrum Twente, Koningsplein 1, Enschede 7512KZ, The Netherlands; 13Department of Internal Medicine, Medical Centre Leeuwarden, Henri Dunantweg 2, Leeuwarden 8934AD, The Netherlands; 14Department of Medical Oncology, University Medical Centre Utrecht, Heidelberglaan 100, Utrecht 3584CX, The Netherlands; 15Department of Pathology, Division of Laboratories, Pharmacy and Biomedical Genetics, University Medical Center Utrecht. Heidelberglaan 100, Utrecht 3584 CX, The Netherlands; 16Department of Internal Medicine, Amphia Hospital, Molengracht 21, Breda 4818CK, The Netherlands; 17Departments of Medical Oncology and Radiology & Nuclear Medicine, Erasmus Medical Centre, ‘s-Gravendijkwal 230, Rotterdam 3015CE, The Netherlands; 18Department of Internal Medicine, Maxima Medical Centre, De Run 4600, Eindhoven 5504DB, The Netherlands and 19Department of Medical Oncology, Radboud University Medical Centre, Geert Grooteplein Zuid 10, Nijmegen 6525GA, The Netherlands Correspondence: Alfonsus J. M. van den Eertwegh (vandeneertwegh@amsterdamumc.nl) British Journal of Cancer https://doi.org/10.1038/s41416-020-01229-1 British Journal of Cancer https://doi.org/10.1038/s41416-020-01229-1 As BRAF/MEK inhibitors have a rapid onset and a high overall response rate (ORR), they are often considered the preferred primary treatment for patients with extensive (symptomatic) disease and a BRAFV600-mutation. However, in such patients, the response duration is relatively short due to acquired resistance.7 As time to response for immunotherapy is generally longer and ORR lower, checkpoint inhibition is usually not the preferred treatment for BRAF-mutant patients with poor prognostic factors such as a poor performance score or elevated LDH.8 Therefore, the perception that checkpoint inhibition leads to more durable responses could at least partially be explained by the fact that patients with better Received: 2 July 2020 Revised: 30 November 2020 Accepted: 10 December 2020 www.nature.com/bjc © The Author(s), under exclusive licence to Cancer Research UK 2021 ARTICLE Clinical Study METHODS: We selected patients diagnosed between 2014 and 2017 with advanced melanoma and a known BRAFV600-mutation treated with ﬁrst-line BRAF/MEK inhibitors or anti-PD-1 antibodies, registered in the Dutch Melanoma Treatment Registry. Patients were matched based on their propensity scores using the nearest neighbour and the optimal matching method. RESULTS: Between 2014 and 2017, a total of 330 and 254 advanced melanoma patients received BRAF/MEK inhibitors and anti-PD- 1 monotherapy as ﬁrst-line systemic therapy. In the matched cohort, patients receiving anti-PD-1 antibodies as a ﬁrst-line treatment had a higher median and 2-year overall survival compared to patients treated with ﬁrst-line BRAF/MEK inhibitors, 42.3 months (95% CI: 37.3-NE) vs. 19.8 months (95% CI: 16.7–24.3) and 85.4% (95% CI: 58.1–73.6) vs. 41.7% (95% CI: 34.2–51.0). CONCLUSIONS: Our data suggest that in the matched BRAFV600-mutant advanced melanoma patients, anti-PD-1 monotherapy is the preferred ﬁrst-line treatment in patients with relatively favourable patient and tumour characteristics. RESULTS: Between 2014 and 2017, a total of 330 and 254 advanced melanoma patients received BRAF/MEK inhibitors and anti-PD- 1 monotherapy as ﬁrst-line systemic therapy. In the matched cohort, patients receiving anti-PD-1 antibodies as a ﬁrst-line treatment had a higher median and 2-year overall survival compared to patients treated with ﬁrst-line BRAF/MEK inhibitors, 42.3 months (95% CI: 37.3-NE) vs. 19.8 months (95% CI: 16.7–24.3) and 85.4% (95% CI: 58.1–73.6) vs. 41.7% (95% CI: 34.2–51.0). CONCLUSIONS: Our data suggest that in the matched BRAFV600-mutant advanced melanoma patients, anti-PD-1 monotherapy is the preferred ﬁrst-line treatment in patients with relatively favourable patient and tumour characteristics. ARTICLE Clinical Study Jesper van Breeschoten1,2, Michel W. J. M. Wouters1,3, Doranne L. Hilarius4, John B. Haanen5, Christian U. Blank5,6, Maureen J. B. Aarts7, Franchette W. P. J. van den Berkmortel8, Jan-Willem B. de Groot9, Geke A. P. Hospers10, Ellen Kapiteijn11, Djura Piersma12, Roos S. van Rijn13, Karijn P. M. Suijkerbuijk14, Willeke A. M. Blokx15, Bert-Jan J. ten Tije16, Astrid A. M. van der Veldt 17, Art Vreugdenhil18, Marye J. Boers-Sonderen19 and Alfonsus J. M. van den Eertwegh2 BACKGROUND: Anti-PD-1 antibodies and BRAF/MEK inhibitors are the two main groups of systemic therapy in the treatment of BRAFV600-mutant advanced melanoma. Until now, data are inconclusive on which therapy to use as ﬁrst-line treatment. The aim of this study was to use propensity score matching to compare ﬁrst-line anti-PD-1 monotherapy vs. BRAF/MEK inhibitors in advanced BRAFV600-mutant melanoma patients. BACKGROUND: Anti-PD-1 antibodies and BRAF/MEK inhibitors are the two main groups of systemic therapy in the treatment of BRAFV600-mutant advanced melanoma. Until now, data are inconclusive on which therapy to use as ﬁrst-line treatment. The aim of this study was to use propensity score matching to compare ﬁrst-line anti-PD-1 monotherapy vs. BRAF/MEK inhibitors in advanced BRAFV600-mutant melanoma patients. METHODS: We selected patients diagnosed between 2014 and 2017 with advanced melanoma and a known BRAFV600-mutation treated with ﬁrst-line BRAF/MEK inhibitors or anti-PD-1 antibodies, registered in the Dutch Melanoma Treatment Registry. Patients were matched based on their propensity scores using the nearest neighbour and the optimal matching method. RESULTS: Between 2014 and 2017, a total of 330 and 254 advanced melanoma patients received BRAF/MEK inhibitors and anti-PD- 1 monotherapy as ﬁrst-line systemic therapy. In the matched cohort, patients receiving anti-PD-1 antibodies as a ﬁrst-line treatment had a higher median and 2-year overall survival compared to patients treated with ﬁrst-line BRAF/MEK inhibitors, 42.3 months (95% CI: 37.3-NE) vs. 19.8 months (95% CI: 16.7–24.3) and 85.4% (95% CI: 58.1–73.6) vs. 41.7% (95% CI: 34.2–51.0). CONCLUSIONS: Our data suggest that in the matched BRAFV600-mutant advanced melanoma patients, anti-PD-1 monotherapy is the preferred ﬁrst-line treatment in patients with relatively favourable patient and tumour characteristics. p METHODS: We selected patients diagnosed between 2014 and 2017 with advanced melanoma and a known BRAFV600-mutation treated with ﬁrst-line BRAF/MEK inhibitors or anti-PD-1 antibodies, registered in the Dutch Melanoma Treatment Registry. Patients were matched based on their propensity scores using the nearest neighbour and the optimal matching method. BACKGROUND van den Eertwegh (vandeneertwegh@amsterdamumc.nl) Received: 2 July 2020 Revised: 30 November 2020 Accepted: 10 December 2020 © The Author(s), under exclusive licence to Cancer Research UK 2021 First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in. . . J van Breeschoten et al. 2 and symptomatic) and liver metastases (yes/no) and use of immunomodulating agents (corticosteroids, azathioprine and interferon). Covariates were chosen based on clinical practice and previous research identifying these prognostic risk factors.14,15 A Monte Carlo simulation study has demonstrated that his method of selecting covariates for propensity score matching works very well.16 prognostic factors are being treated with immunotherapy. Although interstudy comparison suggests that immunotherapy results in more durable responses as compared to targeted therapy,9 these results need to be interpreted with caution due to the potential biases associated with this type of analysis. Due to the confounding by indication bias, a head-to-head comparison between both groups, based on real-world data is difﬁcult. Propensity score matching, a statistical method devel- oped by Rosenbaum and Rubins,10 accounts for the unequal distribution of confounders. A propensity score is the chance of receiving the treatment, based on measured confounders such as age and tumour stage.11,12 This method matches patients based on their propensity score, creating two groups with similar distribution of confounders. A multivariable Cox proportional hazards model was used to assess the association of prognostic factors with OS. Prognostic factors estimated were age at diagnosis (<75, ≥75), baseline ECOG-performance status (0–1 and ≥2), baseline lactate dehy- drogenase (LDH; normal, 250–500 U/L and >500 U/L), distant metastases (<3 organ sites and ≥3 organ sites involved), brain metastases (none, asymptomatic and symptomatic) and liver metastases (yes/no). The proportional hazards assumption was tested with the scaled Schoenfeld residuals. None of the covariates violated the proportional hazards assumption. In this study, we (1) compare OS of BRAF/MEK inhibitors and anti-PD-1 monotherapy in BRAF-mutant advanced melanoma patients, and (2) use propensity score matching to determine the overall survival in the BRAF/MEK inhibitors vs. anti-PD-1 mono- therapy groups after matching. For matching purposes, 1:1 nearest neighbour matching with a calliper set of 0.001 was used for propensity score matching. To assess the quality of matching, standardised mean differences for all covariates were calculated. Standardised mean differences of <0.1 were assumed to be negligible. BACKGROUND Over the last decade, new systemic treatment options have emerged in the treatment of metastatic melanoma. These approved treatments can be divided into two main groups: targeted therapy consisting of BRAF and MEK inhibitors1,2 and immunotherapy, anti-PD-1 and anti-CTLA-4 monoclonal antibo- dies.3–5 Their introduction has increased the overall survival of these patients.2,6 Based on clinical consensus, treatment choice depends on several prognostic and predictive factors such as BRAF-mutation status, lactate dehydrogenase levels (LDH), per- formance status and the tumour stage. 1Dutch Institute for Clinical Auditing, Rijnsburgerweg 10, Leiden 2333AA, The Netherlands; 2Department of Medical Oncology, Amsterdam UMC, VU University Medical Center, Cancer Center Amsterdam, De Boelelaan 1118, Amsterdam 1081HZ, The Netherlands; 3Department of Surgical Oncology, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066CX, The Netherlands; 4Department of Pharmacy, Rode Kruis Ziekenhuis, Vondellaan 13, Beverwijk 1942LE, The Netherlands; 5Department of Medical Oncology and Immunology, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066CX, The Netherlands; 6Department of Molecular Oncology & Immunology, Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam 1066CX, The Netherlands; 7Department of Medical Oncology, Maastricht University Medical Centre, P. Debyelaan 25, Maastricht 6229 HX, The Netherlands; 8Department of Medical Oncology, Zuyderland Medical Centre Sittard, Dr. H. van der Hoffplein 1, Sittard-Geleen 6162BG, The Netherlands; 9Isala Oncology Center, Isala, Dokter van Heesweg 2, Zwolle 8025AB, The Netherlands; 10Department of Medical Oncology, University Medical Centre Groningen, Hanzeplein 1, Groningen 9713GZ, The Netherlands; 11Department of Medical Oncology, Leiden University Medical Centre, Albinusdreef 2, Leiden 2333ZA, The Netherlands; 12Department of Internal Medicine, Medisch Spectrum Twente, Koningsplein 1, Enschede 7512KZ, The Netherlands; 13Department of Internal Medicine, Medical Centre Leeuwarden, Henri Dunantweg 2, Leeuwarden 8934AD, The Netherlands; 14Department of Medical Oncology, University Medical Centre Utrecht, Heidelberglaan 100, Utrecht 3584CX, The Netherlands; 15Department of Pathology, Division of Laboratories, Pharmacy and Biomedical Genetics, University Medical Center Utrecht. Heidelberglaan 100, Utrecht 3584 CX, The Netherlands; 16Department of Internal Medicine, Amphia Hospital, Molengracht 21, Breda 4818CK, The Netherlands; 17Departments of Medical Oncology and Radiology & Nuclear Medicine, Erasmus Medical Centre, ‘s-Gravendijkwal 230, Rotterdam 3015CE, The Netherlands; 18Department of Internal Medicine, Maxima Medical Centre, De Run 4600, Eindhoven 5504DB, The Netherlands and 19Department of Medical Oncology, Radboud University Medical Centre, Geert Grooteplein Zuid 10, Nijmegen 6525GA, The Netherlands Correspondence: Alfonsus J. M. METHODS Study design and population y g p p This longitudinal observational study used data from the Dutch Melanoma Treatment Registry (DMTR), a population-based, prospective registry capturing all patients with unresectable stage IIIc and IV melanoma in the Netherlands. Since the 1 July 2013, all patients with unresectable stage IIIC or stage IV melanoma seen in one of the 14 melanoma centres have been registered in the DMTR. Data are prospectively registered from diagnosis until death. Patients are followed every three months for changes in disease status, patients’ characteristics or treatment characteristics by an independent trained data manager. A detailed description of the DMTR setup has been published by Jochems et al.13 All patients of 18 years and older, diagnosed between 2014 and 2017 (dataset cut-off date was 01-08-2019) with unresectable stage IIIC and IV melanoma, with a known BRAFV600-mutation who used anti-PD-1 monotherapy (pembrolizumab or nivolumab) or BRAF/MEK inhibitors (dabrafenib + trametinib or vemurafenib + cobimetinib) as a ﬁrst-line systemic therapy, were selected for analysis. Combination therapies of encorafenib + binimetinib and ipilimumab + nivolumab were not yet available during this time period. Line of therapy was deﬁned as the start of a new drug class. Patients with uveal or mucosal melanoma were excluded from this analysis. BACKGROUND After matching, a univariable Cox proportional hazards model with a robust variance estimator to account for the matched nature of the sample was used to estimate the relative change in hazard of survival.17,18 To address potential biases due to incomplete matching, we compared our results to a second matched sample set using optimal matching. Optimal matching forms matched pairs to minimise the average within-pair difference in propensity scores.19 We used both matching techniques in order to observe the inﬂuence of well- balanced prognostic factors across both groups in the nearest neighbour-matched group on OS and TTNT compared to the optimal matching method. Due to the matched nature, the use of a log-rank test comparing the survival curves will likely result in type I error rates that are artiﬁcially low,20 stratiﬁed log-rank tests were used to compare the survival curves of the matched samples.18 Statistical software used was R (version 3.5.2; packages car, tidyverse, survival and matchit). Patient characteristics Patient characteristics Between 1-1-2014 and 31-12-2017, a total of 330 and 254 BRAF- mutant patients with unresectable stage IIIc or IV melanoma received BRAF/MEK inhibitors or anti-PD-1 monotherapy, respec- tively, as ﬁrst-line systemic therapy (Fig. 1). Overall, patients receiving BRAF/MEK inhibitors as a ﬁrst-line systemic treatment had a higher ECOG-performance status (≥2: 23.0% vs. 8.7%), elevated LDH levels, (50.3% vs 22.4%), IV-M1c disease (87.6% vs. 67.3%), brain metastases (41.8% vs. 18.5%), liver metastases (33.9% vs. 21.3%) and metastases in more organ sites (≥3 organ sites: 59.4% vs. 42.5%) (Table 1). There was no signiﬁcant difference in age and gender between patients receiving ﬁrst- line treatment with BRAF/MEK inhibitors and anti-PD-1 ligands. Median follow-up time, calculated with a reverse Kaplan–Meier was 28.3 months for the total population. Subsequent therapies are shown in Supplement 1 and subsequent therapies of pro- gressive patients are shown in Supplement 2. Overall, progressive patients that received ﬁrst-line BRAF/MEK inhibitors receive less second and third-line therapy compared to patients receiving ﬁrst-line anti-PD-1, 61.3% vs. 94.2% and 6.5% vs.43.4%. Statistical analysis Overall, nonmatched patients who received BRAF/MEK inhibitors as ﬁrst-line systemic Total number of patients without a V600-BRAF mutation (N = 1602) Total number not treated with anti-PD-1 monotherapy or BRAF-MEK inhibitors as first-line systemic therapy (N = 847) Total number of uveal melanoma (N = 190) or mucosal melanoma (N = 121) Only patients with cutaneous melanoma (N = 3151) Patients with a V600 BRAF- mutation (N = 1549) Patients treated with anti- PD-1monotherapy or BRAF- MEK inhibitors as first-line systemic therapy (N = 702) Patients treated with anti- PD-1monotherapy as first- line therapy (N = 254) Patients treated with BRAF- MEK inhibitors as first-line therapy (N = 330) No missing values in any of the matching variables (N = 584) Fig. 1 Flow chart. Flow chart of the study population. Total number of uveal melanoma (N = 190) or mucosal melanoma (N = 121) Only patients with cutaneous melanoma (N = 3151) Total number of patients without a V600-BRAF mutation (N = 1602) Patients with a V600 BRAF- mutation (N = 1549) Total number not treated with anti-PD-1 monotherapy or BRAF-MEK inhibitors as first-line systemic therapy (N = 847) Patients treated with anti- PD-1monotherapy or BRAF- MEK inhibitors as first-line systemic therapy (N = 702) No missing values in any of the matching variables (N = 584) Patients treated with BRAF- MEK inhibitors as first-line therapy (N = 330) Patients treated with anti- PD-1monotherapy as first- line therapy (N = 254) . 1 Flow chart. Flow chart of the study population. Propensity score matching 95% CI: 2.56–7.12) were all associated with higher odds of receiving ﬁrst-line BRAF/MEK inhibitors. With unresectable stage IIIc as a reference, substage IV-M1b had lower odds of receiving ﬁrst-line BRAF/MEK inhibitors (OR: 0.29, 95% CI: 0.11–0.74). Age ≥75 years, female gender, ≥3 organ sites, asymptomatic brain metastases and liver metastases were not signiﬁcantly associated with receiving ﬁrst-line BRAF/MEK inhibitors (p > 0.05). 95% CI: 2.56–7.12) were all associated with higher odds of receiving ﬁrst-line BRAF/MEK inhibitors. With unresectable stage IIIc as a reference, substage IV-M1b had lower odds of receiving ﬁrst-line BRAF/MEK inhibitors (OR: 0.29, 95% CI: 0.11–0.74). Age ≥75 years, female gender, ≥3 organ sites, asymptomatic brain metastases and liver metastases were not signiﬁcantly associated with receiving ﬁrst-line BRAF/MEK inhibitors (p > 0.05). Statistical analysis 3 3 Total number of patients in the DMTR diagnosed between 2014 and 2017 (N = 3462) 95% CI: 2.56–7.12) were all associated with higher odds of receiving ﬁrst-line BRAF/MEK inhibitors. With unresectable stage IIIc as a reference, substage IV-M1b had lower odds of receiving ﬁrst-line BRAF/MEK inhibitors (OR: 0.29, 95% CI: 0.11–0.74). Age ≥75 years, female gender, ≥3 organ sites, asymptomatic brain metastases and liver metastases were not signiﬁcantly associated with receiving ﬁrst-line BRAF/MEK inhibitors (p > 0.05). Predictors of OS In the multivariable Cox-regression on OS analysis age of ≥75 years (HR: 1.53, 95% CI: 1.10–2.13), ECOG-performance status ≥2 (HR: 2.30, 95% CI: 1.76–3.01), LDH > 500 U/L (HR: 3.38, 95% CI: 2.34–4.89) and symptomatic brain metastases (HR: 1.87, 95% CI: 1.44–2.43) were associated with a higher hazard of death. Female gender, asymptomatic brain metastases, liver metastases and ≥3 organ sites were not associated with a higher hazard of death (Table 3). Propensity score matching After 1:1 nearest neighbour propensity score matching, 310 patients were included in the ﬁnal analysis: 155 patients who received ﬁrst-line BRAF/MEK inhibitors (50%) and 155 patients who received ﬁrst-line anti-PD-1 (50%). Groups did not differ with regards to age (median age: 62 vs. 62 years), gender (47.7% vs 47.7% female), ECOG-performance status ≥2 (7.1% vs. 7.1) elevated LDH > 250 U/L (30.3% vs. 30.3%), stage IV-M1c (77.4% vs. 77.4%), organ sites ≥3 (45.8% vs. 45.2%), brain metastases (22.6% vs. 22.6%), liver metastases (21.9% vs1 21.9%) and immunomodulating agents (13.5% vs. 13.5%). These results conﬁrm satisfactory propensity score matching. In total, 274 patients were not matched: 175 patients who received ﬁrst-line BRAF-MEK and 99 patients receiving anti-PD-1 as a ﬁrst-line treatment (Supplement 3). Statistical analysis y Baseline patient- and disease characteristics of BRAFV600-mutant patients were analysed using descriptive statistics. Categorical variables were compared using the Chi-square test. Overall survival (OS) was deﬁned as time from start systemic treatment until death from any cause with corresponding two-sided 95% conﬁdence intervals and was estimated using the Kaplan–Meier method. TTNT was deﬁned as the time until next treatment of a new drug class or death from any cause. OS and TTNT between subgroups was compared using log-rank tests for categorical variables. Patients alive or lost to follow-up were right-censored at the time of last registered contact. g In order to reduce confounding by indication, propensity score matching was used. As a ﬁrst step, a multivariable logistic regression analysis was used to estimate propensity scores. Covariates used for the multivariable logistic regression were age at diagnosis (<75, ≥75), baseline ECOG-performance status (0–1 and ≥2), baseline lactate dehydrogenase (LDH) (normal, 250–500 U/L and >500 U/L), stage at diagnosis (unresectable IIIc, IV-M1a, M1b and IV-M1c), distant metastasis (<3 organ sites and ≥3 organ sites involved), brain metastases (none, asymptomatic, Predictors of receiving ﬁrst-line BRAF/MEK inhibitors To determine which factors were associated with receiving ﬁrst- line BRAF/MEK inhibitors, a multivariable logistic regression was performed (Table 2). The results show that an ECOG-performance status of ≥2 (OR: 1.93, 95% CI: 1.11–3.44), elevated LDH (OR: 2.84, 95% CI: 1.86–4.38) and symptomatic brain metastases (OR: 3.84, First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in. . . J van Breeschoten et al. First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in. . . J van Breeschoten et al. Total number of patients in the DMTR diagnosed between 2014 and 2017 (N = 3462) Total number of patients without a V600-BRAF mutation (N = 1602) Total number not treated with anti-PD-1 monotherapy or BRAF-MEK inhibitors as first-line systemic therapy (N = 847) Total number of uveal melanoma (N = 190) or mucosal melanoma (N = 121) Only patients with cutaneous melanoma (N = 3151) Patients with a V600 BRAF- mutation (N = 1549) Patients treated with anti- PD-1monotherapy or BRAF- MEK inhibitors as first-line systemic therapy (N = 702) Patients treated with anti- PD-1monotherapy as first- line therapy (N = 254) Patients treated with BRAF- MEK inhibitors as first-line therapy (N = 330) No missing values in any of the matching variables (N = 584) Fig. 1 Flow chart. Flow chart of the study population. J van Breeschoten et al. Statistical analysis Propensity score matching After 1:1 nearest neighbour propensity score matching, 310 patients were included in the ﬁnal analysis: 155 patients who received ﬁrst-line BRAF/MEK inhibitors (50%) and 155 patients who received ﬁrst-line anti-PD-1 (50%). Groups did not differ with regards to age (median age: 62 vs. 62 years), gender (47.7% vs 47.7% female), ECOG-performance status ≥2 (7.1% vs. 7.1), elevated LDH > 250 U/L (30.3% vs. 30.3%), stage IV-M1c (77.4% vs. 77.4%), organ sites ≥3 (45.8% vs. 45.2%), brain metastases (22.6% vs. 22.6%), liver metastases (21.9% vs1 21.9%) and immunomodulating agents (13.5% vs. 13.5%). These results conﬁrm satisfactory propensity score matching. Predictors of OS In the multivariable Cox-regression on OS analysis age of ≥75 years (HR: 1.53, 95% CI: 1.10–2.13), ECOG-performance status ≥2 (HR: 2.30, 95% CI: 1.76–3.01), LDH > 500 U/L (HR: 3.38, 95% CI: 2.34–4.89) and symptomatic brain metastases (HR: 1.87, 95% CI: 1.44–2.43) were associated with a higher hazard of death. Female gender, asymptomatic brain metastases, liver metastases and ≥3 organ sites were not associated with a higher hazard of death (Table 3). y y g In total, 274 patients were not matched: 175 patients who received ﬁrst-line BRAF-MEK and 99 patients receiving anti-PD-1 as a ﬁrst-line treatment (Supplement 3). Overall, nonmatched patients who received BRAF/MEK inhibitors as ﬁrst-line systemic First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in. . . J van Breeschoten et al. First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in. . . J van Breeschoten et al. Table 1. Baseline characteristics. Statistical analysis 51.5%) more ≥3 organ sites affected (73.1% vs. 37.4%), brain metastases (58.8% vs. 12.1%), liver metastases (44.6% vs. 20.2%) and used immunomo- dulating agents (38.3% vs. 9.1%) compared to the unmatched patients who received anti-PD-1 monotherapy. CI: 6.2–8.7) in patients receiving ﬁrst-line BRAF/MEK inhibitors vs. 18.9 months (95%CI: 11.0–24.4) in patients receiving anti-PD-1 ligands as ﬁrst-line therapy. Six and twelve-month OS were also lower for ﬁrst-line BRAF/MEK inhibitors compared to anti-PD-1 monotherapy, 75.9% (95% CI: 71.4–80.7) and 47.7% (95% CI: 42.6–53.4) vs. 91.3% (95% CI: 87.9–94.8) and 81.7% (95% CI: 77.0–86.6) (Table 4). therapy had poorer ECOG-performance status (ECOG-performance status ≥2 37.1% vs. 11.1%) and more often had elevated LDH levels (68.0% vs. 10.1%), stage IV-M1c (96.6% vs. 51.5%) more ≥3 organ sites affected (73.1% vs. 37.4%), brain metastases (58.8% vs. 12.1%), liver metastases (44.6% vs. 20.2%) and used immunomo- dulating agents (38.3% vs. 9.1%) compared to the unmatched patients who received anti-PD-1 monotherapy. Statistical analysis Original sample Nearest neighbour-matched sample BRAF-MEK Anti-PD-1 p value BRAF-MEK Anti-PD-1 SMD Baseline variable (N = 330) (N = 254) (N = 155) (N = 155) Age, median (range) 59 (19–91) 62 (22–87) 0.177 62 (19–91) 62 (25–87) 0.027 Gender Male 178 (53.9) 145 (57.1) 0.500 81 (52.3) 81 (52.3) <0.001 Female 152 (46.1) 109 (42.9) 74 (47.7) 74 (47.7) ECOG-performance status 0–1 254 (77.0) 232 (91.3) <0.001 144 (92.9) 144 (92.9) <0.001 ≥2 76 (23.0) 22 (8.7) 11 (7.1) 11 (7.1) LDH Not determined/normal 164 (49.7) 197 (77.6) <0.001 108 (69.7) 108 (69.7) <0.001 250–500 U/L 108 (32.7) 55 (21.7) 45 (29.0) 45 (29.0) >500 U/L 58 (17.6) 2 (0.8) 2 (1.3) 2 (1.3) Stage (7th edition AJCC) Unresectable IIIc 15 (4.5) 14 (5.5) <0.001 10 (6.5) 10 (6.5) <0.001 IV-M1a 13 (3.9) 29 (11.4) 13 (8.4) 13 (8.4) IV-M1b 13 (3.9) 40 (15.7) 12 (7.7) 12 (7.7) IV-M1c 289 (87.6) 171 (67.3) 120 (77.4) 120 (77.4) Brain metastases No 192 (58.2) 207 (81.5) <0.001 120 (77.4) 120 (77.4) <0.001 Yes. asymptomatic 42 (12.7) 24 (9.4) 15 (9.7) 15 (9.7) Yes. symptomatic 96 (29.1) 23 (9.1) <0.001 20 (12.9) 20 (12.9) Liver metastases No 218 (66.1) 200 (78.7) 121 (78.1) 121 (78.1) <0.001 Yes 112 (33.9) 54 (21.3) 34 (21.9) 34 (21.9) Organ sites 0–2 134 (40.6) 146 (57.5) 0.001 86 (55.5) 83 (53.5) <0.001 ≥3 196 (59.4) 108 (42.5) 69 (44.5) 72 (46.5) Immunomodulating agents No 242 (73.3) 224 (88.2) <0.001 134 (86.5) 134 (86.5) <0.001 Yes 88 (26.7) 30 (11.8) 21 (13.5) 21 (13.5) <0.001 BRAFV600-mutation V600E 274 (83.0) 212 (83.5) 0.833 130 (83.9) 132 (85.2) 0.073 V600K 44 (13.3) 35 (13.8) 19 (12.3) 19 (12.3) V600R/D/E2 12 (3.6) 7 (2.8) 6 (3.9) 4 (2.6) Comparison of baseline characteristics of patients receiving BRAF/MEK inhibitors or anti-PD-1 as a ﬁrst-line treatment in the original sample and the matched sample. CI: 6.2–8.7) in patients receiving ﬁrst-line BRAF/MEK inhibitors vs. 18.9 months (95%CI: 11.0–24.4) in patients receiving anti-PD-1 ligands as ﬁrst-line therapy. Six and twelve-month OS were also lower for ﬁrst-line BRAF/MEK inhibitors compared to anti-PD-1 monotherapy, 75.9% (95% CI: 71.4–80.7) and 47.7% (95% CI: 42.6–53.4) vs. 91.3% (95% CI: 87.9–94.8) and 81.7% (95% CI: 77.0–86.6) (Table 4). therapy had poorer ECOG-performance status (ECOG-performance status ≥2 37.1% vs. 11.1%) and more often had elevated LDH levels (68.0% vs. 10.1%), stage IV-M1c (96.6% vs. Clinical outcomes Nearest neighbour propensity matching. In the nearest neighbour-matched cohort (N = 310), the overall survival of ﬁrst- line BRAF/MEK inhibitors was signiﬁcantly lower compared to anti- PD-1 (stratiﬁed log-rank p < 0.001). This matched cohort only included patients treated with anti-PD-1 and BRAF/MEK inhibitors in which the propensity score of both patients was within the pre- set calliper. Median OS of ﬁrst-line BRAF/MEK inhibitors was lower compared to the anti-PD-1 cohort, 19.8 months (95% CI: 16.7–24.3) vs. 42.3 (95% CI 37.3-NE). Median TTNT of ﬁrst-line BRAF/MEK Clinical outcomes In the cohort before matching, patients treated with BRAF/MEK inhibitors as ﬁrst-line systemic treatment were more likely to die compared to patients treated with anti-PD-1 monotherapy (p < 0.001, log-rank test). Kaplan–Meier estimates are shown in Fig. 2. Median overall survival was 11.0 months (95% CI: 9.9–13.7) in patients receiving ﬁrst-line BRAF/MEK inhibitors vs. 42.3 months (95% CI: 34.8-NE) in patients receiving anti-PD-1 ligands as ﬁrst-line therapy. Median TTNT was 7.0 months (95% First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in. . . J van Breeschoten et al. 5 Table 3. Multivariable Cox-regression analysis of OS. N HR 95% CI p value Age <75 years 520 1 ≥75 years 64 1.53 (1.10–2.13) 0.012 Gender Female 323 1 Male 261 0.94 (0.75–1.17) 0.585 ECOG-performance status 0–1 486 1 ≥2 98 2.30 (1.76–3.01) <0.001 LDH Normal 361 1 250–500 U/L 163 1.28 (0.99–1.65) 0.065 >500 U/L 60 3.38 (2.34–4.89) <0.001 Brain metastases No 399 1 Yes, asymptomatic 66 1.36 (0.97–1.92) 0.076 Yes, symptomatic 119 1.87 (1.44–2.43) <0.001 Liver metastases No 418 1 Yes 166 1.18 (0.9–1.55) 0.241 Number of organ sites 0–2 289 1 ≥3 295 1.25 (0.96–1.62) 0.096 LDH lactate dehydrogenase, HR hazard ratio, CI conﬁdence interval. Table 2. Multivariable logistic regression. Clinical outcomes Variables OR 95% CI p value (Intercept) 0.84 (0.40–1.82) 0.659 Age (categories) <75 years Ref ≥75 years 1.54 (0.86–2.78) 0.149 Gender Male Ref Female 1.34 (0.92–1.95) 0.132 ECOG-performance status 0–1 Ref ≥2 1.59 (0.89–2.89) 0.123 LDH Not determined/normal Ref 250–500 U/L 2.03 (1.86–4.38) 0.002 >500 U/L 30.73 (8.77–195.30) <0.001 Stage (7th edition AJCC) Unresectable IIIc Ref IV-M1a 0.47 (0.17–1.27) 0.140 IV-M1b 0.33 (0.12–0.88) 0.029 IV-M1c 0.58 (0.25–1.34) 0.201 Organ sites 0–2 Ref ≥3 1.52 (0.98–2.35) 0.063 Brain metastases No Ref Yes, asymptomatic 1.40 (0.76–2.60) 0.279 Yes, symptomatic 3.97 (2.22–7.35) <0.001 Liver metastases No Ref Yes 0.89 (0.54–1.45) 0.629 Immunomodulating agents No Ref Yes 1.41 (0.81–2.47) 0.222 Multivariable logistic regression of receiving ﬁrst-line BRAF-MEK inhibitor treatment. LDH lactate dehydrogenase, OR odds ratio, CI conﬁdence interval. Table 2. Multivariable logistic regression. treated with ﬁrst-line BRAF/MEK inhibitors. Median OS of ﬁrst-line BRAF/MEK inhibitors was lower compared to anti-PD-1 mono- therapy, 15.6 months (95% CI: 12.4–19.1). vs. 42.3 months (95% CI: 34.4-NE). Median TTNT of ﬁrst-line BRAF/MEK inhibitors was lower compared to anti-PD-1 monotherapy, 7.6 months (95% CI: 6.1–9.9). vs. 18.9 months (95% CI: 11.0–24.4). Six-, 12-and 24-month OS was also lower for BRAF/MEK inhibitors compared to anti-PD-1 monotherapy, 83.7% (95% CI: 79.3–88.4), 57.1% (95% CI: 51.3–63.6) and 34.4 (95% CI: 28.7–41.3) vs. 91.3% (95% CI: 87.9–94.8), 81.7% (95% CI: 77.0–86.6) and 64.0 (95% CI: 58.2–70.4) (Table 4). Kaplan–Meier estimates of the optimal matched cohort are shown in Fig. 4. The estimated HR with a robust variance estimator was 0.43 (95% CI: 0.34–0.56). Thus, treatment with ﬁrst-line anti-PD-1 reduced the hazard of death by 57% in the optimal matched sample. inhibitors was not signiﬁcantly lower compared to the anti-PD-1 cohort, 10.1 months (95% CI: 7.7–15.0) vs. 14.6 (95% CI 10.1–27.7). Six-, 12-and 24-month OS was also lower for BRAF/MEK inhibitors compared to anti-PD-1 monotherapy, 92.9% (95% CI: 89.9–97.0), 83.0% (95% CI: 77.3–89.2) and 65.4% (58.1–73.6) vs. 84.3% (95% CI: 78.7–90.3), 65.3% (95% CI: (58.2–73.3) and 41.7% (95% CI: 34.2–51.0) (Table 4). Kaplan–Meier estimates of the nearest neighbour-matched cohort are shown in Fig. 3. The estimated HR with a robust variance estimator was 0.50 (95% CI: 0.36–0.70) Thus, treatment with ﬁrst-line anti-PD-1 ligands reduced the hazard of death by 50% in this matched sample. Overall survival of the nonmatched patients receiving BRAF/MEK inhibitors was signiﬁcantly lower compared to anti-PD-1 (log-rank p < 0.001) (Supplement 4). DISCUSSION 6 100% 75% 50% p < 0.0001 Survival probability First-line treatment First-line treatment 25% 0% BRAF-MEK inhibitors Anti-PD-1 monotherapy BRAF-MEK inhibitors Anti-PD-1 monotherapy 0 Number at risk 330 254 248 230 154 203 102 163 58 116 6 12 Time (months) 18 24 0 6 12 Time (months) 18 24 First-line treatment BRAF/MEK inhibitors Anti-PD-1 BRAF/MEK inhibitors Anti-PD-1 100% 75% 50% Survival probability 25% 0% 330 254 253 187 137 164 93 144 64 130 45 110 2 8 Number at risk 0 4 8 12 16 20 24 Time 0 4 8 12 16 20 24 Time Fig. 2 Kaplan–Meier estimate of OS and TTNT of ﬁrst-line BRAF/MEK inhibitors vs. anti-PD-1 monotherapy. Kaplan–Meier estimate of OS (left) and TTNT (right) of ﬁrst-line BRAF/MEK inhibitors vs. anti-PD-1 monotherapy. Conﬁdence interval is displayed by the shadow of both curves. Table 4. TTNT and OS estimates of different methods. BRAF/MEK inhibitors Anti-PD-1 monotherapy Overall Median TTNT (months) Median OS (months) 6 months (%) 12 months (%) 24 months (%) Median TTNT (months) Median (months) 6 months (%) 12 months (%) 24 months (%) HR (95% CI) Kaplan–Meier estimate 7.0 (6.2–8.7) 11.0 (9.9–13.7) 75.9 (71.4–80.7) 47.7 (42.6–53.4) 23.6 (19.0–29.3) 18.9 (11.0–24.4) 42.3 (34.8-NE) 91.3 (87.9–94.8) 81.7 (77.0–86.6) 57.9 (51.8–64.8) 0.36 (0.28–0.45) Nearest neighbour matching 10.2 (7.7–15.0) 19.8 (16.7–24.3) 84.3 (78.7–90.3) 65.3 (58.2–73.3) 41.7 (34.2–51.0) 14.6 (10.1–27.7) 42.3 (37.3-NE) 92.9 (89.9–97.0) 83.0 (77.3–89.2) 65.4 (58.1–73.6) 0.50 (0.36–0.70) Optimal matching 7.6 (6.1–9.9) 15.6 (12.5–19.1) 83.7 (79.3–88.4) 57.1 (51.3–63.6) 34.4 (28.7–41.3) 18.9 (11.0–24.4) 42.3 (34.8-NE) 91.3 (87.9–94.8) 81.7 (77.0–86.6) 64.0 (58.2–70.4) 0.43 (0.34–0.56) TTNT and OS estimates shown as survival rates at 6,12 and 24 months and median overall survival stratiﬁed for ﬁrst-line therapy (BRAF/MEK inhibitors vs. anti- PD-1 monotherapy). NE non estimable. Table 4. TTNT and OS estimates of different methods. TTNT and OS estimates shown as survival rates at 6,12 and 24 months and median overall survival stratiﬁed for ﬁrst-line therapy (BRAF/MEK inhibitors vs. anti- PD-1 monotherapy). NE non estimable. choice. However, the results need to be conﬁrmed in randomised clinical trials to assess optimal front-line-and sequence therapy. Currently, several clinical trials are investigating the optimal front- line treatment of immunotherapy and targeted therapy. The COWBOY study evaluates a planned sequence vemurafenib and cobimetinib, followed by ipilimumab and nivolumab vs. DISCUSSION This report, based on real-world population-based data, describes the overall survival of BRAFV600-mutant advanced melanoma patients after using ﬁrst-line treatment with BRAF-MEK inhibitors or anti-PD-1 monotherapy. In total 310 patients were matched, resulting in 155 matched pairs. In the matched cohort, patients treated with anti-PD-1 monotherapy as a ﬁrst-line treatment showed a higher 2-year survival compared to patients treated with ﬁrst-line BRAF-MEK. Median OS in the anti-PD-1 monotherapy cohort was 42.3 months while the median OS was 19.8 months in patients receiving BRAF/MEK inhibitors as ﬁrst-line treatment. Optimal matching. In the optimal matched cohort (N = 508), overall survival was signiﬁcantly lower for BRAF/MEK inhibitors compared to anti-PD-1 monotherapy (stratiﬁed log-rank p < 0.001). This matched cohort included all patients treated with ﬁrst-line anti-PD-1 matched with the most alike set of patients In the nearest neighbour-matched cohort, patients treated with anti-PD-1 as a ﬁrst-line treatment have higher OS compared to patients treated with BRAF/MEK inhibitors as a ﬁrst-line treatment. There were also patients who could not be matched. Overall, we matched patients with relatively good prognosis factors who First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in. . . J van Breeschoten et al. 100% 75% 50% p < 0.0001 Survival probability First-line treatment First-line treatment 25% 0% BRAF-MEK inhibitors Anti-PD-1 monotherapy BRAF-MEK inhibitors Anti-PD-1 monotherapy First-line treatment BRAF/MEK inhibitors Anti-PD-1 First-line treatment BRAF/MEK inhibitors Anti-PD-1 100% 75% 50% Survival probability 25% 0% 0 Number at risk 330 254 330 254 253 187 137 164 93 144 64 130 45 110 2 8 248 230 154 203 102 163 58 116 Number at risk 0 4 8 12 16 20 24 6 12 Time (months) Time 0 4 8 12 16 20 24 Time 18 24 0 6 12 Time (months) 18 24 Fig. 2 Kaplan–Meier estimate of OS and TTNT of ﬁrst-line BRAF/MEK inhibitors vs. anti-PD-1 monotherapy. Kaplan–Meier estimate of OS (left) and TTNT (right) of ﬁrst-line BRAF/MEK inhibitors vs. anti-PD-1 monotherapy. Conﬁdence interval is displayed by the shadow of both curves. DISCUSSION First-line treatment BRAF/MEK inhibitors Anti-PD-1 100% 75% 50% Survival probability 25% 0% 0 4 8 12 16 20 24 Time 0 4 8 12 16 20 24 Time BRAF/MEK inhibitors Anti-PD-1 254 254 205 187 126 164 87 144 62 130 44 110 2 8 Number at risk First-line treatment BRAF-MEK inhibitors Anti-PD-1 monotherapy 100% 75% 50% Survival probability 25% 0% 0 4 8 12 16 20 24 Time 0 4 8 12 16 20 24 Time First-line treatment BRAF-MEK inhibitors Anti-PD-1 monotherapy Number at risk 254 254 233 237 179 223 142 203 118 174 85 149 56 116 Fig. 4 Kaplan–Meier estimate of the optimal matched sample. Kaplan–Meier estimates of OS (left) and TTNT (right) for the optimal matched cohort. Conﬁdence interval is displayed by the shadow of both curves. In Phase 3 RCT’s, 1-year survival was 74.1% for pembrolizumab and 72.9% for nivolumab.4,25 Comparing the results of this study with the randomised clinical trials of both anti-PD-1 ligands, we ﬁnd a higher 1-year survival (81.7%, 95% CI: 77.0–86.6). This is remarkable because we would expect 1-year survival of patients treated in clinical practice to be similar or worse due to unfavourable patient and tumour characteristics. However, in the CHECKMATE-066 study, only BRAF wild-type patients were included and the KEYNOTE-006 included only 35% of BRAF-mutant patients. These patients lack treatment options with BRAF/MEK inhibitors, Fig. 4 Kaplan–Meier estimate of the optimal matched sample. Kaplan–Meier estimates of OS (left) and TTNT (right) for the optimal matched cohort. Conﬁdence interval is displayed by the shadow of both curves. In Phase 3 RCT’s, 1-year survival was 74.1% for pembrolizumab and 72.9% for nivolumab.4,25 Comparing the results of this study with the randomised clinical trials of both anti-PD-1 ligands, we ﬁnd a higher 1-year survival (81.7%, 95% CI: 77.0–86.6). This is remarkable because we would expect 1-year survival of patients treated in clinical practice to be similar or worse due to unfavourable patient and tumour characteristics. However, in the CHECKMATE-066 study, only BRAF wild-type patients were included and the KEYNOTE-006 included only 35% of BRAF-mutant patients. These patients lack treatment options with BRAF/MEK inhibitors, Fig. 4 Kaplan–Meier estimate of the optimal matched sample. Kaplan–Meier estimates of OS (left) and TTNT (right) for the optimal matched cohort. Conﬁdence interval is displayed by the shadow of both curves. DISCUSSION ipilimu- mab and nivolumab (NCT02968303).22 The DREAMseq study (NCT02224781) evaluates a sequence of dabrafenib and trameti- nib until progression, followed by ipilimumab and nivolumab or ipilimumab and nivolumab until progression followed by dabra- fenib and trametinib. The SECOMBIT (NCT02631447) evaluates the sequence of encorafenib plus binimetinib followed by ipilimumab plus nivolumab or the opposite sequence or encorafenib plus binimetinib for 8 weeks followed by ipilimumab plus nivolumab until progression followed by encorafenib plus binimetinib.23,24 These studies are expected to be completed by the end of 2021 and 2022. received BRAF/MEK inhibitors with patients with relatively poor prognosis factors who received anti-PD-1 monotherapy as ﬁrst-line systemic therapy. The unmatched patients consisted of patients with poor prognostic factors who received BRAF/MEK inhibitors as ﬁrst-line therapy and the patients with good prognostic factors who received anti-PD-1 as ﬁrst-line systemic therapy. In the optimal matching method, we matched all patients receiving ﬁrst-line anti- PD-1, resulting in lower OS for BRAF/MEK inhibitors as sicker patients were matched, which could, in turn, lead to residual confounding.18 Consequently, we cannot compare the efﬁcacy of BRAF/MEK inhibitors or anti-PD-1 in the unmatched group. In BRAF-mutant patients with a very poor prognosis, BRAF-MEK inhibitors might be the preferred choice of ﬁrst-line treatment as this treatment results in quick antitumour response.21 This study suggests that in the matched cohort, consisting of patients with relative favourable patient and tumour character- istics, anti-PD-1 monotherapy is the ﬁrst-line treatment option of First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in. . . J van Breeschoten et al. First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in. . . J van Breeschoten et al. First-line treatment BRAF-MEK inhibitors Anti-PD-1 monotherapy First-line treatment BRAF/MEK inhibitors Anti-PD-1 100% 75% 50% Survival probability 25% 0% 100% 75% 50% Survival probability 25% 0% 0 4 8 12 16 20 24 Time 0 4 8 12 16 20 24 Time 0 4 8 12 16 20 24 Time 0 4 8 12 16 20 24 Time First-line treatment BRAF-MEK inhibitors Anti-PD-1 monotherapy First-line treatment BRAF/MEK inhibitors Anti-PD-1 Number at risk 155 155 155 155 124 113 83 100 63 86 47 78 35 69 2 5 142 146 119 137 98 125 85 107 62 94 40 75 Number at risk Fig. 3 Kaplan–Meier estimate OS and TTNT of the nearest neighbour-matched sample. Kaplan–Meier estimates of OS (left) and TTNT (right) for the nearest neighbour-matched cohort. DISCUSSION Conﬁdence interval is displayed by the shadow of both curves. 7 First-line treatment BRAF-MEK inhibitors Anti-PD-1 monotherapy 100% 75% 50% Survival probability 25% 0% 0 4 8 12 16 20 24 Time 0 4 8 12 16 20 24 Time First-line treatment BRAF-MEK inhibitors Anti-PD-1 monotherapy Number at risk 155 155 142 146 119 137 98 125 85 107 62 94 40 75 First-line treatment BRAF/MEK inhibitors Anti-PD-1 100% 75% 50% Survival probability 25% 0% 0 4 8 12 16 20 24 Time 0 4 8 12 16 20 24 Time BRAF/MEK inhibitors Anti-PD-1 155 155 124 113 83 100 63 86 47 78 35 69 2 5 Number at risk Survival probability Fig. 3 Kaplan–Meier estimate OS and TTNT of the nearest neighbour-matched sample. Kaplan–Meier estimates of OS (left) and TTNT (right) for the nearest neighbour-matched cohort. Conﬁdence interval is displayed by the shadow of both curves. In Phase 3 RCT’s, 1-year survival was 74.1% for pembrolizumab and 72.9% for nivolumab.4,25 Comparing the results of this study with the randomised clinical trials of both anti-PD-1 ligands, we ﬁnd a higher 1-year survival (81.7%, 95% CI: 77.0–86.6). This is remarkable because we would expect 1-year survival of patients treated in clinical practice to be similar or worse due to unfavourable patient and tumour characteristics. However, in the CHECKMATE-066 study, only BRAF wild-type patients were included and the KEYNOTE-006 included only 35% of BRAF-mutant patients. These patients lack treatment options with BRAF/MEK inhibitors, First-line treatment BRAF-MEK inhibitors Anti-PD-1 monotherapy First-line treatment BRAF/MEK inhibitors Anti-PD-1 100% 75% 50% Survival probability 25% 0% 100% 75% 50% Survival probability 25% 0% 0 4 8 12 16 20 24 Time 0 4 8 12 16 20 24 Time 0 4 8 12 16 20 24 Time 0 4 8 12 16 20 24 Time First-line treatment BRAF-MEK inhibitors Anti-PD-1 monotherapy First-line treatment BRAF/MEK inhibitors Anti-PD-1 Number at risk 254 254 254 254 205 187 126 164 87 144 62 130 44 110 2 8 233 237 179 223 142 203 118 174 85 149 56 116 Number at risk Fig. 4 Kaplan–Meier estimate of the optimal matched sample. Kaplan–Meier estimates of OS (left) and TTNT (right) for the optimal matched cohort. Conﬁdence interval is displayed by the shadow of both curves. ACKNOWLEDGEMENTS We thank all physicians and data managers who registered the patient data in the Dutch Melanoma Treatment Registry. DISCUSSION was responsible for the study conception and design, analysis and interpretation of data and drafting of the manuscript. p Another real-world study investigating the outcomes of ﬁrst-line immunotherapy and targeted therapy was performed by Luke et al.27 Opposed to our current study, they ﬁnd a lack of survival beneﬁt for patients receiving ﬁrst-line immunotherapy, despite a favourable imbalance of prognostic factors. These differences could be explained by the percentage of M1c patients (77.4%) in our study vs. 59.7% and 51.6% in the study by Luke et al. A direct comparison between both studies is hard due to differences in patient and tumour characteristics, study design and geographic location. There are limitations to this study. First, this study did not include patients who received ipilimumab monotherapy or ipilimumab combined with nivolumab. Ipilimumab monotherapy is no longer given as ﬁrst-line treatment since anti-PD-1 has proven superior efﬁcacy.28 However, combination therapy of ipilimumab and nivolumab has proven to be effective in metastatic melanoma patients in the CHECKMATE-067 trial.29 We did not have enough patients who received combination therapy of ipilimumab and nivolumab to make a reliable analysis of this group. Second, the observational nature of the DMTR may have introduced bias. However, since the start of this registry, independent data managers have been trained annually and data are checked and conﬁrmed by treating physicians. The online registration platform warns data managers of inconsistent or missing values in the registry. Previous studies have shown the quality and validity of the Dutch population-based registries.30 We therefore argue that the data used in this study is of high quality. The third limitation is that we could not assess how subsequent therapies impact the outcome. By studying patients receiving second-line treatment, we would introduce a selection bias. Only patients ﬁt enough would be able to receive second-line treatment. Studying the impact of subsequent therapies on outcomes would require a randomised controlled trial with several predeﬁned arms. In the real-world, choice of type of subsequent therapy depends on patient and tumour character- istics as well as preference of the oncologist. DISCUSSION In Phase 3 RCT’s, 1-year survival was 74.1% for pembrolizumab and 72.9% for nivolumab.4,25 Comparing the results of this study with the randomised clinical trials of both anti-PD-1 ligands, we ﬁnd a higher 1-year survival (81.7%, 95% CI: 77.0–86.6). This is remarkable because we would expect 1-year survival of patients treated in clinical practice to be similar or worse due to unfavourable patient and tumour characteristics. However, in the CHECKMATE-066 study, only BRAF wild-type patients were included and the KEYNOTE-006 included only 35% of BRAF-mutant patients. These patients lack treatment options with BRAF/MEK inhibitors, treated in clinical practice to be similar or worse due to unfavourable patient and tumour characteristics. However, in the CHECKMATE-066 study, only BRAF wild-type patients were included and the KEYNOTE-006 included only 35% of BRAF-mutant patients. These patients lack treatment options with BRAF/MEK inhibitors, treated in clinical practice to be similar or worse due to unfavourable patient and tumour characteristics. However, in the CHECKMATE-066 study, only BRAF wild-type patients were included and the KEYNOTE-006 included only 35% of BRAF-mutant patients. These patients lack treatment options with BRAF/MEK inhibitors, First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in. . . J van Breeschoten et al. 8 which may result in fewer treatment options available after progression on anti-PD-1 treatment. On the other hand, in our matched anti-PD-1 cohort, 7.1% of patients had a baseline ECOG- performance status of ≥2. These patients have been excluded from the previously mentioned RCT’s and as a result, we would expect that the clinical outcomes of this study would be worse compared to RCT’s. Compared to Phase 3 RCT’s of BRAF/MEK inhibitors, one year survival was 71.2% for dabrafenib and trametinib and 74.5% for vemurafenib and cobimetinib.2,26 Compared to the 1-year survival of this study, we ﬁnd a lower 1-year survival of 65.3% (95% CI: 58.2–73.3). Differences in survival could be related to a higher age (62 vs. 55), inclusion of ECOG ≥2 (7.1%), more patients with IV- M1c disease (77% vs. 62–63%) and inclusion of patients with brain metastases (22.6%) in our matched sample. interpretation of data and drafting of the manuscript. W.B. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. J.H. DISCUSSION performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. C.B. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. M.A. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. F.B. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript, performed critical revision and ﬁnal approval of the manuscript. J.G. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. G.H. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. E.K. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. D.P. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. R.R. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. K.S. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. A.T. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. A.V. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. G.V. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. M.B. performed data acquisition, study conception, helped with the analysis and interpretation of the data, drafted the manuscript and performed critical revision and ﬁnal approval of the manuscript. A.E. ADDITIONAL INFORMATION Ethics approval and consent to participate In compliance with Dutch regulations, the DMTR was approved by a medical ethical committee (METC Leiden University Medical Center, 2013) and is not considered subject to the Medical Research Involving Human Subjects Act. Data availability The datasets generated during and/or analysed during the current study are not publicly available due to privacy regulations in the Netherlands but are available from the corresponding author on reasonable request. Competing interests A.J.M.V.D.E has consulting/advisory relationships with BMS, Roche, MSD, and Novartis. He received a study grant from Roche. J.W.D.G. has received personal fees outside the submitted work from Bristol-Myers Squibb, Pierre Fabre, Servier, MSD, Novartis. GH consultancy/advisory relationships with Amgen, Bristol-Myers Squibb, Roche, MSD, Pﬁzer, Novartis and has received research grants not related to this paper from Bristol-Myers Squibb, Seerave. E.K. has consultancy/ advisory relationships with Amgen, Bristol-Myers Squibb, Novartis, Merck, Pierre Fabre, and received research grants not related to this paper from Bristol-Myers Squibb. K.P.M.S. has consulting/advisory relationships with BMS and MSD. She received honoraria from Novartis, Pierre Fabre, and Roche. A.V.D.V. has consultancy relationships with Bristol-Myers Squibb, MSD, Roche, Novartis, Pierre Fabre, Pﬁzer, Sanoﬁ, Ipsen, Eisai. J.H. has advisory relationships with Amgen, AstraZeneca, Bayer, Bristol-Myers Squibb, Celsius Therapeutics, GSK, Immunocore, Ipsen, MSD, Merck Serono, Novartis, Neon Therapeutics, Pﬁzer, Roche/Genentech, Sanoﬁ, Seattle Genetics and has received research grants not related to this paper from Novartis, Bristol-Myers Squibb, MSD, Neon Therapeutics. C.U.B. has received commercial research grants from Novartis, Bristol-Myers Squibb, and NanoString; is a paid advisory board member for Bristol-Myers Squibb, MSD, Roche, Novartis, GlaxoSmithK- line, AstraZeneca, Pﬁzer, Lilly, GenMab, and Pierre Fabre; and holds ownership interest in Uniti Cars, Neon Therapeutics, and Forty Seven. M.J.B.S. has consultancy relationships with Pierre Fabre, MSD and Novartis. All grants were paid to the institutions. The funders had no role in the writing of this article or decision to submit it for publication. All remaining authors have declared no conﬂicts of interest Based on the matched cohort, our data suggest that in BRAFV600-mutant cutaneous metastatic melanoma patients, anti- PD-1 monotherapy should be the preferred treatment if a quick antitumour response, for example for symptom relief, is not the primary aim of treatment. Additional results from RCTs are necessary to conﬁrm these results to determine optimal front- line and sequential therapy for BRAF-mutant cutaneous metastatic melanoma patients. REFERENCES 19. Austin, P. C. A comparison of 12 algorithms for matching on the propensity score. Stat. Med. 33, 1057–1069 (2014). 1. Chapman, P. B., Hauschild, A., Robert, C., Haanen, J. B., Ascierto, P., Larkin, J. et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N. Engl. J. Med. 364, 2507–2516 (2011). 20. Austin, P. C. Type I error rates, coverage of conﬁdence intervals, and variance estimation in propensity-score matched analyses. Int. J. Biostat. 5, 1–21 (2009). 2. Robert, C., Karaszewska, B., Schachter, J., Rutkowski, P., Mackiewicz, A., Stroia- kovski, D. et al. Improved overall survival in melanoma with combined dabrafenib and trametinib. N. Engl. J. Med. 372, 30–39 (2015). 21. Yushak, M., Chapman, P., Robert, C. & Kudchadkar, R. 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After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0). 16. Austin, P. C., Grootendorst, P. & Anderson, G. M. A comparison of the ability of different propensity score models to balance measured variables between treated and untreated subjects: a Monte Carlo study. Stat. Med. 26, 734–753 (2007). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 17. Lin, D. Y. & Wei, L. J. The Robust Inference for the Cox Proportional Hazards Model. J. Am. Stat. Assoc. 84, 1074 (1989). 18. Austin, P. C. The use of propensity score methods with survival or time-to-event outcomes: reporting measures of effect similar to those used in randomized experiments. Stat. Med. 33, 1242–1258 (2014). AUTHOR CONTRIBUTIONS J.B. performed the analyses and wrote the manuscript. M.W. was responsible for the study conception and design, analysis and interpretation of data and drafting of the manuscript. D.H. was responsible for the study conception and design, analysis and First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in. . . J van Breeschoten et al. 9 quality assurance in the care of patients with metastatic melanoma in the Netherlands. Eur. J. Cancer 72, 156–165, http://www.ncbi.nlm.nih.gov/pubmed/ 28030784 (2017). Funding information For the Dutch Melanoma Treatment Registry (DMTR), the Dutch Institute for Clinical Auditing foundation received a start-up grant from governmental organisation The Netherlands Organization for Health Research and Development (ZonMW, project number 836002002). The DMTR is structurally funded by Bristol-Myers Squibb, Merck Sharpe & Dohme, Novartis and Roche Pharma. Roche Pharma stopped funding in 2019 and Pierre Fabre started funding of the DMTR in 2019. For this work no funding was granted. 14. 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https://openalex.org/W3165006152	https://hal.science/hal-03378111/file/Crevecoeur_2021_Jebel%20Sahaba_SR.pdf	English	null	New insights on interpersonal violence in the Late Pleistocene based on the Nile valley cemetery of Jebel Sahaba	Scientific reports	2,021	cc-by	13,084	New insights on interpersonal violence in the Late Pleistocene based on the Nile valley cemetery of Jebel Sahaba Isabelle Crevecoeur, Marie-Hélène Dias-Meirinho, Antoine Zazzo, Daniel Antoine, François Bon Isabelle Crevecoeur, Marie-Hélène Dias-Meirinho, Antoine Zazzo, Daniel Antoine, François Bon To cite this version: Isabelle Crevecoeur, Marie-Hélène Dias-Meirinho, Antoine Zazzo, Daniel Antoine, François Bon. New insights on interpersonal violence in the Late Pleistocene based on the Nile valley cemetery of Jebel Sahaba. Scientific Reports, 2021, 11, pp.9991. 10.1038/s41598-021-89386-y. hal-03378111 New insights on interpersonal violence in the Late Pleistocene based on the Nile valley cemetery of Jebel Sahaba New insights on interpersonal violence in the Late Pleistocene based on the Nile valley cemetery of Jebel Sahaba OPEN Isabelle Crevecoeur1, Marie‑Hélène Dias‑Meirinho2, Antoine Zazzo3, Daniel Antoine4 & François Bon2 Isabelle Crevecoeur1, François Bon2 Isabelle Crevecoeur1, Marie‑Hélène Dias‑Meirinho2, Antoine Zazzo3, Daniel Antoine4 & François Bon2 The remains of 61 individuals buried in the cemetery of Jebel Sahaba (site 117) offer unique and substantial evidence to the emergence of violence in the Nile Valley at the end of the Late Pleistocene. Excavated and assessed in the 1960s, some of the original findings and interpretations are disputed. A full reanalysis of the timing, nature and extent of the violence was conducted through the microscopic characterization of the nature of each osseous lesion, and the reassessment of the archaeological data. Over 100 previously undocumented healed and unhealed lesions were identified on both new and/or previously identified victims, including several embedded lithic artefacts. Most trauma appears to be the result of projectile weapons and new analyses confirm for the first time the repetitive nature of the interpersonal acts of violence. Indeed, a quarter of the skeletons with lesions exhibit both healed and unhealed trauma. We dismiss the hypothesis that Jebel Sahaba reflects a single warfare event, with the new data supporting sporadic and recurrent episodes of inter-personal violence, probably triggered by major climatic and environmental changes. At least 13.4 ka old, Jebel Sahaba is one of the earliest sites displaying interpersonal violence in the world. The end of the Late Pleistocene and the beginning of the Holocene were marked by major climatic changes whose impact on human populations is still poorly understood (1–3; cf. Supplementary Text S1). In the Nile Valley, cli- matic conditions are depicted as hyper-arid during the second half of the Late Pleistocene4. Around 15–14 ka, the sudden overflow of Lake Victoria into the White Nile establishes the present Nile-flow regime, causing regular and severe flooding all the way down to Egypt5,6. Only after the Younger Dryas (~ 12.9–11.7 ka), do the monsoon conditions of the African Humid Period start to stabilize3. There is little evidence for human occupations at the end of the Late Pleistocene (~ 18–11.7 ka) in the Nile Valley, with sites restricted to the floodplain of Upper Egypt and Nubia7–9 (cf. Fig. S1). Of these, few have yielded complete human remains. HAL Id: hal-03378111 https://hal.science/hal-03378111v1 Submitted on 14 Oct 2021 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. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2021) 11:9991 Results Th d The individuals examined and the occurrence of healed and unhealed lesions and traumas are listed in the Sup- plementary Table S2. A systematic macroscopic and microscopic analysis confirmed most of the lesions origi- nally described by Anderson19 and Butler22, and allowed the identification of a substantial number of additional traumas and lesions in new and previously identified individuals (identified by green and orange dots in Fig. 1). Reassessment of the evidence of interpersonal violence. A further 106 previously unidentified lesions were observed, including 52 that can now be interpreted as Projectile Impact Marks (PIMs). They reveal that a further twenty-one individuals had clear signs of interpersonal trauma in addition to the twenty described by Wendorf23 and Anderson19. Of the sixty-one individuals studied, forty-one (67.2%) exhibit at least one type of healed or unhealed lesion (Table 1). This includes three-quarters of the adults (74.4%, n = 32), and half of the non-adults affected (50%; n = 9). Our analyses also show that out of these sixty-one individual, 26.2% (n = 16) had signs of perimortem traumas (i.e. unhealed traumas and/or PIMs), and 62.3% (n = 38) displayed healed and/ or unhealed traumas. Both sexes have the same percentage of healed and unhealed lesions. Among the adults with traces of injuries, 36.6% (n = 15) display signs of both healed and unhealed lesions, with males (n = 8) and females (n = 8) similarly affected. Only one non-adult, an adolescent [15–19], has both healed and unhealed lesions (Table 1). Most indi- viduals with lesions 92.7% (n = 38) had some that were traumatic in origin, and over half of these individuals had a projectile impact (61.0%; n = 25). This percentage is similar in adults and non-adults, and between males and females. Embedded lithic fragments, among which two-third are newly identified ones (n = 13, out of 20), were recorded in the PIMs of eleven individuals (26.8%, n = 11), and with a higher proportion in males (n = 6).h g p p The location of the lesions also reveals some patterning to the traumas or PIMs (Table 2 and Supplementary Figs. S2 & S3). First, the number of healed fractures are mainly concentrated on the upper limb and the shoul- der girdle (84.8%, n = 28). Results Th d Fifty percent of these upper limb fracture involve the hands, with both the proximal phalanges and the metacarpals affected, and one-third are located on the forearm. Of the latter, defensive parry fractures of the ulna are the most common (cf. Table 2 and Supplementary Fig. S2;33). A significant difference (P(χ2) > 0.05) between males and females was observed, with parry fractures of left and right sides, without favoring a side, mostly found on female individuals (88.9%, n = 8). Although not significant, hand bone fractures are more frequent in male individuals (58%, n = 7). PIMs are most commonly observed on the lower limb and on the pelvic girdle compared to other anatomical areas (44.3%, n = 70; Table 2 and Supplementary Fig. S3). Similarly, this anatomical region has the highest fre- quency of puncture PIMs and embedded lithic artefacts (respectively 50.0%, n = 12; and 55.0%, n = 11). The sex of the individual does not appear to have influenced the frequency of these marks on different part of the body. Drag marks are present on both upper and lower part of the body, with lower limbs marks mostly found on the femur (94.1%, n = 16) and equally distributed across males and females, as well as the left and right sides. In the upper limbs, the clavicles and humeri exhibit the highest number of projectile marks (n = 11). The direction of the strike reveals no differences between males and females, with both displaying a similar number of projectile marks that had entered from the back or the front of the body. In both sexes, several individuals (n = 6) also exhibit marks consistent with both posterior and anterior impacts. Finally, the analysis reveals that all types of traumas were observed on the cranium. However, most of the perforations caused by blunt force traumas and/ or projectile impacts are observed on the cranium of non-adults (87.5% of the perforations, n = 7). Individual case studies. Three cases best illustrate the complexity and range of lesions found in the Jebel Sahaba individuals regardless of their age-at-death, sex or burial.hi The first case concerns the double burial of two children JS 13 and JS 14, who are close to 5 and 4 years of age, respectively, based on dental development and bone growth. Five lithic artefacts were found in association with the two individuals (23, p. 963). www.nature.com/scientificreports/ most emblematic and widely cited example of early widespread violence is the cemetery of Jebel Sahaba. Dated between 13,400 and 18,600 cal BP (10,032 ± 46 BP, UBA-20131, to 13,740 ± 600 BP, Pta-116), the Jebel Sahaba cemetery is the earliest funerary complex from the Nile Valley (cf. Supplementary Text S2 and Table S1). Early analyses of the skeletons by Anderson19 and Butler22 revealed evidence of interpersonal violence on the bones of at least half of the Jebel Sahaba individuals. In addition, abundant lithic artefacts from the Qadan industry were discovered within the physical space of the bodies, where the soft tissues would have once been, or directly embedded in the bones23. Given their position, these lithic artefacts cannot be considered to be grave goods, nor can the Jebel Sahaba individuals be referred to as belonging to a Qadan population, particularly as other cultural entities are present in Lower Nubia during the same period17. p g p Since its discovery and original publication by Wendorf11, the cemetery has been cited as a key example for the emergence of violence and organized warfare triggered by territorial disputes24–28. Many elements of the original findings, particularly the timing, nature and extent of the violence, but also the lithic association, have since been challenged e.g.29–32. However, no holistic study of the traces of violence left on the human remains has been undertaken to reassess the site and provide an updated perspective on violence and human behavior at the end of the Late Pleistocene26. Here we address several unanswered questions that benefit from a full reanalysis of the collection using the latest anthropological and forensic methods. Indeed, it remains unclear whether the cemetery was the result of a single event, of sporadic or repetitive episodes of inter-personal violence, or was used as a place for the burial of specific individuals. Some cutmarks appear to be the result of projectile penetration while others are thought to have been caused by deliberate cuts as part of specific mortuary treatments. Finally, a reassessment of the lithic assemblage would also further our understanding of the site. New insights on interpersonal violence in the Late Pleistocene based on the Nile valley cemetery of Jebel Sahaba OPEN These include Jebel Sahaba (Site 117), Tushka (Site 8905), Wadi Kubbaniya, and site 6-B-36 from Wadi Halfa10–12.fi y Culturally, different lithic industries have been identified with sites associated to the end of the Late Pleisto- cene e.g.13–17. These occur in restricted geographical areas along the Nile, mainly in Upper Egypt. They do not seem to be related to specific activities and are defined by characteristic sets of lithic tools and/or technology that appear to be associated with distinct small hunting-fishing-gathering groups15–17. Each of these lithic groups is believed to represent a cultural tradition that reflects group identity15 (cf. Supplementary Text S1 and Fig. S1). The occurrence of large graveyards at the end of the Late Pleistocene reinforces the idea of strong social units within these residential groups18.i g p Set in a context of possible environmental pressures and geographical constraints, the identification of traces of interpersonal violence on the individuals buried in Jebel Sahaba have attracted much attention18,19. Evidence of conflicts is not uncommon in the Nile valley. The oldest documented case (~ 20 ka) appears to be from Wadi Kubbaniya, where the remains of a partial skeleton belonging to a young adult male provides early evidence of interpersonal violence (12,20). Embedded lithic and healed fractures have also been documented on some indi- viduals buried in the Wadi Halfa cemetery, associated with Qadan lithic industry (site 6-B-36;10,21). However, the 1UMR 5199‑PACEA, CNRS, Université de Bordeaux, B8, Allée Geoffroy Saint‑Hilaire, CS 50023, 33615 Pessac Cedex, France. 2UMR 5608‑TRACES, Université de Toulouse Jean Jaurès, Maison de La Recherche, 5 Allées Antonio Machado, 31058 Toulouse Cedex 9, France. 3UMR 7209‑AASPE, CNRS, MNHN, CP 56 ‑ 43 Rue Buffon, 75005 Paris, France. 4Department of Egypt and Sudan, The British Museum, Great Russell Street, London WC1B 3DG, UK. email: isabelle.crevecoeur@u-bordeaux.fr \| https://doi.org/10.1038/s41598-021-89386-y Scientific Reports \| (2021) 11:9991 www.nature.com/scientificreports/ Results Th d Although no osseous lesion was visible on JS 13, both the cranium and https://doi.org/10.1038/s41598-021-89386-y Scientific Reports \| (2021) 11:9991 \| www.nature.com/scientificreports/ Figure 1. Location of the Jebel Sahaba cemetery, Site 117, in the Nile Valley and map of the excavated area and burials (map following23, Image Courtesy British Museum Wendorf Archive, modified with Adobe Illustrator CS6, https://www.adobe.com/products/illustrator.html). Red dots: individuals exhibiting signs of violence and/ or traumatic lesions19,20,91; orange dots: newly identified lesions in the latter individuals; green dots: individuals newly identified as showing signs of violence and/or traumatic lesions; large dots: individuals discussed in detail in the text. Satellite image: Google Maps, 2020. 21° 58′ 12.0" N 31° 22′ 12.0" E, elevation 21.9 M. [online] Available through: <https://www.google.com/maps/place/>. Figure 1. Location of the Jebel Sahaba cemetery, Site 117, in the Nile Valley and map of the excavated area and burials (map following23, Image Courtesy British Museum Wendorf Archive, modified with Adobe Illustrator CS6, https://www.adobe.com/products/illustrator.html). Red dots: individuals exhibiting signs of violence and/ or traumatic lesions19,20,91; orange dots: newly identified lesions in the latter individuals; green dots: individuals newly identified as showing signs of violence and/or traumatic lesions; large dots: individuals discussed in detail in the text. Satellite image: Google Maps, 2020. 21° 58′ 12.0" N 31° 22′ 12.0" E, elevation 21.9 M. [online] Available through: <https://www.google.com/maps/place/>. infra-cranium of JS 14 have unhealed trauma caused by projectile impacts (Fig. 2). The majority of the lesions are located on the calvaria and none had previously been documented. The frontal bone exhibits a blunt force trauma at the level of the glabella. Several drag marks and an oblong perforation are also present on the left side of the frontal squama, as well as scraping drag marks close to bregma. Both a puncture site with faulting and part of an embedded artifact are visible approximately one centimeter above the left orbit (Fig. 2-1). A perforation is also present on the right parietal and on the occipital. The frontal and occipital perforation exhibit internal bevelling consistent with projectile impacts34. A further set of marks is visible on the left femur, including two groups of drags on the antero-lateral border of the proximal part of the diaphysis (Fig. 2-2). The first group has two subparallel incisions with wide flat floors marked with parallel microstriations. Bone flaking is also present at the end of the trajectory. Results Th d The second drag is located about one centimeter below the proximal one, and oriented slightly more anteriorly, with a bisecting pattern at the end of the marks. Based on these cutmark characteristics, the projectile most probably arrived from the medial side of the femoral diaphysis, in a downwards motion and towards the lateral side. h The second case, JS 31, focuses on the remains of a probable male over 30 years old based on his heavy dental wear and bone remodeling. Seventeen lithic artefacts found in situ were in direct association with his skeletal remains, with two embedded in the bone and fifteen within the physical space of the body (23, p. 973–974). https://doi.org/10.1038/s41598-021-89386-y Scientific Reports \| (2021) 11:9991 \| www.nature.com/scientificreports/ Total (n = 61) Mature (n = 43) Immature (n = 18) Female (n = 19) Male (n = 20) Und. (n = 6) Total [0– < 1] (n = 2) [1–4] (n = 5) [5–9] (n = 6) [10–14] (n = 3) [15–19] (n = 2) n % n % n % n % n % n % n % n % n % n % n % No lesion 20 32.8 11 25.6 9 50.0 2 100.0 3 60.0 4 66.7 0 0.0 0 0.0 5 26.3 5 25.0 1 16.7 Lesions 41 67.2 32 74.4 9 50.0 0 0.0 2 40.0 2 33.3 3 100.0 2 100.0 14 73.7 15 75.0 5 83.3 Healed lesions 38 92.7 32 100.0 5 55.6 0 0.0 0 0.0 1 50.0 2 66.7 2 100.0 14 100.0 15 100.0 5 100.0 Unhealed lesions 19 46.3 15 46.9 5 55.6 0 0.0 2 100.0 1 50.0 1 33.3 1 50.0 8 57.1 8 53.3 0 0.0 H&U lesions 16 39.0 15 46.9 1 11.1 0 0.0 0 0.0 0 0.0 0 0.0 1 50.0 8 57.1 8 53.3 0 0.0 1. Results Th d The percentage in the underlying lines represents the proportion of individuals with healed, unhealed and healed and unhealed lesion occurrence within the numbered line category. n = number; % = percentage; PIM = Projectile Impact Mark; H&U = Healed and Unhealed. Und. = mature individuals whose sexual diagnosis is undeterminate. The embedded chips were originally found in the seventh cervical vertebra and in the left pubis (19,23), with the bone around both lithics showing severe reactive changes (cf. p. 989 in23, and p. 1027 in19). Unfortunately, these bones are not part of the collection donated to the British Museum. The lesions observed on JS 31 are located on the infra-cranial skeleton. Our reassessment revealed previously unidentified healed and unhealed projectile impact marks, as well as healed lesions that are most probably the result of earlier interpersonal injuries. The new unhealed PIMs identified include a puncture with crushing, faulting and flaking of the bone surface on the anterior part of the left scapula (Fig. 3-1) and a deep V-shaped drag (2 cm long) on the posterior-medial side of the humerus. JS 31 also has a healed fracture of the distal extremity of the right first metacarpal. Finally, the right femur offers further evidence of healed lesions, with the presence of a bone callus on the lateral side of the proximal part of the shaft and of a healed projectile wound on the anterior side at midshaft. Three previously unidentified embedded lithic chips were found trapped in the healing bulge of the latter (Fig. 3-2).h i p pp g g g The third case, JS 44, are the remains of a possible female that appears to have been older than 30 years. Twenty-one lithic artefacts were found in close association with the skeleton, one of which was embedded in the fourth rib (23, p. 978). Wendorf also noted two examples of chip and/or flake alignments during the excavation which he interpreted as evidence of composite projectile use23. The fourth rib with embedded “backed flake” is, unfortunately, also not present in the British Museum Wendorf collection. As with JS 31, all the lesions observed on JS 44 are located in the infra-cranial skeleton (Fig. 4), with healed fractures present on the left clavicle, right ulna and radius, and one left rib. Results Th d Traumas & PIMs 38 92.7 30 93.8 8 88.9 0 0.0 2 100.0 2 100.0 3 100.0 1 50.0 14 100.0 15 100.0 3 60.0 Healed Traumas & PIMs 32 84.2 27 90.0 4 50.0 0 0.0 0 0.0 1 50.0 2 66.7 0 0.0 11 78.6 15 100.0 3 100.0 Unhealed Traumas & PIMs 16 42.1 12 40.0 5 62.5 0 0.0 2 100.0 1 50.0 1 33.3 0 0.0 7 50.0 6 40.0 0 0.0 H&U Traumas & PIMs 10 26.3 9 30.0 1 12.5 0 0.0 0 0.0 0 0.0 0 0.0 1 50.0 4 28.6 6 40.0 0 0.0 2. Fractures 22 36.1 21 48.8 1 5.6 0 0.0 0 0.0 0 0.0 0 0.0 1 50.0 9 47.4 11 55.0 2 33.3 3. PIMs 25 61.0 19 59.4 6 66.7 0 0.0 2 100.0 1 50.0 2 66.7 1 50.0 10 71.4 10 66.7 1 20.0 Healed PIMs 12 48.0 10 52.6 1 16.7 0 0.0 0 0.0 0 0.0 1 50.0 0 0.0 4 40.0 6 60.0 1 100.0 Unhealed PIMs 16 64.0 12 63.2 5 83.3 0 0.0 2 100.0 1 100.0 1 50.0 1 100.0 7 70.0 6 60.0 0 0.0 H&U PIMs 3 12.0 3 15.8 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 1 10.0 2 20.0 0 0.0 4. Embedded lithic 11 26.8 9 28.1 2 22.2 0 0.0 1 50 0 0.0 1 50 0 0.0 3 21.4 6 40.0 0 0.0 Healed PIMs 4 36.4 4 44.4 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 4 66.7 0 0.0 Unhealed PIMs 8 72.7 6 66.7 2 100.0 0 0.0 1 100.0 0 0.0 1 100.0 0 0.0 3 100.0 3 50.0 0 0.0 H&U PIMs 1 9.1 1 11.1 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 1 16.7 0 0.0 Table 1. Number of individuals exhibiting at least one type of lesion grouped by age-at-death or sexual diagnosis. The percentage in the two first lines are calculated on the minimal number of individuals for each category, while the percentage in the numbered bold lines are computed based on the recorded number of individual with lesions for each category. Results Th d The fracture of the left clavicle shaft, located on the acromial end of the diaphysis, reveals a slight torsion and a displacement of the bone fragments. The right forearm healed fracture is oblique, with a displacement (translation and rotation) of the two broken pieces (Fig. 4-1). The clavicle and forearm fractures most probably occurred during the same event. Given the oblique nature in the forearm and acromial involvement in the clavicle, they may have been caused by an indirect trauma, such as a bad fall, rather than a defensive parry fracture (see33). The other lesions, however, are clearly the result of projectile impacts. A triangular notch on the lateral face of the ilium, about 1 cm from the greater sciatic notch, has a lithic fragment embedded in the incision. The laminated aspect of the bone overlying the flake suggests there was an attempt to extract the projectile (Fig. 4-2). The morphology of the PIM also indicates the projectile travelled from the postero-medial to the antero-lateral side of the left pelvic bone, which implies the projectile was travelling back to front. PIMs were also observed on the right femur. Two parallel drags less than 1 cm long and approximately 2 cm from each other are visible on the posterior side of the diaphysis. These drags exhibit a flat bottom with parallel microstriations. The most distal one shows flaking marks on the proximal border (Fig. 4-3). Significantly, the angle of penetration into the bone differs for both drags, with the most proximal one being more tangential. These drag marks reflect a projectile trajectory that came from the disto-lateral to the proximo-medial part of https://doi.org/10.1038/s41598-021-89386-y Scientific Reports \| (2021) 11:9991 \| www.nature.com/scientificreports/ Table 2. Number and type of lesions recorded on the Jebel Sahaba individuals. Percentage of each of these lesions in relation to the anatomical parts in bold numbered lines and percentage of infliction to specific Traumas & PIMs Total Lesions Traumas PIMs Total Fractures Perforations/ BFT Drags Punctures Perforations Total Embedded lithic Number of lesions 33 4 40 24 6 70 20 107 139 Number of individuals 22 4 17 14 3 25 11 38 41 % of individuals 36.1 6.6 27.9 23.0 4.9 41.0 18.0 62.3 67.2 Anatomical repartition 1. Results Th d Cranium (%) 3.0 100.0 20.0 25.0 66.7 25.7 15.0 21.5 20.9 % Frontal – 75.0 50.0 50.0 25.0 44.4 33.3 47.8 48.3 % Parietal – – – 33.3 50.0 22.2 66.7 17.4 13.8 % Temporal – 25.0 12.5 16.7 – 11.1 0.0 13.0 13.8 % Occipital – – – – 25.0 5.6 0.0 4.3 10.3 2. Upper limb and shoulder girdle (%) 84.8 – 35.0 8.3 – 22.9 10.0 41.1 36.0 % Shoulder girdle 7.1 – 35.7 50.0 – 37.5 50.0 18.2 20.0 % Humerus 10.7 – 35.7 – – 31.3 50.0 18.2 18.0 % Ulna 28.6 – 14.3 – – 12.5 – 22.7 20.0 % Radius 3.6 – 14.3 – – 12.5 – 6.8 10.0 % Forearm 32.1 – 28.6 – – 25.0 – 29.5 30.0 % Hand bones 50.0 – 0.0 50.0 – 6.3 – 34.1 32.0 3. Trunk (%) 3.0 – 2.5 16.7 – 7.1 20.0 5.6 5.8 4. Lower limb and pelvic girdle (%) 9.1 – 42.5 50.0 33.3 44.3 55.0 31.8 37.4 % Coxal – – 5.9 66.7 – 29.0 63.6 26.5 23.1 % Femur – – 94.1 25.0 – 61.3 27.3 55.9 53.8 % Tibia – – – – – – – – 3.8 % Fibula 33.3 – – 8.3 – 3.2 9.1 5.9 5.8 % Foot bone 66.7 – – – 100.0 6.5 – 11.8 13.5 Table 2. Number and type of lesions recorded on the Jebel Sahaba individuals. Percentage of each of these lesions in relation to the anatomical parts in bold numbered lines, and percentage of infliction to specific bones of these anatomical parts in underlying lines. PIM = Projectile Impact Mark; BFT = Blunt Force Trauma; % = percentage. Table 2. Number and type of lesions recorded on the Jebel Sahaba individuals. Percentage of each of these lesions in relation to the anatomical parts in bold numbered lines, and percentage of infliction to specific bones of these anatomical parts in underlying lines. PIM = Projectile Impact Mark; BFT = Blunt Force Trauma; % = percentage. the bone. This upward direction suggests the individual was hit while running or that the projectile was drawn from a lower position. Finally, the spacing between these two drags and their morphology are consistent with the penetration from a single composite projectile. This hypothesis is strengthened by Wendorf’s field observation of in situ lithic alignments associated with JS 44. Burial selection and mortality profile. Results Th d In view of the high number of individual with evidence of inter- personal violence, the frequency of projectile impact marks, and the presence of several double or multiple buri- als, the site’s mortality profile was analyzed to investigate possible patterns in burial selection (see35,36). Should the cemetery reflect a single warfare event, an unbalanced demographic profile (e.g. the overrepresentation of a certain sex or age group less likely to die otherwise) is probable37. At Jebel Sahaba, the individuals whose sex could be assigned (n = 39) revealed no bias, with 48.7% females and 51.3% males. The age distribution shows a under- representation of non-adults ([< 20] = 29.5%) compared to the theoretical percentage ([< 20] = 54.5% ± 9.5%) for a pre-jennerian population with a life expectancy at birth of between 25 and 35 years38. However, this imbal- ance is mostly due to the lack of perinates, neonates and young children (age cohorts [0–1] and [1–4]) whose mortality quotient stands outside the lower limits of the theoretical values (cf. Supplementary Fig. S4). The small proportion of very young children is not unusual in pre-Neolithic funerary assemblages and may relate to demo- graphic factors, cultural behaviors such as the separate burial of young infants, or poor preservation, although the latter is unlikely at Jebel Sahaba39–42. To account for these possibilities, we focused on the age cohorts over than five years old to assess any biases in age-at-death representations40,43. Both the J:A ratio (JS = 0.200) and the mean childhood mortality value (JS = 0.073) are below the threshold of biased cemetery populations (respec- tively J:A < 0.380 and MCM < 0.135;43). In the event of a mortality crisis linked to a single event, demographic anomalies are usually found in age cohorts less likely to die otherwise (namely the [10–14] and [15–19] cohorts) by way of an overrepresentation in their mortality quotient35,36,44. The Jebel Sahaba cemetery mortality curve does not include such an anomaly. https://doi.org/10.1038/s41598-021-89386-y Scientific Reports \| (2021) 11:9991 \| www.nature.com/scientificreports/ Figure 2. Location and images of the observed osseous lesions on JS 14. Center: schematic scheme of JS 14′s skeletal preservation. Grey parts represent preserved bones, star = blunt force trauma, full star = unhealed puncture, open circle = perforations, yellow diamond = embedded artefact in a puncture, dash = drags traces of projectile impacts, line = cutmark. Box n°1: lesions of the frontal bone on JS 14. Results Th d Left: superior view of the frontal bone with, below, the magnification in frontal view of the red box showing the blunt force trauma and the embedded lithic (white oval) with hinge fractures. Right: left lateral view of the frontal bone displaying the projectile perforation. Red and white stars are reference points for the magnified area; a = hinge fractures at the level of the entrance of the projectile; b = crushing fractures on the border of the perforation; c = endocranial view of the internal beveling. Note the miss-glued piece of bone associated to the perforation, part of the original conservation works. Box n°2: Projectile impact marks on the left femur of JS 14. Left: anterior view of the preserved part of the left femur. a = close up on the two set of drag marks located on the antero-lateral side of the shaft. White star put as reference point for the magnified area. b = detailed view of the superior drag revealing the wide flat bottom of the groove and the parallel microstriations (magnification ×245). Figure 2. Location and images of the observed osseous lesions on JS 14. Center: schematic scheme of JS 14′s skeletal preservation. Grey parts represent preserved bones, star = blunt force trauma, full star = unhealed puncture, open circle = perforations, yellow diamond = embedded artefact in a puncture, dash = drags traces of projectile impacts, line = cutmark. Box n°1: lesions of the frontal bone on JS 14. Left: superior view of the frontal bone with, below, the magnification in frontal view of the red box showing the blunt force trauma and the embedded lithic (white oval) with hinge fractures. Right: left lateral view of the frontal bone displaying the projectile perforation. Red and white stars are reference points for the magnified area; a = hinge fractures at the level of the entrance of the projectile; b = crushing fractures on the border of the perforation; c = endocranial view of the internal beveling. Note the miss-glued piece of bone associated to the perforation, part of the original conservation works. Box n°2: Projectile impact marks on the left femur of JS 14. Left: anterior view of the preserved part of the left femur. a = close up on the two set of drag marks located on the antero-lateral side of the shaft. White star put as reference point for the magnified area. Results Th d b = detailed view of the superior drag revealing the wide flat bottom of the groove and the parallel microstriations (magnification ×245). Reassessment of the lithic assemblage. We identified 13 new pieces embedded in projectile impact marks on the human remains and counted the multiple fragments found in one PIM as one artefact. Based on these findings, a new total of 132 artefacts were found in direct association with 28 individuals (cf. Supplemen- tary Table S3). With the exception of a few flakes and points, the artefacts collected at the surface of the site (n = 72) differ in terms of their typology and the raw material used when compared to the ones found inside the burials and within the physical space of the skeletons (n = 115; Supplementary Text S3 and Fig. S5).i Our reexamination confirms the diversity of artefact shapes with a tendency toward small size pieces. Despite a strong typological variability, most lithic artifacts found inside the burials can be identified as projectiles or armature elements, including the unretouched parts. Significantly, preliminary functional analysis shows that some artefacts have impact fractures. Technological and typological elements fit well with the definition of the Qadan industry23,45,46. The current reassessment also revealed strong similarities to the Tushka 8905-B industry unambiguously attributed to the Qadan (45–47; Supplementary Text S3).ll Based on this reanalysis, almost half of the elements used as weapons are unretouched flakes and micro-flakes that, as noticed by Wendorf23, would have been missed in any other context (cf. Supplementary Fig. S5). In the case of Jebel Sahaba, their association to weaponry is indisputable. Most appear to be laterally shafted composite elements used as part of projectiles. The points would have been mounted at the end of shafts, with crescents laterally shafted. Their diversity in both size and shape suggests the use of several types of weapons, particularly light arrows but also much heavier arrows or spears. Finally, the use of points with oblique or transverse distal cutting edges appears to indicate that one of the main lethal properties sought was to slash and cause blood loss. The fact that many were found inside the volume of the skeleton also indicates their efficiency at penetrating the body. Those found at the site are likely to be the ones that had detached themselves from their shaft and not successfully removed prior to burial. Discussion c = microscopic view of a bony bridge separating two geometric marks indicating the presence of two lost lithic chips (magnification ×50). injuries from slicing cutmarks and taphonomical modifications (see34,48–52). More than half of the individuals with lesions buried at Jebel Sahaba (n = 41) exhibit clear projectile impact marks (61.0%, n = 25), and most show signs of trauma (92.7%, n = 38). Irrespective of age and sex, the majority of those buried at the site exhibit signs of interpersonal violence that involve projectile weapons. The number of individuals with both healed and unhealed traumas also increases with age from adolescence (n = 1), to young adults (n = 2) and adults (n = 13). Importantly, the co-occurrence of ante-mortem and peri-mortem lesions on several Jebel Sahaba individuals had not previously been noted and indicates that some had experienced multiple episodes of interpersonal violence during their life. injuries from slicing cutmarks and taphonomical modifications (see34,48–52). More than half of the individuals with lesions buried at Jebel Sahaba (n = 41) exhibit clear projectile impact marks (61.0%, n = 25), and most show signs of trauma (92.7%, n = 38). Irrespective of age and sex, the majority of those buried at the site exhibit signs of interpersonal violence that involve projectile weapons. The number of individuals with both healed and unhealed traumas also increases with age from adolescence (n = 1), to young adults (n = 2) and adults (n = 13). Importantly, the co-occurrence of ante-mortem and peri-mortem lesions on several Jebel Sahaba individuals had not previously been noted and indicates that some had experienced multiple episodes of interpersonal violence during their life. g As with experimental studies on ungulates53,54, drag marks are the most frequent PIMs observed at Jebel Sahaba followed by punctures, particularly on the appendicular skeleton. Experimental work also reveals that 45.0% of ungulates PIMs include microscopic fragments of the actual weapons that end up embedded in the bones, either at impact or while attempting to remove the weapon (34,53). At Jebel Sahaba, artefacts were found in one third of the drag and puncture impact marks (31.3%, n = 20). Of these, the great majority were in puncture marks (70.8%, n = 17). The PIMs patterns supports the use of composite weapons made of shafted retouched and unretouched flakes, including light and heavy projectiles. Discussion Since its discovery in the 1960′s, the Jebel Sahaba cemetery has been regarded as the oldest evidence of organ- ized warfare caused by environmental constrains e.g.24–28. However, the lesions observed on the Jebel Sahaba skeletons and the nature of the funerary complex had not been reassessed and it remained unclear whether the site was the result of a single conflict, a specific burial place or the evidence of sustained inter-personal violence in Late Pleistocene hunter-gatherer groups18. g g p Wendorf23 and Anderson19 had highlighted the projectile nature of several lesions, particularly those w embedded lithic artefacts. Here, macroscopic and microscopic methods were used to distinguish projec https://doi.org/10.1038/s41598-021-89386-y Scientific Reports \| (2021) 11:9991 \| www.nature.com/scientificreports/ Figure 3. Location and images of the observed osseous lesions on JS 31. Center: schematic scheme of JS 31′s skeletal preservation. Grey parts represent preserved bones, striped areas are missing bone or areas, full star = unhealed puncture, dash = drags traces of projectile impacts, line = cutmark, plus sign = healed lesions, time sign = healed fracture, full circle = healed puncture, yellow diamond = embedded artefact in a puncture, orange diamond = embedded artefact in lost bone. Box n°1: Projectile impact puncture on the left scapula of JS 31. a = red rectangle close up on the subscapular fossa showing the puncture associated with flaking and faulting. b = composite microscopic image of the puncture displaying the crushing of the bone in the lower border of the puncture (magnification ×40). Box n°2: Healed lesions on the right femur of JS 31. Red rectangle = healed projectile lesion, black ellipse = bone callus. a = red rectangle close up of the healed projectile injury with red and white stars as reference points for the magnified area b and c. b = microscopic view of the three embedded lithic chips marked by arrows. c = microscopic view of a bony bridge separating two geometric marks indicating the presence of two lost lithic chips (magnification ×50). Figure 3. Location and images of the observed osseous lesions on JS 31. Center: schematic scheme of JS 31′s skeletal preservation. Discussion Grey parts represent preserved bones, striped areas are missing bone or areas, full star = unhealed puncture, dash = drags traces of projectile impacts, line = cutmark, plus sign = healed lesions, time sign = healed fracture, full circle = healed puncture, yellow diamond = embedded artefact in a puncture, orange diamond = embedded artefact in lost bone. Box n°1: Projectile impact puncture on the left scapula of JS 31. a = red rectangle close up on the subscapular fossa showing the puncture associated with flaking and faulting. b = composite microscopic image of the puncture displaying the crushing of the bone in the lower border of the puncture (magnification ×40). Box n°2: Healed lesions on the right femur of JS 31. Red rectangle = healed projectile lesion, black ellipse = bone callus. a = red rectangle close up of the healed projectile injury with red and white stars as reference points for the magnified area b and c. b = microscopic view of the three embedded lithic chips marked by arrows. c = microscopic view of a bony bridge separating two geometric marks indicating the presence of two lost lithic chips (magnification ×50). Figure 3. Location and images of the observed osseous lesions on JS 31. Center: schematic scheme of JS 31′s skeletal preservation. Grey parts represent preserved bones, striped areas are missing bone or areas, full star = unhealed puncture, dash = drags traces of projectile impacts, line = cutmark, plus sign = healed lesions, time sign = healed fracture, full circle = healed puncture, yellow diamond = embedded artefact in a puncture, orange diamond = embedded artefact in lost bone. Box n°1: Projectile impact puncture on the left scapula of JS 31. a = red rectangle close up on the subscapular fossa showing the puncture associated with flaking and faulting. b = composite microscopic image of the puncture displaying the crushing of the bone in the lower border of the puncture (magnification ×40). Box n°2: Healed lesions on the right femur of JS 31. Red rectangle = healed projectile lesion, black ellipse = bone callus. a = red rectangle close up of the healed projectile injury with red and white stars as reference points for the magnified area b and c. b = microscopic view of the three embedded lithic chips marked by arrows. Discussion Box n°2: Lateral view of the left pelvis of JS 44 with a projectile impact puncture with an embedded lithic flake. a = red rectangle close up of the PMI with white star as reference point for the magnified area b. b = microscopic view of the puncture showing the laminated aspect of the superior border and the lithic artefact inside the puncture indicated by the red arrow (magnification 30x). Box n°3: Double parallel drags on JS 44 located on the posterior surface of the right femur diaphysis, at the level where the lateral supracondylar line, which delimitates the lateral part of the popliteal plane, meets to lateral side of the femoral diaphysis. a = red rectangle close up showing the two parallel drags and the direction of the projectile with the arrows. White star as reference point for the magnified area b. b = microscopic close up on the distal drag showing the flaking of the superior border at the origin of the drag. Red star as reference point for the magnified area c (magnification ×45). c = composite microscopic view of the proximal part of the distal drag displaying the wide flat bottom of the groove and the parallel microstriations (magnification ×235). Figure 4. Location and images of the observed osseous lesions on JS 44. Center: schematic scheme of JS 44′s skeletal preservation. Grey parts represent preserved bones, striped areas are missing bone, crisscross areas are bone whose exact anatomical position is unknown, full star = unhealed puncture, dash = drags traces of projectile impacts, time sign = healed fracture, yellow diamond = embedded artefact in a puncture, orange diamond = embedded artefact in lost bone. Box n°1: Healed fractures on JS 44. From top to bottom, left clavicle superior view, right radius anterior view and right ulna anterior view. Box n°2: Lateral view of the left pelvis of JS 44 with a projectile impact puncture with an embedded lithic flake. a = red rectangle close up of the PMI with white star as reference point for the magnified area b. b = microscopic view of the puncture showing the laminated aspect of the superior border and the lithic artefact inside the puncture indicated by the red arrow (magnification 30x). Discussion This is corroborated by the alignment of flakes and chips within the physical space of the skeletons, the reassessment of the lithic assemblage and cases of parallel drags less than 2 cm apart consistent with ethnographical and experimental spear and arrow shaft diameters54–56.t g p g p p pt Identifying interpersonal violence on skeletal remains is not always straightforward and often relies on the type of trauma and the archaeological context57,58. Clear examples of fatal interpersonal blunt and sharp force trauma go as far back as the Middle Paleolithic59,60, while the oldest Palaeolithic projectile trauma with an embed- ded point date to the Epigravettian period61. Based on the available evidence, the number of projectile injuries appears to increase over time and cases of fatal trauma in Europe become more frequent during the Mesolithic62. In Africa, the site of Nataruk provides the closest parallel of inter-personal violence to Jebel Sahaba63. Situated west of Lake Turkana and dating to around 10.5–9.5 ka, the individuals found in Nataruk appear to exhibits signs of violent death through projectile impact marks (punctures and perforation), sharp and blunt force trauma and fractures. Although this evidence has been debated64, the Nataruk example also differs from Jebel Sahaba in that there is no clear pattern of deliberate burial, no signs of trauma on children and a lack of healed trauma in the adults.l Violent behavior in past and present hunter-gatherer societies appears to vary, in part reflecting the period, culture and the level of organization of mobile and semi-sedentary societies e.g.24,25,65. Several ethno-archaeologi- cal examples suggest that the concept of warfare can encompass all form of antagonistic relationships from feuds, individual murders, ambush attacks, raids and trophy taking to bloody clashes and larger armed conflicts25,27,65. Scientific Reports \| (2021) 11:9991 \| https://doi.org/10.1038/s41598-021-89386-y www.nature.com/scientificreports/ Figure 4. Location and images of the observed osseous lesions on JS 44. Center: schematic scheme of JS 44′s skeletal preservation. Grey parts represent preserved bones, striped areas are missing bone, crisscross areas are bone whose exact anatomical position is unknown, full star = unhealed puncture, dash = drags traces of projectile impacts, time sign = healed fracture, yellow diamond = embedded artefact in a puncture, orange diamond = embedded artefact in lost bone. Box n°1: Healed fractures on JS 44. From top to bottom, left clavicle superior view, right radius anterior view and right ulna anterior view. Discussion Box n°3: Double parallel drags on JS 44 located on the posterior surface of the right femur diaphysis, at the level where the lateral supracondylar line, which delimitates the lateral part of the popliteal plane, meets to lateral side of the femoral diaphysis. a = red rectangle close up showing the two parallel drags and the direction of the projectile with the arrows. White star as reference point for the magnified area b. b = microscopic close up on the distal drag showing the flaking of the superior border at the origin of the drag. Red star as reference point for the magnified area c (magnification ×45). c = composite microscopic view of the proximal part of the distal drag displaying the wide flat bottom of the groove and the parallel microstriations (magnification ×235). The level of warfare can vary, with some conflicts being all-encompassing, constant and deadly, while others are episodic events of various intensity that occur sporadically65. At Jebel Sahaba, the co-occurrence of healed and unhealed lesions strongly supports sporadic and recurrent episodes of interpersonal violence between Nile valley groups at the end of the Late Pleistocene. The projectile nature of at least half of the lesions suggests inter-group attacks, rather than intra-group or domestic conflicts65–69, and the frequency of healed wound confirm that these events were not always lethal and could occur several time during the life of an individual. While the number of parry fractures is higher among female individuals, and the blunt force trauma mostly present on immature individuals, the remaining pattern of lesions on female and immature individuals at Jebel Sahaba is inconsistent with domestic violence70,71. A catastrophic single mass burial is highly unlikely and not supported by the archaeological evidence and the demographic analysis. With the exception of a higher percentage of parry fractures in females, there appears to be no patterning in the distribution of trauma or PIMs by rather age or sex. Based on the lesions, the projectile direction also reveals an equal number of posterior and anterior strikes that do not support face-to-face battles. Rather, the involvement of a range of ages and both sexes, with primary (n = 26), double (n = 4) and multiple (n = 4) burials, including some with evidence of disturbance due to the addition of later individuals23, indicate small episodes of recurring violent events such as raids or ambushes against this community. Materials and methods The British Museum Jebel Sahaba collection. In 2001, Wendorf donated all the archives, artefacts and skeletal remains from his 1965–1966 Nile Valley excavations to the British Museum76,77. Judd’s preliminary oste- ological analysis noted discrepancies between field notes, photographs and associated skeletal remains, including the absence of three individuals, JS 1, JS 3 and JS 30, as well as some of the bones with embedded lithic artefacts described by Anderson19,76. Not part of the British Museum donation, their whereabouts remains uncertain. The three missing individuals could not be included in this reanalysis. Regarding the few missing bones of reassessed individuals, we relied on Anderson’s description of the trauma as they could not be examined microscopically. Judd’s survey of the skeletal remains also noticed the presence of extra bones or teeth from additional individu- als. Excluding the remains of the three missing individuals, our reanalysis also found supernumerary bones and teeth and, with the British Museum collection, the site can now be regarded as including the remains of at least 64 individuals, three of whom are now missing.ii g In addition to the lithic assemblage from the fill around the skeleton that we attributed to the surface find assemblage (n = 72), our reassessment included 115 pieces from the original collection described as directly asso- ciated to the skeletons. Three pieces from burials JS 25, JS 45 and JS 47 are not in the British Museum collection. A supplementary piece was, however, found associated to burial JS 26. This piece was probably mixed with the surface material early on, which could explain its absence in Wendorf’s inventory (although the piece was drawn in p. 987 in23). We also included five pieces found near burials JS 101 to JS 107. Although not directly in contact with the skeletons, their association to the individuals of this multiple burial is suggested by Wendorf (23, p. 988). These artefacts were part of our reassessment but we remained cautious as to their association with the burials. Biological identification. The analysis involved a full reevaluation of the age and sex using the latest anthropological methods. In some individuals, assessments were limited by the state of preservation and com- pleteness of the skeletal remains. Biological sex was based on the morphology and dimensions of the pelvis78–80. Discussion This appears to have taken place on a short timescale given the homogeneity of the burial place and practices. g g y Special burial places for the victims of violence are documented in ethnological and historical records72. At Jebel Sahaba, the percentage of individuals with traces of peri-mortem traumas and/or lithic artefacts found within the physical space of the skeleton is 54%. If multiple burials are treated as simultaneous deaths and individuals without detectable signs of a violent death but buried in direct association with others that have are included, the percentage is closer to 64%. The nearby site of Wadi Halfa (6-B-36) does not seem to document comparable levels of violence as the percentage of individuals with traumas (22.2%, n = 8) is lower than at Jebel Sahaba (62.3%, n = 38). However, an unhealed projectile trauma with an embedded stone point in a cervical vertebra is documented21, and the frequency of the most identifiable lesion in Wadi Halfa, healed parry fractures, is similar to Jebel Sahaba (respectively 8.3%, n = 3, and 9%, n = 6). Therefore, we consider more likely that the level of interpersonal violence observed in Jebel Sahaba reflects broader inter-group behavioral relationships in the Nile valley at the end of the Late Pleistocene rather than specific funerary practices. https://doi.org/10.1038/s41598-021-89386-y Scientific Reports \| (2021) 11:9991 \| www.nature.com/scientificreports/ The high level of interpersonal violence observed at the site may, in part, have been driven by the climatic variability. During the Late Pleistocene, few human remains are recorded in the Nile valley. This is mirrored by a drastic reduction in the archaeological record with little evidence for the presence of humans along the lower Nile from Marine Isotopic Stage 4 (~ 71 ka) to the Last Glacial Maximum9. During this time period, the survival of small groups in the fewer sustainable areas in Upper Egypt and Lower Nubia is supported by the unusual phenotypic diversity, probably related to population fragmentation and isolation, found in the Late Pleistocene fossils of this region19,21,73–75. With variation of lithic industries indicating different cultural traditions and the co-occurrence of large cemetery spaces suggesting some level of sedentism15, severe territorial competition between the region’s hunter-fisher-gatherer groups is likely to have occurred when forced to adapt to the drastic environmental changes recorded at the end of the Last Glacial Maximum and the beginning of the African Humid Period (cf. Supplementary Text S1). Conclusionsi For the first time since Wendorf’s original publication11, a complete reassessment of the Jebel Sahaba cemetery was used to clarify the nature, extent and dating of the violence experience by the individuals buried at the site. First, direct radiocarbon dates, between 13.4 and 18.2 ka, confirm the antiquity of the site, making Jebel Sahaba the oldest cemetery in the Nile valley. Second, using modern approaches and methods, our reappraisal undeni- ably supports the interpersonal nature of the lesions and confirms the projectile origin of most of the trauma. Our analyses also show that out of sixty-one individual, 26.2% of had signs of perimortem traumas and 62.3% displayed healed and/or unhealed traumas (excluding undiagnosed lesions) regardless of the age-at-death or sex, including children as young as 4 years old. Third, the reassessment of the lithic artefacts associated to each burial reveals that most were elements of composite projectile weapons. Fourth, although double and multiple burials are present, most probably indicating simultaneous deaths, demographic data and burial disturbance caused by subsequent interments does not support a single catastrophic event. While acknowledging the possibility that the Jebel Sahaba cemetery may have been a specific place of burial for victims of violence, the presence of numerous healed traumas and the reuse of the funerary space both support the occurrence of recurrent episodes of small scale sporadic interpersonal violence at the end of the Pleistocene. Most are likely to have been the result of skirmishes, raids or ambushes. Territorial and environmental pressures triggered by climate changes are most probably responsible for these frequent conflicts between what appears to be culturally distinct Nile Valley semi- sedentary hunter-fisher-gatherers groups. Discussion Climate change is most likely to have been a driver towards a violent competition for resources over time as documented in the ethno-archaeological record e.g.25,65,68. www.nature.com/scientificreports/ of join remodeling or entheseal changes where observable. In all the other cases, the individual was assigned to the age group [> 30]. We used the J:A ratio between immature individuals aged [5–14] and adults individual over 20 years [> 20] and the mean childhood mortality value (MCM) to address potential bias in the Jebel Sahaba cemetery population40,43. In order to discuss potential demographic anomalies in the Jebel Sahaba cemetery, we grouped the individuals in six conventional age cohorts ([0– < 1], [1–4], [5–9], [10–14], [15–19] and [20– 29 years]) that allow for comparisons with theoretical mortality values of a population with a life expectancy at birth of between 25 and 35 years38. Immature individuals falling into two cohorts based on age-at-death estimate standard deviations were assigned to the most probable one according to Sellier35. Lesions and Projectile Impact Marks (PIMs) characterizations. Extensive and detailed microscopic analyses of the all areas exhibiting taphonomic and/or anthropogenic traces were conducted using a digital microscope (Dino-Lite Premier) with a 5 Megapixels resolution, a polarizer and a 30×–250× magnification range. Following the recommendations of Smith et al.34, each potential lesion was checked for embedded lithic fragments and characterized. Non-anthropogenic traces, mainly due to gnawing and termite activity, were dif- ferentiated using macroscopic and microscopic criteria48,49,52. The Jebel Sahaba individuals were buried in pits, filled by sediment and covered by sandstone slabs (see Supplementary Text S2), and although trampling marks were unlikely, the diagnostic criteria from Domínguez-Rodrigo et al.50 were used to exclude such taphonomic changes.i g Projectile Impacts Marks (PIMs) were characterized using projectile bone damage identification criteria derived from experimental archaeological research34,51,53–55,90. Although based on the hunting of small and large ungulates, these experimental studies provide a clear system of projectile trauma classification that is often lacking in analyses of interpersonal violence34. The terminology and classification used in this study are characterized by the level of hard tissue projectile penetration defined by O’Driscoll & Thompson (51, see Supplementary Text S4 and Fig. S6). In a number of cases, the projectile origin of a lesion could not be identified, sometimes due to poor preservation and uncharacteristic changes, and the term trauma is used. It also covers all the healed or unhealed bone fractures, blunt force trauma and perforations with no PIM signs. The term fracture is defined as a partial or complete break in the continuity of a bone33. www.nature.com/scientificreports/ Finally, the term lesion refers to an injury whose nature or anthropogenic origin could not be determined. The presences of bone callus or abscesses were also recorded. 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Implications for the study of population dynamics. Int. J. Osteoarchaeol. 25, 625–636 (2015). https://doi.org/10.1038/s41598-021-89386-y Scientific Reports \| (2021) 11:9991 \| Author contributions Conceived and designed the experiments: I.C., M.D.M., F.B. Performed the experiments: I.C., M.D.M., F.B. Analyzed the data: I.C., M.D.M., F.B. Radiocarbon dating conceived, designed, performed and analyzed: D.A., A.Z. Contributed reagents/materials/analysis tools: I.C., M.D.M., F.B., A.Z., D.A. Wrote the paper: I.C., D.A., F.B., M.D.M., A.Z. Conceived and designed the experiments: I.C., M.D.M., F.B. Performed the experiments: I.C., M.D.M., F.B. Analyzed the data: I.C., M.D.M., F.B. Radiocarbon dating conceived, designed, performed and analyzed: D.A., A.Z. Contributed reagents/materials/analysis tools: I.C., M.D.M., F.B., A.Z., D.A. Wrote the paper: I.C., D.A., F.B., M.D.M., A.Z. Competing interests The authors declare no competing interests. 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https://openalex.org/W2329012680	https://europepmc.org/articles/pmc4814577?pdf=render	English	null	The cAMP Pathway as Therapeutic Target in Autoimmune and Inflammatory Diseases	Frontiers in immunology	2,016	cc-by	11,266	Review published: 31 March 2016 doi: 10.3389/fimmu.2016.00123 Review published: 31 March 2016 doi: 10.3389/fimmu.2016.00123 Edited by: Edited by: Josef Bodor, Institute of Experimental Medicine, Czech Republic INTRODUCTION Cells must be able to sense and integrate countless extracellular and intracellular signals and adapt their cellular functions. Second messengers serve as initiating components of intracellular signal transduction cascades that transmit signals by cellular messengers that depend on extracellular sign- aling molecules (1). Thereby second messengers serve to greatly amplify the strength of the original first signal. Cyclic adenosine monophosphate (cAMP) was the first discovered intracellular second messenger of extracellular ligand action (2). Within the immune system, cAMP regulates pro- and anti-inflammatory activities: drugs that elevate intracellular cAMP levels reduce the production of pro-inflammatory mediators and increase the production of anti-inflammatory factors in numerous immune cells. This review aims to shed light on the variety of processes influenced by cAMP in the immune system with regard to treatment options in diseases. Reviewed by: Gottfried Baier, Medical University of Innsbruck, Austria Jacques A. Nunes, Centre de Recherche en Cancerologie de Marseille, France Correspondence: Verena Katharina Raker rakerv@uni-mainz.de The cAMP Pathway as Therapeutic Target in Autoimmune and inflammatory Diseases Verena Katharina Raker , Christian Becker and Kerstin Steinbrink Department of Dermatology, University Medical Center Mainz, Johannes Gutenberg-University Mainz, Mainz, Germany Nucleotide signaling molecules contribute to the regulation of cellular pathways. In the immune system, cyclic adenosine monophosphate (cAMP) is well established as a potent regulator of innate and adaptive immune cell functions. Therapeutic strategies to interrupt or enhance cAMP generation or effects have immunoregulatory potential in autoimmune and inflammatory disorders. Here, we provide an overview of the cyclic AMP axis and its role as a regulator of immune functions and discuss the clinical and translational relevance of interventions with these processes. Keywords: cyclic AMP, autoimmunity, targeted therapies, inflammation, T cells, Tregs, T regulatory cells Specialty section: Specialty section: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology Received: 27 January 2016 Accepted: 18 March 2016 Published: 31 March 2016 Specialty section: This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology Cyclic adenosine monophosphate, identified in 1957 (2) as the first intracellular second messenger of extracellular ligand action, is now established as a universal regulator of metabolism and gene expression in all life forms (3). A family of enzymes called adenylate cyclases (AC) catalyzes cAMP formation from ATP. In vertebrates, AC comprise nine membrane-bound isoforms and one soluble isoform (4). AC vary in distribution and developmental expression and their regulation is complex and isozyme specific. In addition to AC expression and activity cAMP homeostasis is regulated by a superfamily of phosphodiesterases (PDE) that degrade intracellular cyclic nucleotides. PDE com- prise more than 100 enzyme variants divided into 11 families (5) based on their structure, specificity for, and modulation by, cyclic nucleotides. Certain PDE increase their activities in response to cAMP and cAMP stimulates the synthesis of new PDE mRNA (6, 7), resulting in a feedback loop between cAMP levels and PDE activity. Contributing to the complexity of the pathway, some PDE families are strictly cAMP-specific (PDE 4, 7, and 8), whereas others are cyclic guanosine monophosphate Received: 27 January 2016 Accepted: 18 March 2016 Published: 31 March 2016 Edited by: Josef Bodor, Institute of Experimental Medicine, Czech Republic Reviewed by: Gottfried Baier, Medical University of Innsbruck, Austria Jacques A. Nunes, Centre de Recherche en Cancerologie de Marseille, France *Correspondence: Verena Katharina Raker rakerv@uni-mainz.de Citation: Raker VK, Becker C and Steinbrink K (2016) The cAMP Pathway as Therapeutic Target in Autoimmune and Inflammatory Diseases. Front. Immunol. 7:123. doi: 10.3389/fimmu.2016.00123 March 2016 \| Volume 7 \| Article 123 1 Frontiers in Immunology \| www.frontiersin.org cAMP as Therapeutic Target in Autoimmunity and Inflammation Raker et al. (cGMP)-specific (PDE 5, 6, and 9) (8, 9). Additional families hydrolyze both cAMP and cGMP (PDE 1, 2, 3, 10, and 11), establishing cross-regulation of both pathways with important implications in the utility of pharmacotherapeutic agents target- ing cyclic nucleotide metabolism (10, 11). can all be phosphorylated by many other kinases, and the action of PKA is counterbalanced by specific protein phosphatases. i Basal cytosolic cAMP levels are in the low micrometer range (19). In the cytosol, cAMP is not evenly distributed but rather forms submembranous spatially discrete pools generated in microdomains containing AC, PDE next to PKA localized by A-kinase-anchoring proteins (AKAPs) (20). Specificity in cAMP signaling and fine and selective tuning of its different tasks is ensured by the differential expression of distinct isoforms and splice variants of anabolic, katabolic, and signaling molecules in various tissues and cell types and by differential composition of cAMP microdomains (21). Although various cAMP activities can have redundant, independent, or opposing effects within the same cell (22), some individual AC and PDE knockout and transgenic mice (23, 24) show specific phenotypes. In particular, individual PDE control select cyclic nucleotide-regulated events by being integrated into non-overlapping multi-molecular regulatory signaling complexes, suggesting cell or tissue-specific interference points (25, 26).t g y As a second messenger, cyclic AMP serves in multiple downstream pathways. Most prominent, it activates the cAMP- dependent protein kinase A I (PKA) (12) (see Figure 1). Upon binding of cAMP to the regulatory subunits, PKA dissociates into its regulatory and catalytic subunits and the catalytic subunits phosphorylate specific Ser and Thr residues on numerous target proteins initiating successive signaling cascades, particularly in nutrient metabolism (13). In addition, cAMP-activated PKA binds and phosphorylates cAMP-responsive transcription fac- tors, including cAMP-response element binding protein (CREB), members of the cAMP-responsive element modulator/inducible cAMP early repressor (CREM/ICER) protein family (14), activat- ing transcription factor-1 (ATF-1), NFκB, and nuclear receptors (see Figure 1). Phosphorylated CREB, CREM, and ATF-1 inter- act with the transcriptional coactivators CREB-binding protein (CBP) and p300 when bound to cAMP-response elements (CREs) in target genes (15). Citation: In addition to PKA activation, cAMP also directly modulates the activity of guanine-nucleotide-exchange factor (GEF) exchange proteins (Epacs) and cyclic nucleotide- gated channels (CNGs) (16) all with important roles in cellular functions (17, 18). In addition to PKA, CREB, CREM, and ATF-1 Eventually, an important, often overlooked aspect of the path- way consists in the secretion of cAMP into extracellular space and its transmission via gap junctions between cells (27). Whereas transmitted cAMP directly contributes to intracellular cAMP levels, excreted cAMP is converted into AMP and adenosine by cell surface bound PDE and ecto-5′-nucleotidases CD39 and CD73. By signaling through A2A and A2B adenosine receptors, extracellular adenosine stimulates AC and increases intracellular cAMP generation (28). Knockout mice with disrupted CD39 and CD73 have underscored the importance of the extracellular cAMP–adenosine feedback mechanism in physiological pro- cesses (29, 30). In the immune system extracellular cAMP may contribute to regulatory T cells (Treg) function (31, 32) and has been shown to promote monocyte differentiation into dendritic cells (DCs) (33). FIGURE 1 \| cAMP as a regulator of immunity. Adenylate cyclases (AC) produce cAMP from adenosin-tri-phosphate (ATP). High levels of cytosolic cAMP lead to activation of protein kinase A (PKA). PKA stimulation induces the phosphorylation of transcription factors, such as CREB, ICER/CREM, ATF-1, and CBP to drive camp-driven genes. Phosphodiesterase 4 (PDE4) decreases intracellular cAMP levels and counterbalances the intracellular cAMP effect. ATF, cAMP-dependent transcription factor; CBP, cAMP-binding protein; CNG, cyclic nucleotide-gated ion channel; CREB, cAMP response element-binding protein; ICER, inducible cAMP early repressor; P, phosphorylation. CYCLIC AMP IN IMMUNE HOMEOSTASIS AND PATHOPHYSIOLOGY Due to its multiple roles in cell physiology cAMP exerts broad modulatory effects on a variety of cells (see Figure 2). In the immune system, cyclic AMP regulates both innate and adaptive immune cell activities (34). Monocytes and Granulocytesh The functional state of monocytes orchestrates inflammatory and reparative phases in inflammatory responses and appears to be accompanied by changes in their intracellular cAMP levels. In the mouse, two major types of monocytes, Ly6Chigh and Ly6Clow, circulate in blood. Ly6Chigh monocytes display pro-inflammatory activity, whereas Ly6Clow monocytes are patrolling cells, monitor tissue integrity, and exert anti-inflammatory and tissue repair activities (35). The orphan nuclear receptor Nr4a1 (Nur77) regu- lates the expression of genes linked to inflammation. Inflammatory stimuli inhibit its expression and induce an inflammatory Ly6Chigh phenotype (36, 37). In turn, Nur77 is upregulated and represses numerous inflammatory genes in the transition from FIGURE 1 \| cAMP as a regulator of immunity. Adenylate cyclases (AC) produce cAMP from adenosin-tri-phosphate (ATP). High levels of cytosolic cAMP lead to activation of protein kinase A (PKA). PKA stimulation induces the phosphorylation of transcription factors, such as CREB, ICER/CREM, ATF-1, and CBP to drive camp-driven genes. Phosphodiesterase 4 (PDE4) decreases intracellular cAMP levels and counterbalances the intracellular cAMP effect. ATF, cAMP-dependent transcription factor; CBP, cAMP-binding protein; CNG, cyclic nucleotide-gated ion channel; CREB, cAMP response element-binding protein; ICER, inducible cAMP early repressor; P, phosphorylation. March 2016 \| Volume 7 \| Article 123 Frontiers in Immunology \| www.frontiersin.org 2 cAMP as Therapeutic Target in Autoimmunity and Inflammation Raker et al. FIGURE 2 \| Effect of cAMP on immune, tumor, and epithelial cells. Impact and function of cyclic adenosin monophopshate (cAMP) on T and B lymphocytes, granulocytes, monocytes, macrophages, dendritic cells, epithelial cells, and melanoma cells. LTB4, leukotriene B4; LTC4, leukotriene C4. FIGURE 2 \| Effect of cAMP on immune, tumor, and epithelial cells. Impact and function of cyclic adenosin monophopshate (cAMP) on T and B lymphocytes, granulocytes, monocytes, macrophages, dendritic cells, epithelial cells, and melanoma cells. LTB4, leukotriene B4; LTC4, leukotriene C4. immune, tumor, and epithelial cells. Impact and function of cyclic adenosin monophopshate (cAMP) on T and B lymphocytes, ophages, dendritic cells, epithelial cells, and melanoma cells. LTB4, leukotriene B4; LTC4, leukotriene C4. GURE 2 \| Effect of cAMP on immune, tumor, and epithelial cells. Impact and function of cyclic adenosin monophopshate (cA anulocytes, monocytes, macrophages, dendritic cells, epithelial cells, and melanoma cells. LTB4, leukotriene B4; LTC4, leukotriene cells, cAMP levels regulate target cell adherence and cytotoxic function. Both pharmacological repression and induction of cAMP inhibit perforin-mediated and CD95 ligand-mediated target cell lysis (56–60). an inflammatory Ly6Chigh to anti-inflammatory Ly6Clow/neg state (38–40). Monocytes and Granulocytesh Elevated cAMP levels induce Nur77 expression (41) and, thus, favor a reparatory monocyte phenotype (42). Through these effects on phagocytes increased cAMP levels affect myeloid cell immunity against pathogen and parasites (43–45) and may also affect the differentiation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs) by repression of TNF-α production. In regard of the latter CREB activation has been shown to upregulate miR-9 expression that promotes the differentiation of the so- called MDSCs with significantly increased immunosuppressive function (46). B and T Cells In addition to innate cell function, cAMP also controls numerous adaptive immune cell activities. In adaptive immune cells, cAMP is essentially required in the induction of antigen-stimulated acti- vation (69–72) but subsequently limits activation by negatively Dendritic Cells As professional antigen-presenting cells of the immune system, DCs are equipped with a unique capability to induce and regulate adaptive immune responses. In DC, cyclic AMP suppresses the release of pro-inflammatory mediators (TNF-α, IL-17, IFN-γ) (61) and promotes the release of anti-inflammatory mediators, such as IL-10 (62). As a functional consequence, cAMP concen- trations in DC regulate T cell immunity (63). Pharmacological inhibition of cyclic nucleotide PDE4, which is highly expressed in DC, for example, suppresses the DC Th1-polarizing capacity (64, 65) and commands secretion of IL-6 and TGF-beta and sub- sequent induction of Th17 differentiation (66). It, thus, appears that cAMP levels differentially regulate cytokine production by DC as a response to changes in the microenvironment. Apart from spatio-temporal fine-tuning of DC activities, cAMP activi- ties in DC depend on the stage of DC maturation: prostaglandin E2 (PGE2), a key inducer of cAMP, exerts a stimulatory function for immature DCs in peripheral tissues (67) but inhibitory func- tion for mature DCs in lymph nodes (68). In sum, increased cAMP levels appear to generally weaken monocyte inflammatory functions (47–50). Interestingly, bacteria and fungi have taken advantage of this effect in the course of evolu- tion. Pathogen capture and programed destruction are among the most important activities of innate immune cells to prevent tissue invasion and pathogen dissemination. Certain microbacteria and fungi have evolved to hijack the host cAMP axis by introducing microbial adenylyl and guanylyl cyclases (51) and by intoxicating the host cell with preformed cAMP or adenylate cyclase toxins (52–54). Bordetella pertussis, for example, suppresses neutrophil extracellular trap (NET) formation by overwhelming leukocytes with supraphysiologic intracellular cAMP levels (55). Likewise, bacterial-derived or -induced cAMP facilitates intracellular bac- terial survival by multiple actions, including CREB-dependent anti-apoptotic signaling and repression of intracellular bacterial killing in invaded monocytes and macrophages. NK Cells Natural killer (NK) cells are capable of destroying tumor cells and virally infected cells (cytolysis) without prior sensitization. In NK March 2016 \| Volume 7 \| Article 123 Frontiers in Immunology \| www.frontiersin.org 3 cAMP as Therapeutic Target in Autoimmunity and Inflammation Raker et al. FIGURE 3 \| The cAMP pathway in Treg and its regulation by IFN-α. Signaling via the T cell receptor (TCR) leads to an activation of adenylate cyclases, resulting in high cAMP levels in regulatory T cells (Treg). cAMP can be transferred via gap junctions into conventional T cells (Tcon), thereby mediating the suppressive activity of Treg (A). Phosphodiesterase 4 (PDE4), which can be activated by MAP kinase ERK-related pathways, reduces cAMP amounts in Treg by enzymatic cleavage, impairing the regulatory activity of Treg (B). IFN-α abolishes the suppressive function of Treg by cAMP reduction, restoring the Tcon activation. Inhibition of the ERK or PDE4 pathway, respectively, results in a renewed suppressive capacity of IFN-α treated Treg (C). regulating signaling through B cell and T cell receptors (TCR). In B cells, it provides an essential signal in the induction of antigen-stimulated proliferation and antibody production (69, 70, 72). Elevation of intracellular cAMP enhances IgE produc- tion by promoting recombination of the Ig heavy chain loci and by favoring Th2 differentiation. In T cells, cAMP participates in the regulation of nearly all functional activities ranging from peripheral maintenance of naïve T cells (73) to their activation via the TCR (74), acquisition of effector function (75, 76), and memory (77). In cognate activation, cAMP acts as a temporary inhibitory feedback signal that limits T cell activation through the cAMP–PKA–Csk signaling pathway (74). Unlike temporary increases, continuously elevated cAMP levels induce an anergy- like state (78, 79). Likewise, anergizing TCR signals result in increased intracellular cAMP concentrations that upregulate the cyclin-dependent kinase (CDK) inhibitor p27kip1, sequester cyclin D2–cdk4, and cyclin E/cdk2 complexes and prevent pro- gression through the G1 restriction point of the cell cycle (80). Furthermore, cAMP levels regulate the acquisition of effector function. Pharmacological upregulation of cAMP by inhibition of PDE activity, for example, prevents the development and function of cytotoxic T lymphocyte (CTL) (81). NK Cells The significance of cAMP in acquisition of effector functions in T cells is also reflected by the observation that CREB mutant mice have normal T cell numbers in the thymus but exhibit a marked defect in peripheral T cell proliferation and IL-2 production, resulting from G1 cell-cycle arrest and apoptotic cell death (82). Most prominent, cAMP forms an essential component of the suppressive mecha- nism in Treg (83–92). Treg contain increased levels of cytosolic cAMP, further upregulate their cAMP level upon activation and consign cAMP to target cells via gap junctions (83, 85). In the target cell, cAMP inhibits the proliferation and differentiation of effector functions, in part by interfering with gene expression via ICER (90). Repression of cAMP accumulation in Treg by either adenylyl cyclase inhibition, application of a cAMP-specific antagonist, or PDE overexpression abrogates murine and human Treg suppression (83, 84, 86, 91, 93). Inversely, blockade of cAMP degradation by PDE inhibition improves Treg-mediated suppres- sion in a murine asthma model (85). In line, non-functional Treg in Foxp3-mutant scurfy mice harbor significantly reduced levels of cytosolic cAMP (94). FIGURE 3 \| The cAMP pathway in Treg and its regulation by IFN-α. Signaling via the T cell receptor (TCR) leads to an activation of adenylate cyclases, resulting in high cAMP levels in regulatory T cells (Treg). cAMP can be transferred via gap junctions into conventional T cells (Tcon), thereby mediating the suppressive activity of Treg (A). Phosphodiesterase 4 (PDE4), which can be activated by MAP kinase ERK-related pathways, reduces cAMP amounts in Treg by enzymatic cleavage, impairing the regulatory activity of Treg (B). IFN-α abolishes the suppressive function of Treg by cAMP reduction, restoring the Tcon activation. Inhibition of the ERK or PDE4 pathway, respectively, results in a renewed suppressive capacity of IFN-α treated Treg (C). FIGURE 3 \| The cAMP pathway in Treg and its regulation by IFN-α. Si li i th T ll t (TCR) l d t ti ti f d l t FIGURE 3 \| The cAMP pathway in Treg and its regulation by IFN-α. Signaling via the T cell receptor (TCR) leads to an activation of adenylate cyclases, resulting in high cAMP levels in regulatory T cells (Treg). cAMP can be transferred via gap junctions into conventional T cells (Tcon), thereby mediating the suppressive activity of Treg (A). Frontiers in Immunology \| www.frontiersin.org NK Cells The significance of cAMP in acquisition of effector functions in T cells is also reflected by the observation that CREB mutant mice have normal T cell numbers in the thymus but exhibit a marked defect in peripheral T cell proliferation and IL-2 production, resulting from G1 cell-cycle arrest and apoptotic cell death (82). Most prominent, cAMP forms an essential component of the suppressive mecha- nism in Treg (83–92). Treg contain increased levels of cytosolic cAMP, further upregulate their cAMP level upon activation and consign cAMP to target cells via gap junctions (83, 85). In the target cell, cAMP inhibits the proliferation and differentiation of effector functions, in part by interfering with gene expression via ICER (90). Repression of cAMP accumulation in Treg by either adenylyl cyclase inhibition, application of a cAMP-specific antagonist, or PDE overexpression abrogates murine and human Treg suppression (83, 84, 86, 91, 93). Inversely, blockade of cAMP degradation by PDE inhibition improves Treg-mediated suppres- sion in a murine asthma model (85). In line, non-functional Treg in Foxp3-mutant scurfy mice harbor significantly reduced levels of cytosolic cAMP (94) regulating signaling through B cell and T cell receptors (TCR). In B cells, it provides an essential signal in the induction of antigen-stimulated proliferation and antibody production (69, 70, 72). Elevation of intracellular cAMP enhances IgE produc- tion by promoting recombination of the Ig heavy chain loci and by favoring Th2 differentiation. In T cells, cAMP participates in the regulation of nearly all functional activities ranging from peripheral maintenance of naïve T cells (73) to their activation via the TCR (74), acquisition of effector function (75, 76), and memory (77). In cognate activation, cAMP acts as a temporary inhibitory feedback signal that limits T cell activation through the cAMP–PKA–Csk signaling pathway (74). Unlike temporary increases, continuously elevated cAMP levels induce an anergy- like state (78, 79). Likewise, anergizing TCR signals result in increased intracellular cAMP concentrations that upregulate the cyclin-dependent kinase (CDK) inhibitor p27kip1, sequester cyclin D2–cdk4, and cyclin E/cdk2 complexes and prevent pro- gression through the G1 restriction point of the cell cycle (80). Furthermore, cAMP levels regulate the acquisition of effector function. Pharmacological upregulation of cAMP by inhibition of PDE activity, for example, prevents the development and function of cytotoxic T lymphocyte (CTL) (81). NK Cells In contrast to Tcon (blue line), Treg (green line) exhibit high levels of cAMP (A). Stimulated Tcon display a high proliferation whereas Treg are characterized by a low proliferative capacity [(B), left panels, single culture]. Treg efficiently inhibit Tcon proliferation in co-culture experiments by cAMP transfer via gap junctions to Tcon [(B), co-culture]. By contrast, IFN-α abrogates the suppressive function of Treg through reduction of cAMP levels [(A), centered panel], resulting in a restored Tcon activation [(B), centered panel]. Blockade of the ERK or PDE4 pathway, respectively, increases intracellular cAMP amounts [(A), right panel], renews the suppressive activity of Treg [(B), right panel]. Tcon, conventional T cells; Treg, regulatory T cells. FIGURE 4 \| Function of cAMP in the interaction of conventional and regulatory T cells. In contrast to Tcon (blue line), Treg (green line) exhibit high levels of cAMP (A). Stimulated Tcon display a high proliferation whereas Treg are characterized by a low proliferative capacity [(B), left panels, single culture]. Treg efficiently inhibit Tcon proliferation in co-culture experiments by cAMP transfer via gap junctions to Tcon [(B), co-culture]. By contrast, IFN-α abrogates the suppressive function of Treg through reduction of cAMP levels [(A), centered panel], resulting in a restored Tcon activation [(B), centered panel]. Blockade of the ERK or PDE4 pathway, respectively, increases intracellular cAMP amounts [(A), right panel], renews the suppressive activity of Treg [(B), right panel]. Tcon, conventional T cells; Treg, regulatory T cells. and enhanced by ligands that increased cAMP or directly activate the PKA (115).i to control autoimmune diseases and inflammation (107). Interestingly, IL-2 may contribute to increased cAMP produc- tion in Treg by increasing adenylate cyclase AC7 activity (88). In conjunction with its role in control of the Treg phenotype, its transmission via gap junctions to and from Treg also appears to play a role in the Treg lifecycle as evidence by the observation that Treg numbers are significantly reduced in connexin 43 knockout mice (108). Together these findings classify cAMP as a key component of immune cell function and disclose cAMP-regulating enzymes as molecular targets for therapeutic intervention with immune activities in pathological processes like allergy and autoimmunity. NK Cells Phosphodiesterase 4 (PDE4), which can be activated by MAP kinase ERK-related pathways, reduces cAMP amounts in Treg by enzymatic cleavage, impairing the regulatory activity of Treg (B). IFN-α abolishes the suppressive function of Treg by cAMP reduction, restoring the Tcon activation. Inhibition of the ERK or PDE4 pathway, respectively, results in a renewed suppressive capacity of IFN-α treated Treg (C). blocking ERK and PDE/PDE4 activity through specific inhibitors (92, 97) (see Figures 3 and 4). These results are in line with the observation that human T cells predominantly express the short PDE4B and PDE4D isoforms, functionally regulated by the ERK2 MAP kinase (98, 99). As PDE have an essential role in the IFN-α-mediated inhibition of Treg, PDE4 interference by specific inhibitors may represent a therapeutic option to restore immune regulation in autoimmune diseases, such as psoriasis or lupus erythematosus, accompanied by reduced Treg function (64, 100). Increased cAMP formation in Treg is a prerequisite for their suppressive activity (95) (see Figures 3 and 4). Constitutively high cAMP levels in Treg appear to be caused by Foxp3-induced decreased PDE3B expression (96) and increased AC9 activity (87) driven by their constitutive active state (95). During Treg- mediated suppression, cAMP is transferred via gap junctions to conventional T cells (Tcon), where it represses IL-2 production and inhibits the proliferative response (83). Pharmacological inhibition of cAMP formation abrogates the suppressive function of Treg (see Figure 3) (91). Next to its role in the Treg-suppressive mechanism cAMP is required for the generation and maintenance of Treg: the cAMP-responsive transcription factor CREB stabilizes FoxP3 expression and promotes and maintains the Treg phenotype (101, 102). Treg essentially depend on IL-2 for their peripheral maintenance and suppressive activity (103, 104) and their number and activity can be therapeutically manipulated by low- dose IL-2 and particular IL-2/anti-IL-2 complexes (105, 106) In this context, Bacher et al. showed that IFN-α, an antineo- plastic agent with well-known autoimmune side effects, disturbs the immunosuppressive activity of human CD4+CD25+Foxp3+ Treg by disabling cAMP upregulation upon activation (92, 97) (see Figure 3 and 4). IFN-α-mediated inhibition of Treg sup- pression can be partially restored by pharmacological inhibitors March 2016 \| Volume 7 \| Article 123 Frontiers in Immunology \| www.frontiersin.org 4 cAMP as Therapeutic Target in Autoimmunity and Inflammation Raker et al. FIGURE 4 \| Function of cAMP in the interaction of conventional and regulatory T cells. Frontiers in Immunology \| www.frontiersin.org MODULATION OF cAMP IN AUTOIMMUNE AND INFLAMMATORY DISEASES Here, the idea is pursued, that selective inhibitors intervene in a tissue-specific manner, but remain ineffective in tissues that express various AC isoforms (118).i AC-specific compounds already reached preclinical stages and others have been approved for particular diseases, such as colforsin daropate hydrochloride (NKH447), a AC5 selective for- skolin (FSK) derivate, for the treatment of advanced congestive heart failure (119, 120). Thus, even though AC isoform-targeted drugs are still in early stages of the development, the finding that AC have clearly separated physiological functions at least suggests AC as pharmacologic targets in a broad spectrum of diseases ranging from neurodegenerative disorders to congestive heart failure and lung diseases as asthma and chronic obstructive pulmonary disease (COPD).if Since their identification in 1958 (2), continuing efforts have been undertaken to advance the understanding of PDE biology and function, and PDE have been considered pharmacological targets in various diseases, such as pulmonary diseases like COPD and asthma, depression, schizophrenia, erectile dysfunc- tion, and autoimmune disease like psoriasis/psoriasis arthritis and rheumatoid arthritis (8, 100, 121–125). Although numerous PDE inhibitors have been developed, their introduction into the clinic has been hampered by their narrow therapeutic window and side effects, such as nausea and emesis, occurring even at sub-therapeutic levels. In the immune system, PDE family 3, 4, and 7 members rep- resent the predominant cAMP-degrading enzymes (126). PDE4 are encoded by four separate genes (PDE4A–D) and each PDE4 controls non-redundant cellular function (127). In addition, more than 20 PDE4 variants arise from alternative mRNA splic- ing or the use of different transcriptional units (5). While PDE4A, PDE4B, and PDE4D are expressed in immune cells (T and B cells, neutrophils, eosinophils, DCs, monocytes, macrophages), PD4C is minimally active or absent (128, 129). PDE3 and PDE7 are detected in most inflammatory cells, including T and B cells, NK, and myeloid cells (6, 59, 127, 130–132). However, PDE4s are the predominant cAMP-degrading isoenzymes (126, 127). In addition, the expression levels of the PDE isoenzymes are differ- entially regulated by a variety of inflammatory stimuli (126, 127). Apart from immune cells, PDE4 members are also expressed in chondrocytes, smooth muscle cells, epithelial cells, and vascular endothelium (127). MODULATION OF cAMP IN AUTOIMMUNE AND INFLAMMATORY DISEASES In view of side effect profile of second-generation PDE4 inhibitors, new strategies for the design of active and non-emetic compounds have been employed to overcome the adverse effects and to improve thera- peutic effects. In this context, despite highly conserved catalytic domains of PDE4 isoenzymes, PDE4 subtype-specific inhibitors have been generated. For example, potent PDE4B inhibitors with more than 100-fold selectivity over PDE4D have been synthesized (134, 135). Compared with the non-selective PDE4 inhibitor cilo- milast (134), selective PDE4B inhibitors demonstrated a potent anti-inflammatory activity and significantly less gastrointestinal side effects. In order to circumvent side effects observed upon oral administration, inhalation (136) and topical application (137) of PDE4 inhibitors have been explored in the treatment of airway inflammation and inflammatory cutaneous diseases. Two phase studies conducted with a PDE4 inhibitor (AN2728) in psoriasis and atopic dermatitis patients showed promising results (138, 139). The interest for PDE4 anti-inflammatory activity arose from early studies with the prototypic PDE4 inhibitor, rolipram (140). However, although PDE4 inhibitors have been mostly developed to treat lung diseases, such as asthma or COPD, no compound has yet reached the market for asthma treatment. By contrast, the orally active PDE4 inhibitor roflumilast (Daliresp®, Forest Pharmaceuticals) has been approved for COPD by the European Medicines Agency in 2010 and the U.S. Food and Drug Administration in 2011 based on four clinical trials. These studies have shown that roflumilast improves lung function and reduces the frequency of COPD exacerbations in patients with chronic bronchitis symptoms (141–144). Although side effects were generally mild to moderate, nausea, diarrhea, weight loss, and headache were still reported (145). Despite these side effects, roflumilast received approval for COPD with severe air flow limitations, symptoms of chronic bronchitis, and a history of exacerbations in several countries (146, 147). Another currently marketed oral PDE4 inhibitor is apre- pharmacologically targeted through agonists or antagonists affecting upstream G-protein-coupled receptors (GPCR) (23, 116). However, AC knockout and transgenic mice revealed individual and clearly distinct physiological functions for AC isoforms (23). The observation that individual isoforms play a dominant role in specific tissues has led to AC being consid- ered as main drug targets (117). In order to achieve selective interference, isoform-selective compounds are required. Such compounds are currently being sought and tested. MODULATION OF cAMP IN AUTOIMMUNE AND INFLAMMATORY DISEASES Some viruses prevent their rejection by the immune system by interfering with the cAMP pathway in T cells. HIV-1 surface glycoprotein gp120 induces anergy in naive T lymphocytes (109, 110) and increases cAMP levels and suppressive activity in Treg (86, 111, 112). In turn, cAMP repression restores antiviral T cell function in HIV patients (113). Cyclic AMP is a central player in the network of signaling pathways underlying pathogenesis of several diseases and several interference points are used therapeutically in a variety of condi- tions. Although the clinical impact of changes in cAMP remains incompletely defined, one fundamental conclusion can neverthe- less be drawn: interventions that enhance cAMP generation or actions have immune dampening potential; conversely, repression of cAMP or cAMP signaling has immunostimulatory capability. Formation of cAMP by AC and degradation by PDE identi- Beyond their role in immune regulation, Treg take on homeo- static functions by regulating metabolic activity in visceral fat and participating in tissue repair. Functionally distinct Treg accumu- late in injured skeletal muscle and contribute to repair processes. Muscle Treg distinctly express the growth factor amphiregulin, which improves muscle repair by directly acting on muscle satellite cells (114). In line with outlined role of cAMP in Treg function, amphiregulin synthesis is inhibited by PKA inhibitors Formation of cAMP by AC and degradation by PDE identi fies AC and PDE as major targets for therapeutic intervention with cAMP levels. To date, the AC activity has been mostly March 2016 \| Volume 7 \| Article 123 5 cAMP as Therapeutic Target in Autoimmunity and Inflammation Raker et al. Numerous selective PDE4 inhibitors have been patented and some of them have been evaluated in clinical trials, including diseases, such as asthma, COPD, atopic dermatitis, rheumatoid arthritis, and psoriasis/psoriasis arthritis. However, most of these compounds had to be discontinued because of narrow therapeu- tic windows. Doses needed for an efficient treatment could not be reached due to side effects, such as nausea, emesis, diarrhea, and abdominal pain being the most common. It has been hypothesized that adverse side effects of the PDE4 inhibitors are a result of their non-selectivity to all four PDE4 subtypes and PDE4 inhibition in non-target tissues at doses similar (or lower) than needed for therapeutic efficacy. It is postulated that blocking of PDE4D in non-target organs promotes emesis (133). Frontiers in Immunology \| www.frontiersin.org MODULATION OF cAMP IN AUTOIMMUNE AND INFLAMMATORY DISEASES By increasing levels of intracellular cAMP, PDE4 inhibitors show anti-inflammatory effects in almost all inflammatory and immune cells and are known to suppress a multitude of inflammatory responses, including proliferation, chemotaxis, phagocytosis, and release of pro-inflammatory mediators, such as cytokine and chemokines, reactive oxygen species, lipid mediators, and hydrolytic enzymes (34, 126, 129). Another currently marketed oral PDE4 inhibitor is apre- milast (Otezla®, Celgene Corporation) that has been approved by the EMA and FDA for psoriasis and psoriasis arthritis, two autoimmune diseases, characterized by chronic inflammation, tissue and organ involvement, and accelerated growth cycle of skin cells. Apremilast was developed based on the rolipram and roflumilast pharmacophore by coupling a series of phthalimide analogs in order to optimize its activity and to decrease side effects (148). The safety and efficacy of apremilast for the treat- ment of patients with plaque psoriasis and psoriasis arthritis were evaluated in numerous multicenter, randomized, double-blind, March 2016 \| Volume 7 \| Article 123 Frontiers in Immunology \| www.frontiersin.org 6 cAMP as Therapeutic Target in Autoimmunity and Inflammation Raker et al. placebo-controlled clinical trials (ESTEEM-1 and -2 for psoriasis, PALACE-1, -2, and -3 for psoriasis arthritis) (149–152). In the two ESTEEM trials, apremilast reduced the severity and extent of moderate-to-severe plaque psoriasis (including nail, scalp, and palmoplantar manifestations) versus placebo in adults. Similarly, in three PALACE trials (PALACE 1–3), apremilast improved the signs and symptoms of psoriasis arthritis relative to placebo in adults with active disease despite treatment with conventional synthetic and/or biologic disease-modifying anti-rheumatic drugs. According to the published clinical trials, apremilast was well tolerated in all study groups analyzed. Throughout phase II and III trials, the most frequently reported side effects consisted of headache, nausea, diarrhea, emesis, and nasopharyngitis and upper respiratory tract infection under continued treatment. However, the studies showed that the gastrointestinal adverse effects usually subside within a month of therapy. expression. A favorable example exists in proteolytic targeting, such as the ubiquitin proteasome system (UPS) (154). Proteolytic targeting chimeric molecules, or PROTACS comprise a UPS rec- ognition motif coupled to an inhibitor via a linker. While a first generation of PROTACs suffered from limited cell-permeability, the second generation has been improved by using a HIF1α peptide fragment as an E3 ubiquitin ligase recognition motif to increase permeability (155). CONCLUSION AND PERSPECTIVE Because of its central importance as a universal regulator of metabolism and gene expression, systemic intervention of the cAMP metabolism is associated with numerous, sometimes con- siderable, side effects. Additionally or alternatively to the devel- opment of isoform-specific AC and PDE inhibitors, new methods need to be found by which these inhibitors may be delivered to tissues and cells specifically. Novel strategies may encompass the development of highly specific agents, new routes of delivery (cutaneous, inhalation) or the use of nanoparticles for tissue or even cell-specific drug delivery. Since cAMP signaling controls very different processes in different cells, a better understanding of the cAMP-mediated activities in particular cell types could help to pave the way to more specific interventions in cell func- tion. Unlike anabolic and catabolic cAMP metabolism, very few drugs engage in signal transduction yet and, thus, the potential use of such actions remains unclear. Although known for over 60 years, the cAMP signaling still reveals new functional details. Therapeutic intervention of its activities, thus, requires further elucidation of its role in individual cell types and its entangle- ments with other signaling and metabolic pathways. It is an interesting result of the clinical studies that improved inhibitor specificity does not prevent side effects. This result sug- gests that the same or overlapping cell populations caused both wanted and unwanted effects. In view of recent research results regarding the expression and activities of anabolic and catabolic cAMP enzymes in immune cells, the question arises whether par- ticular PDE4 inhibitor effects are caused by alteration of immune cell functions. This question is underlined by the similarity of side effects in PDE4 inhibitor studies and some immunotherapeutic approaches. Unfortunately, effects in individual immune cell populations have not been considered in clinical studies with PDE inhibitors so far. For a better understanding of the underly- ing causes of wanted and unwanted effects, such studies appear urgently needed. Alongside their specificity, effective interference with the cAMP pathway through inhibitors depends on their mechanism of action. Basically, inhibitors may act reversibly or irreversibly. Irreversible inhibitors bind to enzymes through covalent bonds. Covalent inhibitors have many desirable features, including increased biochemical efficiency of target disruption, reduced sensitivity toward pharmacokinetic parameters and increased duration of action that outlasts the pharmacokinetics of the compound. CONCLUSION AND PERSPECTIVE Only few inhibitors of this type, however, exist for anabolic and catabolic cAMP enzymes with the common ADCY inhibitor MDL-12,330A, a cyclo-alkyllactamide deriva- tive supposedly representing an exception (153). Most inhibitors are reversible, bind to enzyme through non-covalent bonds, and typically address the ATP-binding site or the catalytic portion. With non-covalent inhibitors, cells can quickly become insensi- tive by recovering enzyme activity. To increase their activity, how- ever, inhibitors can be coupled to proteins that regulate protein 4. Dessauer CW. Adenylyl cyclase – A-kinase anchoring protein complexes: the next dimension in cAMP signaling. Mol Pharmacol (2009) 76:935–41. doi:10.1124/mol.109.059345 5. Conti M, Beavo J. Biochemistry and physiology of cyclic nucleotide phospho- diesterases: essential components in cyclic nucleotide signaling. Annu Rev Biochem (2007) 76:481–511. doi:10.1146/annurev.biochem.76.060305.150444 6. Erdogan S, Houslay MD. Challenge of human Jurkat T-cells with the ade- nylate cyclase activator forskolin elicits major changes in cAMP phospho- diesterase (PDE) expression by up-regulating PDE3 and inducing PDE4D1 and PDE4D2 splice variants as well as down-regulating a novel PDE4A splice variant. Biochem J (1997) 321(Pt 1):165–75. FUNDING This work was supported by the German Research Foundation (DFG): STE791/6-1, STE791/9-1, CRC 1066/B6, TR156/A4/C5; by the German Cancer Aid (110631), and by grants from the University Medical Center Mainz (all to KS). 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Reduction of exacerbations by the PDE4 inhibitor roflumilast – the importance of defining different subsets of patients with COPD. Respir Res (2011) 12:18. doi:10.1186/1465-9921-12-18 Copyright © 2016 Raker, Becker and Steinbrink. 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. Copyright © 2016 Raker, Becker and Steinbrink. 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. 142. Rabe KF, Bateman ED, O’Donnell D, Witte S, Bredenbröker D, Bethke TD. Roflumilast – an oral anti-inflammatory treatment for chronic obstructive pulmonary disease: a randomised controlled trial. Lancet (2005) 366:563–71. doi:10.1016/S0140-6736(05)67100-0 143. Calverley PMA, Sanchez-Toril F, McIvor A, Teichmann P, Bredenbroeker D, Fabbri LM. Effect of 1-year treatment with roflumilast in severe chronic March 2016 \| Volume 7 \| Article 123 Frontiers in Immunology \| www.frontiersin.org 11
https://openalex.org/W4361941548	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_from_Neoadjuvant_Therapy_Induces_a_Potent_Immune_Response_to_Sarcoma_Dominated_by_Myeloid_and_B_Cells/22488248/1/files/39939854.pdf	English	null	Supplementary Figure from Neoadjuvant Therapy Induces a Potent Immune Response to Sarcoma, Dominated by Myeloid and B Cells	null	2,023	cc-by	2,905	Supplemental Fig. S1. Percentage of immune cell populations detected by mIHC pre- and post- neoadjuvant therapy by sarcoma subtype. Individual samples pre-/post- paired are shown. (A) CD3+ cells, (B) CD4+ cells, (C) FOXP3+ T cells, (D) CD8+ cells, (E) CD68+/CD163+ cells and (F) CD206+ cells. UPS: Undifferentiated pleomorphic sarcoma (n=12); LMS: Leiomyosarcoma (n=2); MRCL: Myxoid/round cell liposarcoma (n=3); SS: Synovial Sarcoma (n=2). Based on the small sample sizes, these represent descriptive comparisons only. A B C D E F UPS LMS MRCL SS 0 5 10 15 20 CD3+ % of Total Cells PRE POST UPS LMS MRCL SS 0 5 10 15 20 CD4+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD3+ FoxP3+ % of CD3+ T Cells PRE POST UPS LMS MRCL SS 0 2 4 6 8 10 CD8+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD68+/CD163+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 100 CD206+ % of CD68+/CD163+ POST PRE Supplemental Fig. S1. Percentage of immune cell populations detected by mIHC pre- and post- neoadjuvant therapy by sarcoma subtype. Individual samples pre-/post- paired are shown. (A) CD3+ cells, (B) CD4+ cells, (C) FOXP3+ T cells, (D) CD8+ cells, (E) CD68+/CD163+ cells and (F) CD206+ cells. UPS: Undifferentiated pleomorphic sarcoma (n=12); LMS: Leiomyosarcoma (n=2); MRCL: Myxoid/round cell liposarcoma (n=3); SS: Synovial Sarcoma (n=2). Based on the small sample sizes, these represent descriptive comparisons only. A B C D E F UPS LMS MRCL SS 0 5 10 15 20 CD3+ % of Total Cells PRE POST UPS LMS MRCL SS 0 5 10 15 20 CD4+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD3+ FoxP3+ % of CD3+ T Cells PRE POST UPS LMS MRCL SS 0 2 4 6 8 10 CD8+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD68+/CD163+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 100 CD206+ % of CD68+/CD163+ POST PRE A UPS LMS MRCL SS 0 5 10 15 20 CD3+ % of Total Cells PRE POST B UPS LMS MRCL SS 0 5 10 15 20 CD4+ % of Total Cells PRE POST B A C UPS LMS MRCL SS 0 20 40 60 80 CD3+ FoxP3+ % of CD3+ T Cells PRE POST D UPS LMS MRCL SS 0 2 4 6 8 10 CD8+ % of Total Cells PRE POST C D C E UPS LMS MRCL SS 0 20 40 60 80 CD68+/CD163+ % of Total Cells PRE POST F UPS LMS MRCL SS 0 20 40 60 80 100 CD206+ % of CD68+/CD163+ POST PRE F E F Supplemental Fig. S1. Percentage of immune cell populations detected by mIHC pre- and post- neoadjuvant therapy by sarcoma subtype. Individual samples pre-/post- paired are shown. (A) CD3+ cells, (B) CD4+ cells, (C) FOXP3+ T cells, (D) CD8+ cells, (E) CD68+/CD163+ cells and (F) CD206+ cells. UPS: Undifferentiated pleomorphic sarcoma (n=12); LMS: Leiomyosarcoma (n=2); MRCL: Myxoid/round cell liposarcoma (n=3); SS: Synovial Sarcoma (n=2). Based on the small sample sizes, these represent descriptive comparisons only. Supplemental Fig. S2. Percentage of immune cell populations analyzed by ImmunoPrism pre- and post- neoadjuvant therapy by sarcoma subtype. Individual samples pre-/post- paired are shown. Supplemental Fig. S1. Percentage of immune cell populations detected by mIHC pre- and post- neoadjuvant therapy by sarcoma subtype. Individual samples pre-/post- paired are shown. (A) CD3+ cells, (B) CD4+ cells, (C) FOXP3+ T cells, (D) CD8+ cells, (E) CD68+/CD163+ cells and (F) CD206+ cells. UPS: Undifferentiated pleomorphic sarcoma (n=12); LMS: Leiomyosarcoma (n=2); MRCL: Myxoid/round cell liposarcoma (n=3); SS: Synovial Sarcoma (n=2). Based on the small sample sizes, these represent descriptive comparisons only. A B C D E F UPS LMS MRCL SS 0 5 10 15 20 CD3+ % of Total Cells PRE POST UPS LMS MRCL SS 0 5 10 15 20 CD4+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD3+ FoxP3+ % of CD3+ T Cells PRE POST UPS LMS MRCL SS 0 2 4 6 8 10 CD8+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD68+/CD163+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 100 CD206+ % of CD68+/CD163+ POST PRE A B C D E F G H I UPS LMS MRCL SS EW/ PNET Other 0 5 10 15 20 T-cells % of Total Cells PRE POST UPS LMS MRCL SS EW/ PNET Other 0 5 10 15 20 CD4 T-cells % of Total Cells PRE POST UPS LMS MRCL SS EW/ PNET Other 0 1 2 3 4 5 Treg cells % of Total Cells PRE POST UPS LMS MRCL SS EW/ PNET Other 0 5 10 15 20 CD8 T-cells % of Total Cells PRE POST UPS LMS MRCL SS EW/ PNET Other 0 20 40 60 B-cells % of Total Cells PRE POST UPS LMS MRCL SS EW/ PNET Other 0 2 4 6 8 10 NK-cells % of Total Cells PRE POST UPS LMS MRCL SS EW/ PNET Other 0 20 40 60 80 Monocytes % of Total Cells PRE POST UPS LMS MRCL SS EW/ PNET Other 0 2 4 6 8 10 M1 Macrophages % of Total Cells PRE POST UPS LMS MRCL SS EW/ PNET Other 0 5 10 15 20 M2 Macrophages % of Total Cells PRE POST B UPS LMS MRCL SS EW/ PNET Other 0 5 10 15 20 CD4 T-cells % of Total Cells PRE POST A UPS LMS MRCL SS EW/ PNET Other 0 5 10 15 20 T-cells % of Total Cells PRE POST C UPS LMS MRCL SS EW/ PNET Other 0 1 2 3 4 5 Treg cells % of Total Cells PRE POST B B C A E UPS LMS MRCL SS EW/ PNET Other 0 20 40 60 B-cells % of Total Cells PRE POST D UPS LMS MRCL SS EW/ PNET Other 0 5 10 15 20 CD8 T-cells % of Total Cells PRE POST F UPS LMS MRCL SS EW/ PNET Other 0 2 4 6 8 10 NK-cells % of Total Cells PRE POST F D E E D H I PNET PNET ther PRE POST UPS LMS MRCL SS EW/ PNET Other 0 2 4 6 8 10 M1 Macrophages % of Total Cells PRE POST UPS LMS MRCL SS EW/ PNET Other 0 5 10 15 20 M2 Macrophages % of Total Cells PRE POST I I UPS LMS MRCL SS EW/ PNET Other 0 5 10 15 20 M2 Macrophages % of Total Cells PRE POST G UPS LMS MRCL SS EW/ PNET Other 0 20 40 60 80 Monocytes % of Total Cells PRE POST H UPS LMS MRCL SS EW/ PNET Other 0 2 4 6 8 10 M1 Macrophages % of Total Cells PRE POST G H Supplemental Fig. Supplemental Fig. S1. Percentage of immune cell populations detected by mIHC pre- and post- neoadjuvant therapy by sarcoma subtype. Individual samples pre-/post- paired are shown. (A) CD3+ cells, (B) CD4+ cells, (C) FOXP3+ T cells, (D) CD8+ cells, (E) CD68+/CD163+ cells and (F) CD206+ cells. UPS: Undifferentiated pleomorphic sarcoma (n=12); LMS: Leiomyosarcoma (n=2); MRCL: Myxoid/round cell liposarcoma (n=3); SS: Synovial Sarcoma (n=2). Based on the small sample sizes, these represent descriptive comparisons only. A B C D E F UPS LMS MRCL SS 0 5 10 15 20 CD3+ % of Total Cells PRE POST UPS LMS MRCL SS 0 5 10 15 20 CD4+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD3+ FoxP3+ % of CD3+ T Cells PRE POST UPS LMS MRCL SS 0 2 4 6 8 10 CD8+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD68+/CD163+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 100 CD206+ % of CD68+/CD163+ POST PRE (A) Total T- cells; (B) CD4 T- cells, (C) T regulatory (Treg) cells; (D) CD8 T cells; (E) B cells; (F) NK cells; (G) Monocytes; (H) M1 Macrophages; and (I) M2 Macrophages. UPS: Undifferentiated pleomorphic sarcoma (n=12); LMS: Leiomyosarcoma (n=2); MRCL: Myxoid/round cell liposarcoma (n=3); SS: Synovial Sarcoma (n=2); EW: Ewing sarcoma (n=2) and other (n=5). Based on the small sample sizes, these represent descriptive comparisons only. Supplemental Fig. S1. Percentage of immune cell populations detected by mIHC pre- and post- neoadjuvant therapy by sarcoma subtype. Individual samples pre-/post- paired are shown. (A) CD3+ cells, (B) CD4+ cells, (C) FOXP3+ T cells, (D) CD8+ cells, (E) CD68+/CD163+ cells and (F) CD206+ cells. UPS: Undifferentiated pleomorphic sarcoma (n=12); LMS: Leiomyosarcoma (n=2); MRCL: Myxoid/round cell liposarcoma (n=3); SS: Synovial Sarcoma (n=2). Based on the small sample sizes, these represent descriptive comparisons only. A B C D E F UPS LMS MRCL SS 0 5 10 15 20 CD3+ % of Total Cells PRE POST UPS LMS MRCL SS 0 5 10 15 20 CD4+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD3+ FoxP3+ % of CD3+ T Cells PRE POST UPS LMS MRCL SS 0 2 4 6 8 10 CD8+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD68+/CD163+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 100 CD206+ % of CD68+/CD163+ POST PRE S2. Percentage of immune cell populations analyzed by ImmunoPrism pre- and post- neoadjuvant therapy by sarcoma subtype. Individual samples pre-/post- paired are shown. (A) Total T- cells; (B) CD4 T- cells, (C) T regulatory (Treg) cells; (D) CD8 T cells; (E) B cells; (F) NK cells; (G) Monocytes; (H) M1 Macrophages; and (I) M2 Macrophages. UPS: Undifferentiated pleomorphic sarcoma (n=12); LMS: Leiomyosarcoma (n=2); MRCL: Myxoid/round cell liposarcoma (n=3); SS: Synovial Sarcoma (n=2); EW: Ewing sarcoma (n=2) and other (n=5). Based on the small sample sizes, these represent descriptive comparisons only. Supplemental Fig. S3. Changes in immune modulatory gene expression in the sarcoma tumor immune microenvironme pre- to post-neoadjuvant therapy as determined by (A) multiplex IHC assaying the percentage of CD3+ cells that are pos PD-1, and transcriptional profiling with (B) ImmunoPrism, and (C-D) NanoString. Results are reported fold-change (po neoadjuvant therapy) for ImmunoPrism and NanoString data with statistical analyses utilizing paired t-test and the Ben Hochberg q-values, respectively. Supplemental Fig. S1. Percentage of immune cell populations detected by mIHC pre- and post- neoadjuvant therapy by sarcoma subtype. Individual samples pre-/post- paired are shown. (A) CD3+ cells, (B) CD4+ cells, (C) FOXP3+ T cells, (D) CD8+ cells, (E) CD68+/CD163+ cells and (F) CD206+ cells. UPS: Undifferentiated pleomorphic sarcoma (n=12); LMS: Leiomyosarcoma (n=2); MRCL: Myxoid/round cell liposarcoma (n=3); SS: Synovial Sarcoma (n=2). Based on the small sample sizes, these represent descriptive comparisons only. A B C D E F UPS LMS MRCL SS 0 5 10 15 20 CD3+ % of Total Cells PRE POST UPS LMS MRCL SS 0 5 10 15 20 CD4+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD3+ FoxP3+ % of CD3+ T Cells PRE POST UPS LMS MRCL SS 0 2 4 6 8 10 CD8+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD68+/CD163+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 100 CD206+ % of CD68+/CD163+ POST PRE A C D B PRE POST 0 20 40 60 80 100 % of CD3+ T Cells CD3+ PD-1+ ImmunoPrism Gene Expression Gene ID Fold change (post / pre) Paired t- test Significant OX40 0.90 0.0321 * TIM-3 3.70 0.0002 * CTLA4 1.70 0.1170 ns PD-1 3.10 0.3347 ns PD-L1 1.40 0.7070 ns BTLA 4.40 0.6345 ns ICOS 1.90 0.6821 ns CD47 1.30 0.1127 ns IDO1 2.30 0.2239 ns ARG1 1.40 0.9466 ns NanoString Immune Stimulating Molecules Gene ID Fold change (post / pre) q-value Significant TNFRSF4 (OX40) 0.45 0.0003 * TNFSF18 1.37 0.0527 * CD86 (B7-2) 1.64 0.0003 * CD28 1.37 0.2657 ns CD226 0.89 0.2906 ns TNFSF4 (OX-40L) 0.87 0.3957 ns ICOS 0.74 0.4032 ns TNFRSF9 (CD137) 0.66 0.4555 ns CD40 0.89 0.7071 ns CD27 0.67 0.7870 ns TNFRSF18 0.98 0.8110 ns TNFSF9 (CD137L) 1.08 0.8276 ns CD80 (B7-1) 0.92 0.8472 ns CD40LG 1.07 0.9539 ns NanoString Immune Inhibitory Molecules Gene ID Fold change (post / pre) q-value Significant CD276 (B7-H3) 0.58 0.0013 * PDCD1LG2 (PD-L2) 1.43 0.0027 * ADORA2A 0.66 0.0132 * HAVCR2 (TIM3) 1.34 0.0047 * CD96 1.60 0.0528 ns CTLA4 0.62 0.4929 ns CD274 (PD-L1) 0.96 0.6662 ns LAG3 0.97 0.9218 ns TIGIT 0.98 0.8343 ns PDCD1 (PD1) 0.89 0.9855 ns A B B ImmunoPrism Gene Expression Gene ID Fold change (post / pre) Paired t- test Significant OX40 0.90 0.0321 * TIM-3 3.70 0.0002 * CTLA4 1.70 0.1170 ns PD-1 3.10 0.3347 ns PD-L1 1.40 0.7070 ns BTLA 4.40 0.6345 ns ICOS 1.90 0.6821 ns CD47 1.30 0.1127 ns IDO1 2.30 0.2239 ns ARG1 1.40 0.9466 ns A PRE POST 0 20 40 60 80 100 % of CD3+ T Cells CD3+ PD-1+ C NanoString Immune Inhibitory Molecules Gene ID Fold change (post / pre) q-value Significant CD276 (B7-H3) 0.58 0.0013 * PDCD1LG2 (PD-L2) 1.43 0.0027 * ADORA2A 0.66 0.0132 * HAVCR2 (TIM3) 1.34 0.0047 * CD96 1.60 0.0528 ns CTLA4 0.62 0.4929 ns CD274 (PD-L1) 0.96 0.6662 ns LAG3 0.97 0.9218 ns TIGIT 0.98 0.8343 ns PDCD1 (PD1) 0.89 0.9855 ns D NanoString Immune Stimulating Molecules Gene ID Fold change (post / pre) q-value Significant TNFRSF4 (OX40) 0.45 0.0003 * TNFSF18 1.37 0.0527 * CD86 (B7-2) 1.64 0.0003 * CD28 1.37 0.2657 ns CD226 0.89 0.2906 ns TNFSF4 (OX-40L) 0.87 0.3957 ns ICOS 0.74 0.4032 ns TNFRSF9 (CD137) 0.66 0.4555 ns CD40 0.89 0.7071 ns CD27 0.67 0.7870 ns TNFRSF18 0.98 0.8110 ns TNFSF9 (CD137L) 1.08 0.8276 ns CD80 (B7-1) 0.92 0.8472 ns CD40LG 1.07 0.9539 ns C D Supplemental Fig. Supplemental Fig. S1. Percentage of immune cell populations detected by mIHC pre- and post- neoadjuvant therapy by sarcoma subtype. Individual samples pre-/post- paired are shown. (A) CD3+ cells, (B) CD4+ cells, (C) FOXP3+ T cells, (D) CD8+ cells, (E) CD68+/CD163+ cells and (F) CD206+ cells. UPS: Undifferentiated pleomorphic sarcoma (n=12); LMS: Leiomyosarcoma (n=2); MRCL: Myxoid/round cell liposarcoma (n=3); SS: Synovial Sarcoma (n=2). Based on the small sample sizes, these represent descriptive comparisons only. A B C D E F UPS LMS MRCL SS 0 5 10 15 20 CD3+ % of Total Cells PRE POST UPS LMS MRCL SS 0 5 10 15 20 CD4+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD3+ FoxP3+ % of CD3+ T Cells PRE POST UPS LMS MRCL SS 0 2 4 6 8 10 CD8+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 CD68+/CD163+ % of Total Cells PRE POST UPS LMS MRCL SS 0 20 40 60 80 100 CD206+ % of CD68+/CD163+ POST PRE S3. Changes in immune modulatory gene expression in the sarcoma tumor immune microenvironment from pre- to post-neoadjuvant therapy as determined by (A) multiplex IHC assaying the percentage of CD3+ cells that are positive for PD-1, and transcriptional profiling with (B) ImmunoPrism, and (C-D) NanoString. Results are reported fold-change (post / pre neoadjuvant therapy) for ImmunoPrism and NanoString data with statistical analyses utilizing paired t-test and the Benjamini- Hochberg q-values, respectively. Supplemental Fig. S3. Changes in immune modulatory gene expression in the sarcoma tumor immune microenvironment from pre- to post-neoadjuvant therapy as determined by (A) multiplex IHC assaying the percentage of CD3+ cells that are positive for PD-1, and transcriptional profiling with (B) ImmunoPrism, and (C-D) NanoString. Results are reported fold-change (post / pre neoadjuvant therapy) for ImmunoPrism and NanoString data with statistical analyses utilizing paired t-test and the Benjamini- Hochberg q-values, respectively. Supplemental Fig. S3. Changes in immune modulatory gene expression in the sarcoma tumor immune microenvironment from pre- to post-neoadjuvant therapy as determined by (A) multiplex IHC assaying the percentage of CD3+ cells that are positive for PD-1, and transcriptional profiling with (B) ImmunoPrism, and (C-D) NanoString. Results are reported fold-change (post / pre neoadjuvant therapy) for ImmunoPrism and NanoString data with statistical analyses utilizing paired t-test and the Benjamini- Hochberg q-values, respectively. -5 -4 -3 -2 -1 0 1 2 3 -8 -7 -6 -5 -4 -3 -2 -1 0 Log2 FC Log10 p-value IGJ FCER1A IGSF6 CTSB CXCL2 S100A8 DUSP1 PILRA CTSS S100A9 CD300LF CD37 CDKN1A SIGLEC9 SYK CD163 CLEC10A MRC1 MARCO FCGR2B LILRB4 HLA.DRA HCLS1 HLA.DMB IRF8 AMICA1 TYROBP CD4 FCGR2A HLA.DMA HCST PTPRC HLA.DPB1 FOLR2 HLA.G CD52 SASH3 CD86 MDM2 SAMHD1 CD14 IL10RA ITGAM PTPN13 BRAF INSR IgSF8 ANXA2 MSH2 SART3 PILRB TNFRSF13C DIRAS3 CCNB2 DCHS1 NCR3 TNFRSF4 CDCA2 MKI67 PRC1 CDCA3 PVRIG UBE2C SEMA6A BIRC5 Supplemental Fig. S4. Volcano plot showing genes significantly up-regulated (≥1.5, n=43, orange) or down-regulat (≤0.67, n=22, blue) pre- versus post- neoadjuvant therapy. Symbols for the genes with p-value <0.0000643 (Bonferr cut-off 0.05/778) are indicated. Supplemental Fig. S4. Volcano plot showing genes significantly up-regulated (≥1.5, n=43, orange) or down-regulated (≤0.67, n=22, blue) pre- versus post- neoadjuvant therapy. Symbols for the genes with p-value <0.0000643 (Bonferroni cut-off 0.05/778) are indicated.
https://openalex.org/W1973293989	https://www.econstor.eu/bitstream/10419/158529/1/799679712.pdf	English	null	The rise of monogamy	Series	2,014	cc-by	11,148	Article The rise of monogamy SERIEs - Journal of the Spanish Economic Association Provided in Cooperation with: Spanish Economic Association Suggested Citation: Citci, Sadettin Haluk (2014) : The rise of monogamy, SERIEs - Journal of the Spanish Economic Association, ISSN 1869-4195, Springer, Heidelberg, Vol. 5, Iss. 4, pp. 377-397, https://doi.org/10.1007/s13209-014-0113-y Terms of use: Documents in EconStor may be saved and copied for your personal and scholarly purposes. Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen Zwecken und zum Privatgebrauch gespeichert und kopiert werden. You are not to copy documents for public or commercial purposes, to exhibit the documents publicly, to make them publicly available on the internet, or to distribute or otherwise use the documents in public. Sie dürfen die Dokumente nicht für öffentliche oder kommerzielle Zwecke vervielfältigen, öffentlich ausstellen, öffentlich zugänglich machen, vertreiben oder anderweitig nutzen. If the documents have been made available under an Open Content Licence (especially Creative Commons Licences), you may exercise further usage rights as specified in the indicated licence. Sofern die Verfasser die Dokumente unter Open-Content-Lizenzen (insbesondere CC-Lizenzen) zur Verfügung gestellt haben sollten, gelten abweichend von diesen Nutzungsbedingungen die in der dort genannten Lizenz gewährten Nutzungsrechte. http://creativecommons.org/licenses/by/2.0/ http://creativecommons.org/lice Citci, Sadettin Haluk Citci, Sadettin Haluk The rise of monogamy Sadettin Haluk Citci Received: 11 September 2013 / Accepted: 14 June 2014 / Published online: 5 July 2014 © The Author(s) 2014. This article is published with open access at SpringerLink.com Abstract Why has polygyny, marriage of a man to multiple women, common in most societies throughout history, almost disappeared in modern industrialized countries? Do women play a role in its disappearance? A simple theoretical model suggests that at the later stage of the transition from polygyny to monogamy, the spread of human capital and the increase of labor income have led to the rise of monogamy. A general equilibrium model of the marriage market illustrates that the spread of human capital results in an increase in women’s income opportunities and lowers the importance of bequests in determining their sons’ incomes. Both effects improve women’s outside option, mating monogamously, and decrease the marginal beneﬁt of the economic advantages provided by polygynous unions. This, in turn, reduces polygyny by increasing the cost of polygynous mating for men. When the human capital level is sufﬁciently high, men ﬁnd it optimal to marry monogamously instead of bearing the high cost of polygynous mating. Keywords Marriage · Polygyny · Monogamy · Development · Human capital JEL Classiﬁcation J12 · J24 · O10 · O40 · Z13 JEL Classiﬁcation J12 · J24 · O10 · O40 · Z13 S. H. Citci (B) Faculty of Business Administration, Gebze Institute of Technology, Gebze, Kocaeli, Turkey e-mail: hcitci@gyte.edu.tr SERIEs (2014) 5:377–397 DOI 10.1007/s13209-014-0113-y ORIGINAL ARTICLE 1 Introduction Polygynous mating is a global phenomenon in the sense that it has occurred in most societies throughout history. For example, Murdock’s Ethnographic Atlas men- tions that polygyny exists in 850 of 1,170 societies although to varying degrees (Hartung et al. 1982). Similarly, human relations area ﬁles documents that 93% of 12 3 3 378 SERIEs (2014) 5:377–397 1,154 recorded societies recognize some degree of polygyny (Clark 1998). Moreover, polygyny is not an issue of the past. We still observe polygynous marriage at a rate of up to 55% in many sub-Saharan African countries (Tertilt 2005). For example, the percentage of married men in a polygynous union is 55% in Cameroon and 40% in Senegal and Burkina Faso. However, polygynous mating is almost nonexistent in developed countries. This is referred to as the mystery of monogamy (Gould et al. 2008). These observations lead to the following questions: Why did the marriage type evolve into monogamy during the course of economic development? Which factors determine the form of marriage in a given society? Existing studies answering these questions commonly focus on changes in the demand for females. Lagerlof (2005) illustrates decreasing quantity of demand as a result of the decline in income inequality among men. Becker (1991) explains the nonexistence of polygyny in advanced economies with the shift in men’s demand from quantity of children to quality of children. Similar to Becker (1991) and Gould et al. (2008) explain the nonexistence of polygyny as a result of the trade-off between the quantity and quality of children. However, they present a different mechanism and propose that when the return on human capital is high, a rich man may choose to marry one well-educated, high skilled woman who can provide human capital to his children, rather than mating with many women to increase the number of chil- dren.1 Possibly because of the fact that women did not have much decision-making power in marriages or access to economic resources in the early stage of the transi- tion from polygyny to monogamy, the existing literature focuses on the demand side changes. Yet, does it mean that women played no role in the virtual disappearance of polygyny? p ygy y Historical accounts of the transition from polygyny to monogamy in the Western World suggest that polygyny was prevalent up to the industrialization. 1 This issue is also a common research ﬁeld for anthropology and sociology. In anthropology literature, Melotti (1981) explains the transition to monogamy as a result of evolution. He argues that monogamous mating is evolutionarily superior to polygyny when considering altruism among children. MacDonald (1990), Betzig (1986) and Alexander (1987) argue that this phenomenon is the result of egalitarianism or the need for cohesion in democratic—industrialized countries where the division of labor or the ’rule of law’ is prominent. In a recent paper, Lagerlof (2010) extends and formalizes ideas discussed in Alexander (1987). In sociology literature, Kanazawa and Still (1999) assert that women choose to marry polygynously when wealth inequality among men is high and choose monogamy if the inequality declines sufﬁciently. In a political economy framework, Croix and Mariani (2012) argue that decreasing inequality among males can lead poor women and poor men to form a coalition supporting monogamy as the socially imposed marriage regime. 1 Introduction Industrilization increased women’s autonomy in marriages and access to economic resources, espe- cially their labor income, signiﬁcantly. Actually, it is not easy to establish the exact timing of the virtual disappearance of polygynous mating. However, many sociologists and anthropologists argue that in medieval Europe rich men, although married monog- amously, had sexual access to servants, concubines and mistresses, so they continued to mate polygynously (e.g., Gould et al. 2008; Stone 1961, 1977; Goody 1983; Grimal 1986; Betzig 1992, 1995, 2002; Scheidel 2009, 2011). For example, Stone (1961) writes about this widespread practice in England in the late medieval period that “In the sixteenth century the provision in wills for illegitimate children indicates that the 12 123 123 379 SERIEs (2014) 5:377–397 maintenance by a peer of a lower-class mistress was compatible with a stable marriage, and that it was fairly frequent occurrence.” Anthropologist Laura Betzig argues that the same pattern continued up to the twentieth century when, after a longer gradual decline, polygynous mating died out. Particularly, Betzig (1995) writes that “When, why did polygyny and despotism end, and monogamy and democracy begin?...It seems to me that one event changed all that: the switch to an industrial economy in Europe in the past few centuries.” This timing ﬁts well with the spread of human capital and the increasing role of women both in the economy and in marriage decisions. Moreover, several studies employing cross-sectional and cross-cultural data show that women’s human capital level, their socio-economic position and the degree of polygyny go hand in hand. Lesthaeghe and Surkyn (1988) and Lesthaeghe et al. (1989) conducted cross-sectional comparative analyses of polygyny in Africa and found that polygyny is less prevalent in societies where more adult women are literate. Similarly, Kaufmann and Meekers (1998), based on their ﬁndings researching marriage patterns in Sub-Saharan Africa, argue that “where women have higher status on their own right and therefore possess a measure of autonomy through inheritance rights, education, or matriliny, polygyny is not as common.” Although these ﬁndings do not necessitate causality, they open the door for the potential role of women in the transition from polygyny to monogamy.2 Findings of ﬁeld studies and research based on micro-data are more suggestive concerning the role of women in the transition. For example, study of Ahmed (1986) on Yoruba women in Western Nigeria shows that women’s education and the occupation of women’s parents have a strong inﬂuence on the type of marriage women enter. Comparing the differential impact of individual characteristics on the type of marriage women enter, he ﬁnds that higher educated women are less likely to be involved in a polygynous union, and the higher the occupational level of a woman’s parents, the less likely she is to marry a polygynist. There is also supporting evidence for the aforementioned role of women in the transition to monogamy in Clignet (1970), Grossbard (1976), Gould et al. (2008), Ware (1979) and Armstrong et al. (1993). Based on these observations, the goal of this paper is to focus on the supply side changes and to analyze women’s role in the virtual disappearance of polygyny. 2 Some papers in the related literature argue that the causality between economic development and the degree of polygyny might be reverse. For example, Tertilt (2005) shows that enforcing monogamy may lower fertility and result in an increase in saving. She estimates that banning polygyny may lead GDP per capita to go up by 170%. Similarly, Tertilt (2006) shows that increasing female autonomy in marital choices may increase GDP per capita by lowering the degree of polygyny. 123 I argue thatatthelaterstageofthetransitionfrompolygynytomonogamy,thespreadofhuman capital and the increase of the value of women’s income opportunities have led to the virtual disappearance of polygyny in modern industrialized countries. Speciﬁcally, I demonstrate that above a sufﬁciently high level of human capital, polygynous mating disappears. I build a framework where there are two groups of men with different income levels, while all women are identical. Each individual values consumption, spending time together (the amount of marital interaction with the mate), number of offspring and future incomes of children. 12 3 3 380 SERIEs (2014) 5:377–397 Men and women differ in their reproductive ability.3 Women are assumed to be biologically constrained to have a ﬁnite number of children and they cannot increase their offspring by increasing the number of their spouses. On the other hand, men can increase the number of their children by increasing the number of their mates. This gives men an incentive to marry polygynously and it yields competition among men for mates. All else equal, a woman’s utility decreases with the number of women in the household since, as the number of woman increases, the marital interaction per wife decreases. In order to marry polygynously, men have to compensate for the loss in women’s utility due to lower marital interaction. The compensation takes the form of transfers to each wife and higher bequests to children, more than they would get in a monogamous marriage. These economic advantages that need to be offered in polygynous marriage also constitute the cost of polygyny for men. An increase in the level of human capital alters the cost of polygynous mating in various ways. First, the spread of human capital results in an increase in women’s incomeopportunities,mainlythroughitseffectontheleveloflaborincome.Asaresult, women allocate more resources for their own consumption and leave larger bequests to their children. Therefore, economic advantages provided by men in polygynous mating become relatively less important and men need to increase the amount of economic resources to compensate for women’s forgone utility resulting from sharing their husbands with co-wives. This makes polygynous mating more costly for men and decreases the degree of polygyny in the society. Second, an increase in the level of humancapitalleadswomentoanticipatethattheimportanceofbequestsindetermining their sons’ income will be lower. Thus, the marginal beneﬁt that they derive from the bequests left to their sons diminishes. 3 Siow (1998) studies a model of differential fecundity and how it can affect labor and marriage market outcomes. 5 In the model economy, all women participate in the labor force. However, in Sect. 4, I show that when female labor force participation is zero, the second channel that resembles the quantity-quality trade-off is still at work and the main result extends to the partial female labor force participation. 6 Instead, one can consider that the human capital investment is costly, but the return of the human capital investment is sufﬁciently high that parents choose to invest in their children’s human capital. 7 Polyandry marriage is the mating of a woman with multiple men at the same time. 8 The phylogenetic approach argues that parents transfer a greater amount of wealth to their sons in order to maximize their reproductive-success (Hartung et al. 1982). The speciﬁed utility functions in the model can be considered a reduced form of a more general utility function where agents care about their reproductive success. Although in the model, the gender inequality in bequests is in extreme form, relaxing this assumption does not change the results of the paper as long as male biased inequality in bequests is present. 123 Consequently, as the level of human capital increases, a man who tries to convince a woman to participate in polygynous mating needs to increase the amount of economic resources to her or to increase the amount of the bequest left to her son. Hence, the incidence of polygyny declines as the human capital of children increases. It is worth noting that this change does not happen due to men’s demand shift from the quantity to quality of children, known as the quantity- quality trade-off, but rather due to the increased cost of polygynous mating. In other words, in an economy where the level of human capital and, relatedly, the value of women’s income opportunities (excluding the economic transfers provided by their mates) such as labor income are low, a rich man can persuade a woman to become involved in a polygynous union instead of marrying monogamously by offering few economic advantages for her or her children. Therefore, inequality in economic resources translates into inequality in the number of wives in the competitive marriage market of this economy. However, the increase of return on human capital and labor income decreases the marginal beneﬁt of the economic advantages provided in polygynous unions and leads to a substantial increase in the cost of polygyny for men. Consequently, in an economy with a sufﬁciently high human capital level men ﬁnd it optimal to marry monogamously instead of bearing the high cost of polygynous mating, which gives rise to monogamy even in the presence of high income inequality. 12 3 SERIEs (2014) 5:377–397 381 This paper is the ﬁrst to introduce a supply-side explanation for the phenomenon. I show that the virtual disappearance of polygyny and the presence of the quantity- quality trade-off for children arise from the increasing cost of polygyny that occurs with the spread of human capital. Hence, the hypothesis offers a new perspective and complements the previous demand side explanations. The rest of the paper is organized as follows. Section2 introduces the model. Sec- tion 3 examines the model, presents the results and discusses existing anthropological and cross-country ﬁndings on the issue. Section 4 analyzes the robustness of the the- ory. In this section, I show that the main result extends for partial female labor force participation and discuss the implications of female autonomy. Section 5 provides a concluding summary. 4 In Appendix C I show that the results also carry over to a dynamic overlapping model with an inﬁni horizon. 2 The model The model is a variation of Gould et al. (2008). I consider a static general equilibrium of the marriage market with a continuum of men and women.4 Population sizes of both genders are equal and normalized to 1. The economy produces a single homogeneous good, using efﬁciency units of labor as its sole input. Output of a man and a woman is equal to h, which is exogenously given and equivalent to the human capital of a person. Human capital could be any skill that makes a person more productive in the labor market.5 For simplicity, I normalize the cost of human capital investment to zero.6 Marriage in the model can be thought of as an agreement between the husband and the wife over the division of household resources among them and their children. A man can marry a woman if he provides her with the equilibrium utility level, to be described in more detail below, determined in the competitive marriage market, uw. Marriage is not restricted to monogamy. However, since the analysis focuses on polygyny and monogamy, I specify the utility functions so that staying single and polyandrous mating are not possible in equilibrium.7 Men and women have similar preferences. Each man and woman gets utility from consumption, cm and cw respectively. They also get utility from the number and the total income of own children. Speciﬁcally, both men and women get utility from human capital, h, of both sons and daughters. However, each parent gets utility from the total bequest left to sons only.8 The bequests left from the father and from the mother are denoted by, bm and bw, respectively. 12 3 3 382 SERIEs (2014) 5:377–397 A central assumption of my model is that time spent together with the mate (emo- tional and sexual interaction) is a normal good for agents and the amount of marital interaction negatively depends on the number of co-wives in the household. There is a substantial empirical literature supporting this assumption. For example, estimates for jointleisureconsumptionbycouplesindicatethatbothmenandwomendemandspend- ing time together with their spouses (Hamermesh 2002). 9 Parallel to these, several empirical studies also indicate that in polygynous marriages, co-wives compete, conﬂict for and are jealous of the sexual and emotional attention of their husbands (Meekers and Franklin 1990; Mulder 1990; Farrell 1987; Solway 1990; Aluko and Aransiola 2003). 10 This assumption also results in men’s and women’s utility functions being asymmetric in the benchmark model. Nonetheless, the asymmetry in men’s and women’s utility functions is relaxed in Appendix B. Speciﬁcally, I assume in Appendix B that (all else equal) a man’s and a woman’s utility is a continuous function of time spent together with the mate (both of them value marital interaction) and a man divides his limited time equally among his wives. I show that under these assumptions, the second channel is still at work and the main result holds. 11 The size of the upper bound on women’s fertility is not critical. Thus, the number of children of a woman can be assumed to be more than two. The results are valid as long as there exists a limit on women’s fertility. 2 The model Similarly, Sullivan (1996), Hallberg (2003), Jenkins and Osberg (2005) and Connelly and Kimmel (2009) ﬁnd evidence of this desire for spending time with one’s spouse.9 The literature on marital happiness and marital stability also provides evidence for the causal affect of amount of marital interaction on marital happiness (White 1983; Hill 1988; Zuo 1992 and ref- erences therein). To simplify the analysis, I assume in the benchmark model that only women value marital interaction and the difference between a woman’s utility derived from time spent with her mate in monogamous marriage and of that in polygynous marriage is constant, denoted by λ ∈ℜ+.10 Men choose their quantity of children implicitly by choosing how many wives to marry, n. In order to simplify the model, I follow Becker (1991) and assume that n is a continuous variable. Contrary to males, females are biologically constrained to have two children.11 Although women cannot choose the number of their mates, they can choose whether to mate monogamously or polygynously. The mating decision of a woman is denoted by x ∈{0, 1}, where x = 0 if the mating is monogamous, and x = 1 if the mating is polygynous (n > 1). In particular, preferences of men are represented by the following utility function In particular, preferences of men are represented by the following utility function ln cm + ln [n (2h + bw + bm)] , (1) (1) 9 Parallel to these, several empirical studies also indicate that in polygynous marriages, co-wives compete, conﬂict for and are jealous of the sexual and emotional attention of their husbands (Meekers and Franklin 1990; Mulder 1990; Farrell 1987; Solway 1990; Aluko and Aransiola 2003). where Im ∈{Ir, Ip}. Finally, the bequest decisions of men and women are sequential. First, the husband decides the bequest level per son, bm, and the income transfer level per wife, y. In the second stage, each wife takes the bequest and income transfer decision of the husband as given and then decides the amount of bequest, bw, to her son.12 In order to simplify the analysis by avoiding corner solutions, I allow negative values of the bequest left by a woman, which may be interpreted as an income transfer from son to his mother after he starts to work in the labor market. whereas a woman’s utility function is given by whereas a woman’s utility function is given by ln cw + ln (2h + bw + bm) −λx (2) (2) A woman earns income in the labor market (Iw = h). Moreover, she receives a transfer from her husband, y. A man’s income is the sum of bequests received from his parents and his labor income. There are two groups of men according to their income levels, rich with income Ir and poor with income Ip. The proportion of poor is given by θ and that of rich is given by (1 −θ). 12 123 SERIEs (2014) 5:377–397 383 A woman’s and a man’s budget constraints are given by A woman’s and a man’s budget constraints are given by cw + bw = Iw + y (3) cm + n (bm + y) = Im, (4) (3) (4) ( ) (4) (4) cm + n (bm + y) = Im, where Im ∈{Ir, Ip}. 3 Analysis Each woman chooses her consumption, cw, the bequest transfer to her son, bw, and the type of mating to enter into, x, to maximize (2) subject to (3), given the amount of income transfer and the bequest left to her son by her husband: max {x, cw, bw} ln cw + ln (2h + bw + bm) −λx s.t. cw + bw = Iw + y Each man chooses his consumption, cm, the number of wives, n, the amount of income transfer to each wife, y, and the bequests for each of his sons, bm, to maximize (1) subject to (4) and non-negativity constraints, given women’s equilibrium utility, uw: max {cm, n, y, bm} ln cm + ln [n (2h + bw + bm)] s.t. cm + n (bm + y) = Im, bm, y ≥0, ln cw + ln (2h + bw + bm) −λx ≥uw The last constraint can be considered as the participation constraint of a woman to enter into a polygynous marriage. The last constraint can be considered as the participation constraint of a woman to enter into a polygynous marriage. Finally, market clearance implies that all women are married in equilibrium. Lemma 1 In polygynous mating the sum of the income transfer to each woman and bequest to her son is higher than the sum of those in monogamous mating. The differ- ence is equal to 2 exp uw 2 exp λ 2 −1 2 exp uw 2 exp λ 2 −1 12 Compared to simultaneous decision-making this bequest decision pattern is more in line with patriarchy. Thus, I intentionally kept the pattern in this form. However, changing the pattern does not affect the results. 3 384 SERIEs (2014) 5:377–397 Proof Substituting (3) into (2) and deriving the ﬁrst-order condition with respect to bw yields bw = Iw + y −2h −bm 2 (5) (5) By substituting (5) and (3) into the participation constraint, one can rewrite this constraint in the following form: 2 ln Iw + y + 2h + bm 2 −λx ≥uw (6) (6) In the polygynous equilibrium, men’s optimization requires that (6) holds with equality. Thus, (6) and x ∈{0, 1} together imply that In the polygynous equilibrium, men’s optimization requires that (6) holds with equality. 3 Analysis Thus, (6) and x ∈{0, 1} together imply that y + bm = 2Uk −Iw −2h if n > 1 (7) y + bm = 2U −Iw −2h if n ≤1 (8) (7) (8) (7) (7) (8) (8) where U ≡exp( uw 2 ) and k ≡exp( λ 2). (7) and (8) represent the sum of the income transfer to each woman and the bequest to her son in polygynous mating and the sum of those in monogamous mating, respectively. Subtracting (8) from (7) produces the result. The intuition underlying Lemma 1 is straightforward. A man who marries polyg- ynously provides less marital interaction to each of his wives compared to a man in a monogamous marriage. Hence, the man in a polygynous marriage has to compensate for each of his wives’ forgone utility that results from sharing him with co-wives. There are two ways he can do this. He could offer a higher income transfer to his wife or a higher amount of bequest to her son than she would receive in a monogamous marriage. The difference can be interpreted as the cost of polygynous mating for men. Figure 1 represents the relation between the cost of polygyny and women’s equilib- rium utility level, uw. It shows that as uw increases, men need to offer a higher amount of income transfer to a woman, y, and bequest, bm, for her son in order to compensate for her forgone utility in polygyny, λ. The cost of polygynous mating increases with uw at an increasing rate. As a result, any factor that improves women’s utility also increases the cost of polygynous mating for men. Figure 1 shows that concavity and monotonicity properties of utility functions, which are standard assumptions, imply this result. The intuition underlying Lemma 1 is straightforward. A man who marries polyg- ynously provides less marital interaction to each of his wives compared to a man in a monogamous marriage. Hence, the man in a polygynous marriage has to compensate for each of his wives’ forgone utility that results from sharing him with co-wives. There are two ways he can do this. He could offer a higher income transfer to his wife or a higher amount of bequest to her son than she would receive in a monogamous marriage. The difference can be interpreted as the cost of polygynous mating for men. 3 Analysis Figure 1 represents the relation between the cost of polygyny and women’s equilib- rium utility level, uw. It shows that as uw increases, men need to offer a higher amount of income transfer to a woman, y, and bequest, bm, for her son in order to compensate for her forgone utility in polygyny, λ. The cost of polygynous mating increases with uw at an increasing rate. As a result, any factor that improves women’s utility also increases the cost of polygynous mating for men. Figure 1 shows that concavity and monotonicity properties of utility functions, which are standard assumptions, imply this result. Lemma 2 Rich men have at least as many wives as poor men. Lemma 2 Rich men have at least as many wives as poor men. Proposition 1 The degree of polygyny is (i) Positively associated with income inequality among males, (i) Positively associated with income inequality among males, (ii) negatively associated with human capital and income level of women. Lemma 2 Rich men have at least as many wives as poor men. Proof After substituting (4), (5) and (6) into (1), men’s maximization problem boils down to max {n} ln Im −n 2Ukx −Iw −2h + ln nUkx s.t. bm, y ≥0 (9) 123 123 SERIEs (2014) 5:377–397 385 Deriving the ﬁrst-order condition with respect to n produces the following conditions after rearranging: n = Im 2(2Uk−Iw−2h) if n > 1 Im 2(2U−Iw−2h) if n ≤1 (10) (10) In the polygynous equilibrium, the characterization of n together with the assumption Ir > Ip produces the result that a rich man has more wives than a poor man. Observing that every man has one wife in the monogamous equilibrium completes the proof.13 The result of the cost of polygyny and the competition among males for women is that if polygynous mating exists in equilibrium, only men with adequate resources can afford it. The assumption of a balanced sex ratio in the model implies that only rich men can marry polygynously. This result is also in line with Becker and Lewis (1973), Bergstrom (1994b), Wright (1994) and Gould et al. (2008). The next proposition presents two important factors that affect the degree of polyg- yny. Proposition 1 The degree of polygyny is 13 Women’s equilibrium utility, uw, and number of wives for rich and poor are explicitly derived in Appen- dix A. 14 Income inequality between rich and poor men is measured as Ir −Ip Ip . 13 Women’s equilibrium utility, uw, and number of wives for rich and poor are explicitly derived in Appe dix A. 14 Income inequality between rich and poor men is measured as Ir −Ip Ip . 15 The threshold of the human capital level is explicitly given in the appendix. Proof See Appendix A. The intuition behind the ﬁrst statement of Proposition 1 is as follows.14 Holding the income of poor men constant, an increase in the income level of rich men enlarges their choice sets. Concavity and monotonicity of the utility function imply that rich men are willing to increase their number of wives. On the other hand, holding the income of rich men constant, a decrease in poor men’s income leads them to leave smaller bequests to their sons and to transfer fewer resources to their wives. This makes monogamous mating less appealing for women. As a result, more women engage in polygynous mating. The proposition also states that an increase in the human capital level and in the labor income of women decreases the degree of polygyny. As the income of women increases, the marginal utility of extra economic resources provided in polygynous mating diminishes. As a result, rich men have to increase the sum of income transfers and the amount of bequests, if they want to marry polygynously. The intuition for this statement follows from Lemma 1. As I show in Lemma 1, anything that increases women’s equilibrium utility also increases the cost of polygyny. Thus, the increase of women’s human capital level and labor income increases the cost of polygyny through its effect on uw. 12 3 SERIEs (2014) 5:377–397 386 Fig. 1 Women’s utility level vs. the cost of polygyny Proposition 2 The degree of polygyny declines with h and if h is sufﬁciently high, polygynous mating is nonexistent in equilibrium. Fig. 1 Women’s utility level vs. the cost of polygyny Proposition 2 The degree of polygyny declines with h and if h is sufﬁciently high, polygynous mating is nonexistent in equilibrium. Proposition 2 The degree of polygyny declines with h and if h is sufﬁciently high, polygynous mating is nonexistent in equilibrium. Proposition 2 The degree of polygyny declines with h and if h is sufﬁciently high, polygynous mating is nonexistent in equilibrium. Proposition 2 The degree of polygyny declines with h and if h is sufﬁciently high, polygynous mating is nonexistent in equilibrium. Proof See Appendix A. Proof See Appendix A. Proposition 2 states the main result of the paper. Above a critical level of human capital and labor income, monogamy turns out to be the unique mating type in equi- librium.15 There are two channels in which the increase in human capital and labor income decreases the degree of polygyny, although each of these channels alone might lead to the disappearance of polygyny. The ﬁrst one is the effect of the spread of human capital through the increase of labor income of women. As I establish in the second statement of Proposition 1, women’s human capital and the degree of polygyny are inversely related. As women’s human capital and, relatedly, labor income increase, so do their equilibrium utility level and the cost of polygyny (see Fig. 1). Second, an increase in the level of human capital leads women to anticipate that their sons will earn a higher income in the labor market. As a result, the beneﬁt that women derive from the total bequest left to their sons diminishes. This effect coerces men to provide higher economic advantages in order to compensate for women’s utility loss from polygynous mating. Hence, the cost of polygyny increases and the degree of polygyny falls. The second channel resembles a well-known phenomenon: the quantity-quality trade-off for children. However, in contrast to the existing literature, the shift from the quantity to quality in this setting occurs due to supply-side reasons. Both Becker (1991) and Gould et al. (2008) argue that as the return on human capital increases, men prefer to have fewer but higher quality children. As a result, the degree of polygyny 12 123 SERIEs (2014) 5:377–397 387 falls. On the other hand, Proposition 2 states that the decline arises from the increasing cost of polygyny rather than the demand shift of men. falls. On the other hand, Proposition 2 states that the decline arises from the increasing cost of polygyny rather than the demand shift of men. Results of an informative survey carried out in Kenya support the effectiveness of the emphasized cost-side changes in explaining the decline of polygyny. To analyze changing family and kinship in Kenya, Adams and Mburugu (1994) surveyed the Kikuyu who the authors regard as the most changed or urbanized of the Kenyan peoples. 16 I do not provide a theory of female labor force participation here. Such an analysis is beyond the scope of this study. Proof See Appendix A. According to their survey, there was a fall in polygyny at the start of 1990 (from 28% of the respondents’ fathers to an expectation of 16% of the 30–40years olds respondents) and the primary reason for this fall is the increasing cost of polygyny and rising standard of living expectations. Based on their ﬁndings, the authors report that “cost is central” for monogamy and they conclude that “In fact, if the results of this pilot study are generalizable, cost plus the effects of higher education may be sufﬁcient to limit polygyny...” 4 Robustness In the previous section, I show the transition from polygynous equilibrium to monog- amous equilibrium under implicit assumptions of full female labor force participation and ultimate female autonomy in marriage decisions. Although these assumptions are innocuous for current advanced countries, one can question their validity in the pre- industrial world. Nonetheless, I argue that the mechanism behind the main result is still effective even when these assumptions are relaxed, as I show below. First, I investigate whether the main result holds if only a fraction of females participates in the labor force. For this purpose, I modify the model by assuming an exogenous female labor force participation rate, ε, which is less than unity. The next proposition states that the main result of the paper extends under the assumption of partial female labor force participation.16 Proposition 3 For all ε ∈[0, 1), the degree of polygyny declines with h and if h is sufﬁciently high, polygynous mating is nonexistent in equilibrium. Proof See Appendix A. The intuition behind Proposition 3 follows from the discussion in Sect. 2. Even if some females do not participate in the labor force, an increase in labor income in line with the spread of human capital still increases the equilibrium utility level of all women. As the human capital level increases, women anticipate that the labor income of their sons will increase and so the beneﬁt of the total bequest left to sons diminishes for women. In other words, the quantity versus quality channel is effective even when most of females do not work. Moreover, an increase in labor income increases the utility level of women who participate in the labor force. As argued with Lemma 1, anything that increases the equilibrium utility level of women also increases the cost of polygyny. Hence, the ﬁndings are robust to the labor force participation of women. 12 3 3 388 SERIEs (2014) 5:377–397 The mechanism described in the paper is still effective in the absence of ultimate female autonomy in marital choices, as long as at least one of the following assump- tions holds: (i) women have some degree of decision-making power over their marital choices or (ii) parents have some degree of altruism towards their daughters. The validity of the hypothesis under these assumptions is more apparent. Thus, I discuss the mechanism without presenting a formal proof. As it is in the main model, women’s marginal utility derived from the economic resources provided in polygynous mating still diminishes with the increase of the human capital level. Since the two mecha- nisms in the model (the corresponding increase of labor income of women and their sons) still have the same effects on women’s utility. Here, the key role of assump- tions (i) and (ii) is that under either assumption women’s declining incentives to enter into a polygynous marriage, accompanied with the spread of human capital, will be reﬂected in marital decisions. Consequently, the cost of polygynous mating for men increases with an increase in human capital and, once again, the degree of polygyny being negatively correlated with the level of human capital and labor income remains as a result. Moreover, these assumptions are justiﬁed with evidence. There are supportive stud- ies showing that women exert considerable inﬂuence over their marital decisions. Proof See Appendix A. For instance, in Togo where more than 40% of women have been involved in polygy- nous unions, it is documented that over 70% of women have decision-making power over their marital decisions (Gage 1995). Similarly, parental altruism, that can pro- vide alignment of interests on marital choices between daughters and their parents, is also supported by existing studies. For example, in her analysis of 133 societies, Small (1992) concludes that the interests of females in arranged marriages are not necessarily different from the interests of their parents. Hence, the main conclusion of the paper holds even when I relax the assumptions regarding female autonomy and labor force participation. Crucially the results follow from the concavity and monotonicity properties of utility functions and women’s demandformaritalinteraction,whichimplieswomenprefermonogamyoverpolygyny if all else is equal. The ﬁrst two assumptions are standard in economic theory and the last assumption is well-supported by existing empirical evidence. Consequently, the underlying mechanism works in a more general environment as long as these assumptions are preserved. 123 5 Conclusion This paper examines why advanced countries are more monogamous than less devel- oped countries. A common feature of developed economies is that the level of human capital and, relatedly, the average labor income in these countries are much higher than they are in less developed countries. I argue that this characteristic is the main reason for the variations in the degree of polygyny among advanced and less developed countries. I further assert that, through the process of industrialization and economic development, the spread of human capital and the increase of labor income have led to the virtual disappearance of polygyny at the later stage of the transition. The theory is simple and intuitive. I argue that a woman values time spent with her spouse. If she is involved in polygyny, she enjoys less utility due to a lower amount 123 389 SERIEs (2014) 5:377–397 of marital interaction compared to what she would enjoy in a monogamous marriage. Thus, in order to persuade women to enter into a polygynous marriage, men have to offer more economic resources than those provided in a monogamous marriage. These extra payments constitute the cost of polygynous mating for men. The cost of polygynous mating for men increases with the spread of human capital and the increase of labor income through two channels: ﬁrst, the spread of human capital results in an increase of women’s income opportunities independent of their mates’ incomes, mainly through its effect on the level of labor income. This, in turn, decreases the marginal beneﬁt of the economic advantages provided in a polygynous union. As a result, women demand more beneﬁts to enter into a polygynous marriage. Second, the increase of the human capital level leads women to anticipate that their children can receive higher labor income, and so it decreases the marginal contribution of bequests to the total income. Thus, the incentives for women, who care about the income of their sons, to enter into a polygynous marriage diminish as well. Consequently, an increase in human capital and labor income leads to a decline in the number of wives and children of rich men. The second channel implies that the decline in quantity of children, accompanied with the increase of quality of them, arises from the supply-side changes rather than from men’s demand shift from quantity of children to quality of them. 5 Conclusion Finally, this paper emphasizes that women’s roles are also important in the deter- mination of observed marriage types in a society. Hence, the results suggest that poli- cies favoring women, such as encouraging their labor force participation, subsidizing female education and securing more gender equal inheritance should be considered as alternative instruments for the prevention of polygyny. Acknowledgments I would like to thank my advisors Remzi Kaygusuz and Eren Inci for their advice and support, as well as Ayse Imrohoroglu, Esra Durceylan Kaygusuz, Hakki Yazici, Inci Gumus, Nuray Akin, Philip J. Reny, Sebnem K. Ozcan, Ted Bergstrom, Ufuk Akcigit and seminar participants at HSE-SU Workshop 2010, Public Economic Theory Conference 2010, TEA International Conference on Economics 2010 and Koc University Winter Workshop in Economics 2010 for their helpful comments and suggestions. I also thank Editor Nezih Guner and an anonymous referee for a detailed review and valuable comments. Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Appendix A: Proofs Let n be an element of the set {nr, n p} where nr and n p are the numbers of wives of a rich man and a poor man, respectively. Let L and M denote the exogenous bequests received by a rich and a poor man, respectively. Proof of Proposition 1 Substituting (10) into the market clearing condition (θn p + (1 −θ)nr = 1) produces the following equation: U = (k + 1) (4h + 2Iw) + kθ Ip + (1 −θ) Ir ± √ A 8k (11) (11) 3 390 SERIEs (2014) 5:377–397 where A ≡[(1 −θ)Ir −kθ Ip −(k −1)(4h + 2Iw)]2 + 4Irkθ Ip(1 −θ). It gives an expression of U in terms of the exogenous variables for the polygynous equilib- rium. Notice that although U has two roots, the root with negative √ A violates (10). Therefore, the positive root represents U. where A ≡[(1 −θ)Ir −kθ Ip −(k −1)(4h + 2Iw)]2 + 4Irkθ Ip(1 −θ). It gives an expression of U in terms of the exogenous variables for the polygynous equilib- rium. Notice that although U has two roots, the root with negative √ A violates (10). Therefore, the positive root represents U. bstituting (11) into (10) yields Substituting (11) into (10) yields n = Im (k+1)(4h+2Iw)+kθ Ip+(1−θ)Ir+ √ A 2 −2Iw −4h if n > 1 In Lemma 2, I establish that if the equilibrium is polygynous, only rich men marry polygynously. Thus, the number of a rich man’s wives is equal to the following: nr = Ir (k+1)(4h+2Iw)+kθ Ip+(1−θ)Ir+ √ A 2 −2Iw −4h (12) (12) Taking the derivative of nr with respect to Ir and Ip shows that ∂nr/∂Ir > 0 and ∂nr/∂Ip < 0 . Therefore, nr is negatively related to income inequality. Taking the derivative of nr with respect to Iw yields ∂nr/∂Iw < 0 which proves the second statement of Proposition 1.17 Proof of Proposition 2 First, I need to show that ∂nr/∂h < 0. 17 The derivatives are calculated with Maple and the program codes are available. Appendix A: Proofs This implies ∂n p ∂h = 2U + 3M −2(M + h)( ∂U ∂h ) 2(3h −2U)2 < 0 (15) (15) Equation (15) implies ∂U ∂h > 2U + 3M 2(M + h) (16) Equation (15) implies (16) 123 ERIEs (2014) 5:377–397 391 SERIEs (2014) 5:377–397 391 Taking the derivative of nr with respect to h yields Taking the derivative of nr with respect to h yields Taking the derivative of nr with respect to h yields ∂nr ∂h = −2(L + h)( ∂U ∂h ) + 2kU + 3L 2(2Uk −3h)2 > 0 (17) (17) Equation (17) implies ∂U ∂h < 2Uk + 3L 2k(L + h) (18) Equation (17) implies ∂U ∂h < 2Uk + 3L 2k(L + h) (18) (18) quations (16) and (18) together yield Equations (16) and (18) together yield Equations (16) and (18) together yield 2U + 3M 2(M + h) < 2Uk + 3L 2k(L + h) (19) (19) However, (11) implies that U > 6(k + 1)h/8k. Also, k > 1 implies (L −M) > (L −kM) . These two properties together result in However, (11) implies that U > 6(k + 1)h/8k. Also, k > 1 implies (L −M) > (L −kM) . These two properties together result in 2U + 3M 2(M + h) ≮2Uk + 3L 2k(L + h) (20) (20) which is a contradiction. Equating ∂nr/∂h to zero yields a similar contradiction. Hence, the sign of the derivative, ∂nr/∂h, is negative. In the second part of the proof, I show that if h is higher than a critical level, the marriage market equilibrium is monogamous. Equation (10) together with Lemma 2 imply the following two conditions. Ir 2 > 2Uk −Iw −2h (21) Ip 2 ≤2U −Iw −2h (22) (21) (22) Multiplying each side of (22) with k and combining it with (21) yields L + h 2 + h + 2h > 2Uk ≥ M + h 2 + h + 2h k (23) (23) Hence, in the polygynous equilibrium, in addition to (10), (23) should hold. Otherwise, nr cannot be larger than 1. Now, rearranging (23) yields L −kM 7 (k −1) > h (24) (24) Consequently, if h ≥(L −kM)/7(k −1), the marriage market equilibrium cannot be polygynous. This proves the proposition. ⊓⊔ Consequently, if h ≥(L −kM)/7(k −1), the marriage market equilibrium cannot be polygynous. This proves the proposition. Appendix A: Proofs After replacing Iw with its equivalent h, Ir with L + h and Ip with M + h, nr can be written as follows: nr = L + h 4Uk −6h (13) (13) Similarly, the number of wives of a poor man in the polygynous equilibrium is equal to Similarly, the number of wives of a poor man in the polygynous equilibrium is equal to to n p = M + h 4U −6h (14) (14) The exact expression of the derivative, ∂nr/∂h, is quite complex and it is hard to determine the sign of it. Thus, I prove that ∂nr/∂h < 0 with a proof by contradiction. The exact expression of the derivative, ∂nr/∂h, is quite complex and it is hard to determine the sign of it. Thus, I prove that ∂nr/∂h < 0 with a proof by contradiction. First, notice that ∂nr/∂h and ∂n p/∂h have opposite signs because the proportions of rich and poor men and the sum of n and n is constant Now for a contradiction The exact expression of the derivative, ∂nr/∂h, is quite complex and it is hard to determine the sign of it. Thus, I prove that ∂nr/∂h < 0 with a proof by contradiction. First, notice that ∂nr/∂h and ∂n p/∂h have opposite signs because the proportions of rich and poor men and the sum of nr and n p is constant. Now, for a contradiction, assume that ∂nr/∂h > 0. Ir nr = Ir (k+1)4h+kθ(M+h)+(1−θ)(L+h)+ √ D 2 −4h (25) (25) where D ≡[(1 −θ)Ir −kθ Ip −(k −1)(4h)]2 + 4Irkθ Ip(1 −θ). where D ≡[(1 −θ)Ir −kθ Ip −(k −1)(4h)]2 + 4Irkθ Ip(1 −θ). Taking the derivative of nr with respect to h yields ∂nr/∂h < 0. This shows that the degree of polygyny falls with the increase of h even if female labor force participation is assumed to be zero. Similarly, equating Iw to zero in (21) and (22) produces a necessary condition for the existence of the polygynous equilibrium when none of the females participates in the labor force. L + h 2 + 2h > 2Uk ≥ M + h 2 + 2h k (26) (26) Rearranging (26) yields L −kM 5 (k −1) > h (27) (27) Equation (27) shows that in the case of zero female labor force participation, if h ≥ (L −kM)/5(k −1), the marriage market equilibrium is monogamous. Hence, for all ε ∈[0, 1], there exists h∗∈ℜ+ such that if h ≥h∗, polygynous mating is nonexistent in equilibrium. This completes the proof. Equation (27) shows that in the case of zero female labor force participation, if h ≥ (L −kM)/5(k −1), the marriage market equilibrium is monogamous. Hence, for all ε ∈[0, 1], there exists h∗∈ℜ+ such that if h ≥h∗, polygynous mating is nonexistent in equilibrium. This completes the proof. Appendix A: Proofs ⊓⊔ Proof of Proposition 3 In the previous proposition I establish that in the case with full female labor force participation (ε = 1), if h is sufﬁciently high, polygynous mating is 12 3 392 SERIEs (2014) 5:377–397 nonexistent in equilibrium. Now, let the female labor force participation rate be zero. This corresponds to equating Iw to zero in (13) and yields nonexistent in equilibrium. Now, let the female labor force participation rate be zero. This corresponds to equating Iw to zero in (13) and yields Appendix B: Spouses’ shared time In this section, I argue that keeping the difference constant between utility derived from the amount of marital interaction in monogamous mating and in polygynous mating is harmless. Speciﬁcally, I show that the second channel is still at work and the main result holds in a model where a woman’s and a man’s utility is a logarithmic function of time spent together with the mate and men divide their limited time equally among their wives. The new utility function of a woman takes the following form: ln cw + ln 2h + bw + bm + T n (28) (28) where T ∈ℜ+ denotes the amount of a man’s time endowment and n denotes the number of women in the household. Similarly, the new utility function of a man takes the following form: ln cm + ln n 2h + bw + bm + T n (29) (29) 123 12 123 SERIEs (2014) 5:377–397 393 Other speciﬁcations of the main model remain the same. Substituting (3) into (28) and deriving the ﬁrst-order condition with respect to bw yields bw = Iw + y −2h −bm −T n 2 (30) (30) A man can marry a woman if he provides her with the equilibrium utility level deter- mined in the competitive marriage market. This implies the following constraint: A man can marry a woman if he provides her with the equilibrium utility level deter- mined in the competitive marriage market. This implies the following constraint: ln cw + ln 2h + bw + bm + T n ≥uw (31) (31) Substituting (3) and (30) into (31) yields Substituting (3) and (30) into (31) yields Substituting (3) and (30) into (31) yields Substituting (3) and (30) into (31) yields 2 ln Iw + y + 2h + bm + T n 2 ≥uw (32) (32) Men’s optimization requires that (32) holds with equality. After arranging, this implies: y + bm = 2 exp(uw 2 ) −Iw −2h −T n (33) (33) Equation (33) indicates that the sum of the income transfer to each woman and bequest to her son increases with the number of women in the household. Hence, in polygynous mating, the sum of the income transfer to each woman and the bequest to her son is higher than the sum of that in a monogamous marriage. Appendix B: Spouses’ shared time After substituting (4), (30) and (33) into (29), the men’s maximization problem boils down to max n ln Im −n 2 exp(uw 2 )−Iw−2h−T n +ln exp(uw 2 )n s.t. bm, y ≥0 (34) (34) Deriving the ﬁrst-order condition with respect to n produces the following conditions after rearranging: n = Im + T 2 2 exp( uw 2 ) −Iw −2h This yields the following equations: This yields the following equations: Ir + T 2 2 exp( uw 2 ) −Iw −2h (35) Ip + T 2 2 exp( uw 2 ) −Iw −2h (36) (35) (36) 394 SERIEs (2014) 5:377–397 The result of the characterization of n together with the assumption Ir > Ip is that, in the polygynous equilibrium, a rich man has more wives than a poor man. The result of the characterization of n together with the assumption Ir > Ip is that, in the polygynous equilibrium, a rich man has more wives than a poor man. p ygy q p Substituting (35) and (36) into the market clearing condition yields: θ Ip + T 2 2 exp( uw 2 ) −Iw −2h + (1 −θ) Ir + T 2 2 exp( uw 2 ) −Iw −2h = 1 (37) (37) The market clearing condition implies that exp( uw 2 ) is equal to exp(uw 2 ) = θ Ip + (1 −θ)Ir + T + 2Iw + 4h 4 (38) (38) After replacing Iw with its equivalent h, Ir with L +h, Ip with M +h and substituting (38) into the (35), the equation that characterizes nr can be written as follows: After replacing Iw with its equivalent h, Ir with L +h, Ip with M +h and substituting (38) into the (35), the equation that characterizes nr can be written as follows: nr = L + h + T θ M + (1 −θ)L + h + T Taking the derivative of nr with respect to h results in ∂nr/∂h < 0. This indicates that the degree of polygyny declines with the increase in labor income, h. Taking the derivative of nr with respect to h results in ∂nr/∂h < 0. This indicates that the degree of polygyny declines with the increase in labor income, h. 18 Primogeniture is the custom that the eldest son receives nearly all of total bequest left. See Bergstrom (1994a), DeLong (2003) and Bertocchi (2006) for an extensive discussion of the issue. Appendix C: Dynamic extension However, the pace of the transition depends on the structure of wealth transmission and speciﬁcation of the utility functions. Next, I show that the endogenous income inequality is preserved in both polygynous and monogamous equilibriums. First, in the polygynous equilibrium, (40) and (41) imply that income inequality among males at time t is equal to U t−1(k −1)/(U t−1 − 2ht−1 + ht) and it is greater than zero. In the monogamous equilibrium, the solution of the women’s problem results in bt−1 w = ht−1 + yt−1 −2ht −bt−1 m 2 (43) (43) and given n = 1, the solution of the men’s problem implies yt−1 + bt−1 m = I t−1 m −ht−1 −2ht 2 (44) (44) Substituting (43) into (44) yields Substituting (43) into (44) yields Substituting (43) into (44) yields bt−1 w + bt−1 m = ht−1 −6ht + I t−1 m 4 (45) (45) This shows that if the equilibrium is monogamous, the inequality in period t is equal to I t−1 r −I t−1 p / 4I t p and, once again, it is greater than zero. Hence, I establish that the endogenously determined income inequality is preserved both in polygynous and monogamous equilibriums. The simple model that I present above does not provide a good representation of the distribution and intergenerational transmission of wealth. One needs a more sophisticated model to do this. One possibility is to allow for bequest inequalities among sons, i.e., modeling primogeniture.18 However, my analysis is on marriage types and not on the transmission of wealth. Also, this simple model still shows that inferences of the static model hold in the dynamic extension. Hence, in order to preserve simplicity, I intentionally kept the model in this simple form. Appendix C: Dynamic extension In this section, I present a dynamic extension for the model. I show that my results carry through to a dynamic overlapping generations model with an inﬁnite horizon. Let superscript t denote period t and L0 and M0, where L0 > M0, denote the assets of initial rich and poor, respectively. I assume that h increases with time, i.e. ht+1 > ht. At time t, a woman’s income is equal to ht and a man’s income is equal to the sum of the human capital return in that period, ht and the bequests that he receives from his parents, bt−1 w and bt−1 m , where bt−1 m ∈{bt−1 r , bt−1 p }. p First, notice that the results stated in Lemma 1 and 2 and Proposition 1 are time invariant. Thus, the results are still valid. Moreover, the following equation, which is the counterpart of (24), shows the necessary condition for the existence of the polygynous equilibrium in the dynamic model. Lt−1 −kMt−1 7 (k −1) > ht (39) (39) Substituting (5) into (7) and (8) implies that in the polygynous equilibrium, total bequests received by rich and poor men at time t are equal to Substituting (5) into (7) and (8) implies that in the polygynous equilibrium, total bequests received by rich and poor men at time t are equal to bt−1 w + bt−1 m = U t−1k −2ht−1 if nt−1 > 1 (40) bt−1 w + bt−1 m = U t−1 −2ht−1 if nt−1 ≤1 (41) (40) ( ) (41) (41) 123 123 12 SERIEs (2014) 5:377–397 395 Multiplying (41) with k and subtracting the outcome from (40) yields Lt−1 −kMt−1 = 2(k −1)ht−1 (42) (42) Equation (42) implies that at time t, the necessary condition for the polygynous equilib- rium, (39), is not satisﬁed and as a result, polygynous mating disappears. I should note that the transition to the monogamous equilibrium is rapid. After the initial period, polygynous mating disappears. However, the pace of the transition depends on the structure of wealth transmission and speciﬁcation of the utility functions. Equation (42) implies that at time t, the necessary condition for the polygynous equilib- rium, (39), is not satisﬁed and as a result, polygynous mating disappears. I should note that the transition to the monogamous equilibrium is rapid. After the initial period, polygynous mating disappears. Adams B, Mburugu E (1994) Kikuyu bridewealth and polygyny today. J Comp Fam Stud 25(2):159–166 Adams B, Mburugu E (1994) Kikuyu bridewealth and polygyny today. J Comp Fam Stud 25(2):159–166 18 Adams B, Mburugu E (1994) Kikuyu bridewealth and polygyny today. J Comp Fam Stud 25(2):159–166 18 Primogeniture is the custom that the eldest son receives nearly all of total bequest left. See Bergstrom (1994a), DeLong (2003) and Bertocchi (2006) for an extensive discussion of the issue. 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https://openalex.org/W1978268933	https://europepmc.org/articles/pmc2100170?pdf=render	English	null	Geometric Triangular Chiral Hexagon Crystal-Like Complexes Organization in Pathological Tissues Biological Collision Order	PloS one	2,007	cc-by	7,046	INTRODUCTION A methodological analysis of 3000 images has allowed the authors to discover that the complexes are not uncommon or random findings on the contrary they appear as self –assembled structures that fit cohesively inside a morphological pattern, which assembles in space-time intervals, and consequently the authors believe they have a biological meaning . The present study describes and documents self-assembly of geometric triangular chiral hexagon crystal like complexes organizations (GTCHC) in human pathological tissues. This architectural geometric expression has been found at macroscopic and microscopic levels mainly in cancer processes. In the literature, geometric triangular tissue morphological patterns have been documented at different levels: By analyzing these complexes, repetitive morphological patterns that are inherent objectively to the configuration of each formation, and that belong to the field of physics are identified. The authors mention concepts like polarization, chirality, helicoidal pattern and geometric expression as a convergent characteristic of the images. Moreover, the authors in this study At microscopic level: Triangular morphology of neoplastic cells in vitro was seen in rat cell sarcoma culture lines in relation with malignancy. [1] At macroscopic level: Videoscopic triangular pattern images from the viewpoint of three-dimensional configuration of isolated crypts constitute an important factor in the gross assembly of colorectal tumours, [2] principally with a type IIIL. [3] Academic Editor: Claus-Werner Franzke, University of Freiburg, Germany Received December 9, 2006; Accepted November 9, 2007; Published December 12, 2007 Triangular microcalcifications configuration [4] and stellate mammography images (triangular aggregation) are closely linked to malignancy [5]. Copyright: 2007 Diaz 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. Despite these observations there is not corresponding references in human structural pathology about geometric self- assembly of triangular chiral hexagon complex organizations in pathologic tissues principally in malignant lessions, and it is not unknown how these images and their biological meaning are configurate. In this study the hexagonal morphology is evoked not from a simple six sides geometrical structure, but based on our images the authors consider it like a perfect organizational structure formatted from three organized triangular units with their respective mirror images . Geometric Triangular Chiral Hexagon Crystal-Like Complexes Organization in Pathological Tissues Biological Collision Order Jairo A. Dı´az, Natalia A. Jaramillo, Mauricio F. Murillo Laboratory of Pathology, Department of Pathology, Clinic Health Social Entity Policarpa Salavarrieta, University Cooperativa of Colombia, Medicine School, Villavicencio, Meta, Colombia The present study describes and documents self-assembly of geometric triangular chiral hexagon crystal like complex organizations (GTCHC) in human pathological tissues.The authors have found this architectural geometric expression at macroscopic and microscopic levels mainly in cancer processes. This study is based essentially on macroscopic and histopathologic analyses of 3000 surgical specimens: 2600 inflammatory lesions and 400 malignant tumours. Geometric complexes identified photographically at macroscopic level were located in the gross surgical specimen, and these areas were carefully dissected. Samples were taken to carry out histologic analysis. Based on the hypothesis of a collision genesis mechanism and because it is difficult to carry out an appropriate methodological observation in biological systems, the authors designed a model base on other dynamic systems to obtain indirect information in which a strong white flash wave light discharge, generated by an electronic device, hits over the lines of electrical conductance structured in helicoidal pattern. In their experimental model, the authors were able to reproduce and to predict polarity, chirality, helicoid geometry, triangular and hexagonal clusters through electromagnetic sequential collisions. They determined that similar events among constituents of extracelular matrix which drive and produce piezoelectric activity are responsible for the genesis of GTCHC complexes in pathological tissues. This research suggests that molecular crystals represented by triangular chiral hexagons derived from a collision-attraction event against collagen type I fibrils emerge at microscopic and macroscopic scales presenting a lateral assembly of each side of hypertrophy helicoid fibers, that represent energy flow in cooperative hierarchically chiral electromagnetic interaction in pathological tissues and arises as a geometry of the equilibrium in perturbed biological systems. Further interdisciplinary studies must be carried out to reproduce, manipulate and amplify their activity and probably use them as a base to develop new therapeutic strategies in cancer. Citation: Dı´az JA, Jaramillo NA, Murillo MF (2007) Geometric Triangular Chiral Hexagon Crystal-Like Complexes Organization in Pathological Tissues Biological Collision Order. PLoS ONE 2(12): e1282. doi:10.1371/journal.pone.0001282 itation: Dı´az JA, Jaramillo NA, Murillo MF (2007) Geometric Triangular Chiral Hexagon Crystal-Like Complexes Organiz iological Collision Order. PLoS ONE 2(12): e1282. doi:10.1371/journal.pone.0001282 PLoS ONE \| www.plosone.org INTRODUCTION (Figure 1A, 1B, 1C, 1D, 1F) Funding: This investigation was possible thanks to the logistical and economic support of the Medicine School, Pathology Department, Cooperative University of Colombia and the Clinical Carlos Hugo Estrada, State social company Policarpa Salavarrieta, Villavicencio, Colombia. Competing Interests: The authors have declared that no competing interests exist. To whom correspondence should be addressed. E-mail: jaditod@hotmail.com * To whom correspondence should be addressed. E-mail: jaditod@hotmail.com December 2007 \| Issue 12 \| e1282 1 December 2007 \| Issue 12 \| e1282 PLoS ONE \| www.plosone.org Geometric Biological Order Geometric Biological Order Figure 1. Geometric Triangular chiral hexagon cristal like complexes. Panel A. GTCHC complexes found in malignant peritoneal effusion, observe the helical pattern associate. Panel B. Breast malignant cells hexagonal clusters. Panel C. Colon polypoid juvenile tumor. Icosahedron pattern structuring by hexagon geometric stuctucture. Panel D. Macroscopic GTCHC complex in leiomyosarcoma. Panel E. Smear show triangular mirror images of atipical endocervix cells, that biopsy demostrated in situ adenocarcinoma. Panel F. Macroscopic GTCHC complex in lung cancer. doi:10.1371/journal.pone.0001282.g001 Figure 1. Geometric Triangular chiral hexagon cristal like complexes. Panel A. GTCHC complexes found in malignant peritoneal effusion, observe the helical pattern associate. Panel B. Breast malignant cells hexagonal clusters. Panel C. Colon polypoid juvenile tumor. Icosahedron pattern structuring by hexagon geometric stuctucture. Panel D. Macroscopic GTCHC complex in leiomyosarcoma. Panel E. Smear show triangular mirror images of atipical endocervix cells, that biopsy demostrated in situ adenocarcinoma. Panel F. Macroscopic GTCHC complex in lung cancer. doi:10.1371/journal.pone.0001282.g001 propose that the complex GTCHC identified in pathological tissues emerges from sequential collision processes that generate electromagnetic fields. Polarized photon collision of elementary particles like fermion produces chirality [6]. pleural effusions of malignant tumors; when a pattern was detected, it was also documented. For histological observation, all the materials were stained with hematoxylin and eosin routine and Tricromico of Masson coloration. Malignant smears were stained with papanicolaou. A) Observational support The present research is based essentially on macroscopic and histopathologic analysis of 3000 surgical specimens: 2600 inflammatory lesions and 400 malignant tumours taken from the authors’ patients received at the Public Health Clinic Carlos Hugo Estrada Pathology Service Laboratory in Villavicencio Meta during the last three years. It is necessary to consider that GTCHC complexes do not emerge alone; rather, it is necessary to look for them. The search of the authors is similar to that of an archeologist, it is necessary to look carefully amid necrosis, hemorrhages and cystic changes of the tissue to conserve the original structure. When areas were cleaned a documentation process was developed by a photo- graphic session, and it was here that the expression of an impressive geometric order emerged. For the first time, the identification of GTCHC configurations was made on the center of a giant tumor of retroperitoneal localization (Figure 1D) under the premise of determining whether this strange formation was a casual discovery or if it represented a real biological fact. The authors decided to ‘‘look for this structures’’ in different pathological entities, to do so interesting specimens, were photographed, the pictures from different rotationary angles were analyzed, and the images were amplified two to three times. PLoS ONE \| www.plosone.org MATERIALS AND METHODS Geometric complexes were considered valid if they presented volumetric assembling, if it was possible to find patterns at different levels of the surgical specimen, or if they appeared in more than four histology sections. Observational results propagates through a field; chirality, magnetism, and light are intimately correlated [7]. The authors detected GTCHC complexes at macroscopic and microscopic levels in human pathologic tissues. Under the hypothesis of a collision mechanism, the authors designed a model in which a strong white flash wave light discharge, generated by an electronic device, hits over lines of electrical conductance structured in helicoidal patterns, generating an electromagnetic field. From 2600 random inflammatory–immunologic processes ana- lyzed, the authors identified images of triangular chiral counterpart tissue configuration in 312 cases (12%). From 400 cancer processes analyzed, geometric complexes were identified in 216 cases (54%). These percentages show identification of highly ordered structures in more than 50% of the analyzed malignant tumor tissues statistically significant (P= 0.00001) (Table 1). The organizations were observed with more frequency under tumor conditions (Figures 1–8) however also under benign condition (Figures 7D,7E ,7F, ). Sarcoma lesions of greater size were those that evidence more clear complex GTCHC (Figures 1D, 2B, 2F, 3A, 3B, 4D). The objective of this experiment was to determine if the authors were able to reproduce polarization and chirality patterns, or triangular hexagonal geometrical configurations as a result of collision of two interdependent fields: in this case, light and electromagnetism. For such effect, the authors use an electronic flash device attached to a Sony camera model DSC-S600. Strong discharges of light were made over electric conduction lines (150 volts) of helical pattern in time intervals of 3 to 4 minutes during cicles of 60 minutes from 3 to 4 m distance in an atmospheric environment The histopathologic analysis of the tissue at the interior of the complex showed accented cellular decrease, helicoid hypertrophy of collagen type I fibers, the presence of mirror images in lateral Figure 2. Geometric Triangular chiral hexagon cristal like complexes assembled from helicoid pattern. Panel A. Microscopic GTCHC complex in prostate adenocarcinoma. Panel B. Macroscopic GTCHC complex in liposarcoma. Panel C. Microscopic GTCHC complex in lipoma .Helicoid border. Panel D. Macroscopic GTCHC complex in breast cancer. Observe how the straight lines are assembled from helicoid border pattern. Panel E. Full view of GTCHC complex, this is the same as panel D after demarcating and cleaning the area. It is possible equally to identify ‘‘hole’’ microcavities in their surface. Panel F. Macroscopic GTCHC complex in filloid breast tumor with helicoid border pattern. (B) Experimental design It is difficult to carry out an appropriate methodological observation for collision processes when studing biological systems. But one can obtain indirect information through models in other dynamic systems. What the authors were looking for mainly was to determine if one can reproduce and predict components of polarity, chirality, helicoid patterns, and geometric expressions through electromagnetic sequential collisions. Geometric complexes identified photographically at the mac- roscopic level were located in the gross surgical specimen; these areas were carefully dissected and samples were taken from the border lines, inside and outside of the GTCHC complexes, to carry out histological analysis and make a comparison with the macroscopic findings. The authors also worked with peritoneal, Collision event Light and magnetism are reflections of the same substance. Light is an electromagnetic disturbance that December 2007 \| Issue 12 \| e1282 2 Geometric Biological Order Table 1. GTCHC complexes distribution in pathological tissues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cases No GTCHC % Inflammatory Benign conditions 2600 312 12 Cancer 400 216 54 doi:10.1371/journal.pone.0001282.t001 . . . . . . . . . . . . . . . . . . . . . . . . . . . Table 1. GTCHC complexes distribution in pathological tissues. . . . . . . . and a low temperature of 4uC . To avoid lens flare, the experiment was performed in complete darkness. There were photographic sessions of 1 h during a 9-day time interval. Statistical analysis References between the two groups inflammatory benign and tumors malignant conditions where TGCHC complexes appear were determined using the Student test. Statistical analysis References between the two groups inflammatory benign and tumors malignant conditions where TGCHC complexes appear were determined using the Student test. Observational results We appreciate the initial formation of hole microcavities concluding in changes of cystic degeneration at the interior of the geometric complex. doi:10.1371/journal.pone.0001282.g002 Figure 2. Geometric Triangular chiral hexagon cristal like complexes assembled from helicoid pattern. Panel A. Microscopic GTCHC complex in prostate adenocarcinoma. Panel B. Macroscopic GTCHC complex in liposarcoma. Panel C. Microscopic GTCHC complex in lipoma .Helicoid border. Panel D. Macroscopic GTCHC complex in breast cancer. Observe how the straight lines are assembled from helicoid border pattern. Panel E. Full view of GTCHC complex, this is the same as panel D after demarcating and cleaning the area. It is possible equally to identify ‘‘hole’’ microcavities in their surface. Panel F. Macroscopic GTCHC complex in filloid breast tumor with helicoid border pattern. We appreciate the initial formation of hole microcavities concluding in changes of cystic degeneration at the interior of the geometric complex. doi:10.1371/journal.pone.0001282.g002 December 2007 \| Issue 12 \| e1282 PLoS ONE \| www.plosone.org 3 Geometric Biological Order Figure 3. Geometric Triangular chira lhexagon cristal like complexes. Panel A. Macroscopic GTCHC complex in fibrosarcoma. Panel B. Macroscopic GTCHC complex in liposarcoma that induces surprisingly clear tissue mirror images around it: ABC––A’B’C’. Panel C. Microscopic GTCHC complex in basal cell carcinoma that induces surprisingly close tissue mirror images around it. ABCDE––A’B’C’D’E’. Panel D. Microscopic GTCHC in malignant peritoneal effusion. Panel E. Macroscopic GTCHC complex in malignant solid ovary tumor. Panel F. Microscopic fractional crystal hexagon from a cluster of malignant cells in peritoneal malignant effusion. doi:10.1371/journal.pone.0001282.g003 Figure 3. Geometric Triangular chira lhexagon cristal like complexes. Panel A. Macroscopic GTCHC complex in fibrosarcoma. Panel B. Macroscopic GTCHC complex in liposarcoma that induces surprisingly clear tissue mirror images around it: ABC––A’B’C’. Panel C. Microscopic GTCHC complex in basal cell carcinoma that induces surprisingly close tissue mirror images around it. ABCDE––A’B’C’D’E’. Panel D. Microscopic GTCHC in malignant peritoneal effusion. Panel E. Macroscopic GTCHC complex in malignant solid ovary tumor. Panel F. Microscopic fractional crystal hexagon from a cluster of malignant cells in peritoneal malignant effusion. doi:10.1371/journal.pone.0001282.g003 position, and apoptosis changes (Figures 6E,7A, 7B ). Observational results The lineal borders of the macroscopic complexes GTCHC also showed a pattern of helical hypertrophy in most of the cases (Figure 1D, 2C, 2F, 3E, 7C, 7F), additionally authors were able to discern two diferent morphological subtrates, dark condensation, or authofagic vacuoles depending on magnetic spiral sub patterns direction of the tringular complex and their counterpart (Figure 7C, 8 A, 8B, 8C, 8D, 8E) providing the fundamental proof that complexes are real. High cellular activity and frequent mitosis were the typical characteristics of malignancy outside of the complex GTCHC (Figure 6F) and were shown to have a completely opposite biological behavior. Cytological analysis of malignant peritoneal or pleural effusions evidenced the presence of crystals with triangular or hexagonal geometric composition (Figures 1A, 3F, 5A, 5B, 5E, 6A). These crystals are visible clearly in smears but difficult to observe in the cellular blocks of paraffin. From this the authors deduce that probably formaldehyde or the high temperature of the paraffin in the moment of inclusion of the material makes the crystals in histologic sections less evident. process, in the interaction region result ejected particles of wave light that splitting into two components that take opposite directions in helicoid flow pattern with polarization and mirror images. The luminous energy trajectory is not in a straight line, but follows a helical pattern (Figure 9B). Triangular and hexagonal light patterns arises on interleaving of 15–20 sub patterns of indefinite light clusters and defined left handed and right handed spirals ( Figures 10D,10E), lateral to each side of the conduction lines, with ‘‘hole’’ microcavities in their surface (Figure 9C, 9D, 9E, 9F, 10A, 10B, 10C) within a space time intervals sequence discharges of 1 hour cicles. Images moved toward the boundaries of the perturbed system, diminishing their luminous intensity and appear distant. In this process, the authors observe a twisted of 360 degrees in invested position one from another (Figure 10 F). DISCUSSION Pathological tissues are an excellent means of analyzing chaos states and molecular disorder, and identifying order expression through geometry and mirror images in the core of this biological environment, it is a unique fact, and it is to be located in the chaos–order interface. All systems in which their particles are in continuous dynamic movement submit to laws of physics. This means that during the period space–time interval of life of any system, there exists the absolute potential to enter into a collision state at a given moment. PLoS ONE \| www.plosone.org Experimental result Collision event In the experimental model, the collision of a strong flash from a white light against an electromagnetic field of electronic conduction lines produced the following morpho- dynamics sequential images. This logical premise is objective and real when one analyzes the structural pathological morphology from simple inflammatory At the initial momentum of collision, the authors were able to appreciate a great white brightness (Figure 9A). In a dynamic PLoS ONE \| www.plosone.org December 2007 \| Issue 12 \| e1282 4 Geometric Biological Order Figure 4. Geometric Triangular chiral hexagon cristal like complexes. Panel A. Hexagon pattern of breast cancer. Panel B. Breast cancer tissue surface cut hexagonal pattern. Panel C. Malignant breast cells trapped by a network of hexagons. Panel D. Hexagon pattern of fibrosarcoma nodule. Panel E. GTCHC complexes obtained from peritoneal malignant effusion. Panel F. Microscopic hexagonal pattern .Papilar thyroid tumor doi:10.1371/journal.pone.0001282.g004 Figure 4. Geometric Triangular chiral hexagon cristal like complexes. Panel A. Hexagon pattern of breast cancer. Panel B. Breast cancer tissue surface cut hexagonal pattern. Panel C. Malignant breast cells trapped by a network of hexagons. Panel D. Hexagon pattern of fibrosarcoma nodule. Panel E. GTCHC complexes obtained from peritoneal malignant effusion. Panel F. Microscopic hexagonal pattern .Papilar thyroid tumor doi:10.1371/journal.pone.0001282.g004 complex associate directly with the helicoid pattern of the lateral borders. The authors believe that this represents the flow of energy through the fiber (Figure 7C). processes such as the edema, the vasodilatation, and the migration of leukocytes, to malign proliferative processes where masses are generated and increase volumes, disorganizing extracellular matrix at extreme. There are many examples of such orientation induction on elongated supra/macromolecular structures in the literature using electric fields [11] or magnetic fields [12]. Chirality seems to be intimately associated with the growth and the stability of self- assembled fibrillar networks, and additionally molecular chirality is expressed in a scale of nanometers or micrometers, and helps twisted or coiled structures. There exists a specific link between helicoid patterns and chirality [13]. Collagen type I is the main protein of the extracellular matrix and has a crystal structure [8], and this characteristic allows collagen-type fibrils to arrange into a parallel order by regulatory functions of fibroblast cells, being this way in charge of the dielectric properties of natural tissues. The piezoelectric effect is a phenomenon resulting from a coupling between the electrical and the mechanical properties of a material. PLoS ONE \| www.plosone.org Experimental result When mechanical stress is applied to a piezoelectric material, electric potential will be produced. Likewise, when an electric potential is applied to the material, a mechanical change will occur [9]. Piezoelectric crystals involve a nonuniform charge distribution within the cell unit of the crystal. When exposed to an electric field, this charge distribution shifts and the crystal changes its shape. The same polarization mechanism can cause a voltage to develop across the crystal in response to mechanical force; in pathological states of great disorder like cancer tissues, the extracellular matrix and most of the constituents of the system experience an enormous interaction impact that is expressed in piezoelectric activity. The collagen type I has a molecular structure based on quasihexagons [10]. In their experimental model, the authors were able to reproduce and predict polarity, chirality, helicoid geometry, triangular and hexagonal clusters, starting from the impact of a wave of light on an electromagnetic field generated from an electric conduction line (Figures 9 and 10). A similar event of collision this time among constituents of extracellular matrix that drives and produces piezoelectric activity are responsible for the genesis of GTCHC complexes in pathological tissues. In a collision state, electromagnetic field generates electrons, and ions split-up and take opposite trajectories and travel to the boundaries of the system where the particles twisted 360u one of another, and appear as mirror images. In physics this is determined as chirality, and chirality is a property of most of the biomolecules. This means the biomolecules are able to produce mirror images. This is one of the vital characteristics that are seen in pathological tissues, polarization from its side induces chirality, and polarization collision of elementary particles like fermion produces chirality [6]. Polarization formation in organisms shows Based on the observed images, the authors believe that stimulated collagen type I fiber at the moment of collision results in hypertrophy of helicoid patterns and their molecular crystal components are fractioned, being positioned to each side of the bundle fibril axis generating polarization and chirality (Figure 1A, 3D 3F, 4E, 4F, 6A, 6D, 7A, 7B). Straight lines of GTCHC PLoS ONE \| www.plosone.org December 2007 \| Issue 12 \| e1282 5 Geometric Biological Order Figure 5. Morphodynamic sequence of malignant cells trapped at the interior of hexagonal cristal structure. GTCHC complex. Panel A–B. Malignant cells trapped inside crystal hexagons. Observe authofagic vacuolization. Panel C. Experimental result Disintegrated malignant cells inside hexagons in sweat carcinoma with apoptosis changes. Panel D–E–F. Disintegrated malignant cells trapped inside crystal hexagons doi:10.1371/journal.pone.0001282.g005 Figure 5. Morphodynamic sequence of malignant cells trapped at the interior of hexagonal cristal structure. GTCHC complex. Panel A–B. Malignant cells trapped inside crystal hexagons. Observe authofagic vacuolization. Panel C. Disintegrated malignant cells inside hexagons in sweat carcinoma with apoptosis changes. Panel D–E–F. Disintegrated malignant cells trapped inside crystal hexagons doi:10.1371/journal.pone.0001282.g005 similarity to an effect reported in crystals [14]. These data are coherent with the morphological findings. An experimental design was realized at low-temperature atmospheric conditions in this environment with water molecules of oxygen and hydrogen structured as ice crystals in hexagonal shapes. In this medium, when light dielectric discharges are in collision with this type of crystals, refraction deviation appears and generates clear hexag- onal geometry (figure 9C, 9D). Spatio temporal patterns in dielectric discharge in air at atmospheric pressure have shown a rich variety, including traveling hexagon, traveling square, quasicrystals static hexagon, and stripe geometry [15]. By this way it is proved that polarization, chirality, helicoid patterns, and geometric expression can be in intimate relation with collision events and the electromagnetic field that it generates. The laws of the physics are amazingly universal as they serve from common denominators to dissimilar systems. And when geometric images obtained in the experimental pattern of collision are compared, they are amazingly similar to the complex GTCHC identified in pathological tissues, clearly in the point of collision of two systems geometry of equilibrium is generated. similarity to an effect reported in crystals [14]. These data are coherent with the morphological findings. An experimental design was realized at low-temperature atmospheric conditions in this environment with water molecules of oxygen and hydrogen structured as ice crystals in hexagonal shapes. In this medium, when light dielectric discharges are in collision with this type of crystals, refraction deviation appears and generates clear hexag- onal geometry (figure 9C, 9D). Spatio temporal patterns in dielectric discharge in air at atmospheric pressure have shown a rich variety, including traveling hexagon, traveling square, quasicrystals static hexagon, and stripe geometry [15]. By this way it is proved that polarization, chirality, helicoid patterns, and geometric expression can be in intimate relation with collision events and the electromagnetic field that it generates. The laws of the physics are amazingly universal as they serve from common denominators to dissimilar systems. PLoS ONE \| www.plosone.org Experimental result And when geometric images obtained in the experimental pattern of collision are compared, they are amazingly similar to the complex GTCHC identified in pathological tissues, clearly in the point of collision of two systems geometry of equilibrium is generated. successive and sequential impacts inside a density of particles in movement similar to the angular trajectory of a billiard ball that rotates freely in a flat surface and collides with obstacles. The authors consider definitively that complex GTCHC is a mechanism of structural reorganization in perturbed biological systems modulated through geometry and represent orderly spatio- temporal distribution of positive and negative electric discharges inside the system. The authors document macroscopic and microscopic images that demonstrate how clear mirror images form in proximity to the complex GTCHC, and it seems to originate from the geometrical basis of this complex (Figure 3B, 3C, 8A) the same one that indicates that these structures would have magnetic properties, links between magnetism, and chirality exist [7]. Inside their crystal structure, GTCHC complexes can trapped a virus (Figure 7D) an anomalous lipoprotein (Figure 7E), or a malignant cell, and they disintegrate it. In malignant lesions, this morphodynamic sequence can tracked microscopically the cells are somehow caught in a complex (Figure 4C), or disintegrated at the interior of the crystal structure (Figure 5A, 5B, 5E, 5F, 6A, 6B). The complex GTCHC multiplies inside the perturbed system, similar to fullerene carbon structure (Figure 6B, 6C). The essential base of this geometry is the triangulation and the generation of mirror images that conclude in hexagons. Here hexagons are not a simple six-side structure; they are a complete state, net balance structured in the base of three triangular units, with their respective mirror images as a state of molecular order. The authors state that the crystal structure of water, liver tissue, alveolar walls, and the structuring of the fullerene particle of carbon is based on hexagonal geometric patterns that represent definitively a structural reorganization. Triangulation is then a consequence of How did the authors interpret these images? All biomolecules under normal conditions, that is to say in stable balance, have their respective mirror images; in biological disorder state this balance gets lost and their chiral counterpart disappears. This loss of mirror images imbalances the system to more chaos and bigger imbalance of structural polarity. This is what happens in malignant tumors that have lost polarity and have architectural How did the authors interpret these images? December 2007 \| Issue 12 \| e1282 6 Figure 6. Final evolution step of GTCHC complexes. Panel A–B–C–D. Final step of massive malignant cell disintegration by hexagonal crystal complexes that remember carbon fullerene structure. Panel E. Histopa- thology findings inside macroscopic GTCHC: Low cellular population. Apo- ptosis, helicoidal hypertrophy of indi- vidual collagen type I bundles, and mirror images. Panel F. High cellular population, elevated mitosis typical characteristics of malignant behavior outside and near GTCHC complexes. doi:10.1371/journal.pone.0001282.g006 Figure 7. Collagen type I helicoid hypertrophy in GTCHC complexes. Panel A–B. Microscopic helicoid hyper- trophy of individual collagen type I bundle, observed in malignant perito- neal effusion. Panel C. Macroscopic Triangular complex organization ob- served in breast cancer associate with helicoid hypertrophy of individual collagen type I bundle and spiral sub patterns. Panel D. GTCHC complex in benign lesion. Cells with herpes trapped inside triangular crystal struc- ture with their chiral counterpart. Panel E. GTCHC complex in atheroscle- rosis. Panel F. GTCHC complex in superficial mycosis doi:10.1371/journal.pone.0001282.g007 Geometric Biological Order PLoS ONE \| www.plosone.org 7 December 2007 \| Issue 12 \| e1282 Geometric Biological Order Figure 6. Final evolution step of GTCHC complexes. Panel A–B–C–D. Final step of massive malignant cell disintegration by hexagonal crystal complexes that remember carbon fullerene structure. Panel E. Histopa- thology findings inside macroscopic GTCHC: Low cellular population. Apo- ptosis, helicoidal hypertrophy of indi- vidual collagen type I bundles, and mirror images. Panel F. High cellular population, elevated mitosis typical characteristics of malignant behavior outside and near GTCHC complexes. doi:10.1371/journal.pone.0001282.g006 Figure 6. Final evolution step of GTCHC complexes. Panel A–B–C–D. Final step of massive malignant cell disintegration by hexagonal crystal complexes that remember carbon fullerene structure. Panel E. Histopa- thology findings inside macroscopic GTCHC: Low cellular population. Apo- ptosis, helicoidal hypertrophy of indi- vidual collagen type I bundles, and mirror images. Panel F. High cellular population, elevated mitosis typical characteristics of malignant behavior outside and near GTCHC complexes. doi:10.1371/journal.pone.0001282.g006 Figure GTCHC Final s disinte comple fulleren thology GTCHC ptosis, vidual mirror popula charact outside doi:10.1 Figure 6. Final evolution step of GTCHC complexes. Panel A–B–C–D. Final step of massive malignant cell disintegration by hexagonal crystal complexes that remember carbon fullerene structure. Panel E. Histopa- thology findings inside macroscopic GTCHC: Low cellular population. Apo- ptosis, helicoidal hypertrophy of indi- vidual collagen type I bundles, and mirror images. Panel F. How did the authors interpret these images? High cellular population, elevated mitosis typical characteristics of malignant behavior outside and near GTCHC complexes. doi:10.1371/journal.pone.0001282.g006 Figure 7. Collagen type I helicoid hypertrophy in GTCHC complexes. Panel A–B. Microscopic helicoid hyper- trophy of individual collagen type I bundle, observed in malignant perito- neal effusion. Panel C. Macroscopic Triangular complex organization ob- served in breast cancer associate with helicoid hypertrophy of individual collagen type I bundle and spiral sub patterns. Panel D. GTCHC complex in benign lesion. Cells with herpes trapped inside triangular crystal struc- ture with their chiral counterpart. Panel E. GTCHC complex in atheroscle- rosis. Panel F. GTCHC complex in superficial mycosis doi:10.1371/journal.pone.0001282.g007 PLoS ONE \| www.plosone.org 7 December 2007 \| Issue 12 \| e1282 Figure 7. Collagen type I helico hypertrophy in GTCHC complexe Panel A–B. Microscopic helicoid hyp trophy of individual collagen type bundle, observed in malignant perit neal effusion. Panel C. Macroscop Triangular complex organization o served in breast cancer associate w helicoid hypertrophy of individu collagen type I bundle and spiral s patterns. Panel D. GTCHC complex benign lesion. Cells with herp trapped inside triangular crystal stru ture with their chiral counterpa Panel E. GTCHC complex in atherosc rosis. Panel F. GTCHC complex superficial mycosis doi:10.1371/journal.pone.0001282.g0 7 December 2007 \| Issue 12 \| e12 Figure 7. Collagen type I helicoid hypertrophy in GTCHC complexes. Panel A–B. Microscopic helicoid hyper- trophy of individual collagen type I bundle, observed in malignant perito- neal effusion. Panel C. Macroscopic Triangular complex organization ob- served in breast cancer associate with helicoid hypertrophy of individual collagen type I bundle and spiral sub patterns. Panel D. GTCHC complex in benign lesion. Cells with herpes trapped inside triangular crystal struc- ture with their chiral counterpart. Panel E. GTCHC complex in atheroscle- rosis. Panel F. GTCHC complex in superficial mycosis d i 10 1371/j l 0001282 007 Figure 7. Collagen type I helicoid hypertrophy in GTCHC complexes. Panel A–B. Microscopic helicoid hyper- trophy of individual collagen type I bundle, observed in malignant perito- neal effusion. Panel C. Macroscopic Triangular complex organization ob- served in breast cancer associate with helicoid hypertrophy of individual collagen type I bundle and spiral sub patterns. Panel D. GTCHC complex in benign lesion. Cells with herpes trapped inside triangular crystal struc- ture with their chiral counterpart. Panel E. GTCHC complex in atheroscle- rosis. Panel F. How did the authors interpret these images? GTCHC complex in superficial mycosis d i 10 1371/j l 0001282 007 7 y doi:10.1371/journal.pone.0001282.g007 PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org 7 December 2007 \| Issue 12 \| e1282 Geometric Biological Order Figure 8. Spiral left handed , right handed sub patterns of GTCHC Complexes. Panel A. Microscopic view of breast ductal infiltrating adenocarcinoma. Observe how the images are plenty of mirror images and spiral sub patterns associated to GTCHC complex. Hypertrophic helicoid collagen fiber divides in defined left hand and right hand this specific zone of the tumor. Panel B. Smear from carcinomatosis abdominal ascitis .observe triangular mirror assembly of lymphocytes amazingly linked to spiral sub pattern of red Cells. Panel C. Smear from cervical displasia: Triangular mirror images with spiral sub pattern observe opposite dark cromatine condensation, white crystalization substrates of the geometries. Panel D. Prostate adenocarcinoma microscopic view. Opposite dark nuclear condensation, white authofagic vacuoles of triangular units. Panel E. Leiomiosarcoma macroscopic view. Nodules with spiral sub patterns. doi:10.1371/journal.pone.0001282.g008 Geometric Biological Order Figure 8. Spiral left handed , right handed sub patterns of GTCHC Complexes. Panel A. Microscopic view of breast ductal infiltrating adenocarcinoma. Observe how the images are plenty of mirror images and spiral sub patterns associated to GTCHC complex. Hypertrophic helicoid collagen fiber divides in defined left hand and right hand this specific zone of the tumor. Panel B. Smear from carcinomatosis abdominal ascitis .observe triangular mirror assembly of lymphocytes amazingly linked to spiral sub pattern of red Cells. Panel C. Smear from cervical displasia: Triangular mirror images with spiral sub pattern observe opposite dark cromatine condensation, white crystalization substrates of the geometries. Panel D. Prostate adenocarcinoma microscopic view. Opposite dark nuclear condensation, white authofagic vacuoles of triangular units. Panel E. Leiomiosarcoma macroscopic view. Nodules with spiral sub patterns. doi:10.1371/journal.pone.0001282.g008 Figure 9. Morphodynamics sequential images of electro-optical dynamic system. Initial Momentum. Panel A. It illustrates a radian brigth that result from the collision interaction between the flash light and electromagnetic field. Panel B. In the interaction region result ejected particles of wave light that splitting into two components that take opposite directions in helicoid flow pattern with polarization and mirror image. Panel C–D. Each opossite light particles clusters begin to express hexagonal geometric configuration and ‘‘hole’’ microcav- ities in their surface.Higher magnification. Panel E–F. Triangular–hexagonal light pattern assem- bly in mirror images. How did the authors interpret these images? doi:10.1371/journal.pone.0001282.g009 PLoS ONE \| www.plosone.org 8 December 2007 \| Issue 12 \| e1282 PLoS ONE \| www.plosone.org Figure 9. Morphodynamics sequential images of electro-optical dynamic system. Initial Momentum. Panel A. It illustrates a radian brigth that result from the collision interaction between the flash light and electromagnetic field. Panel B. In the interaction region result ejected particles of wave light that splitting into two components that take opposite directions in helicoid flow pattern with polarization and mirror image. Panel C–D. Each opossite light particles clusters begin to express hexagonal geometric configuration and ‘‘hole’’ microcav- ities in their surface.Higher magnification. Panel E–F. Triangular–hexagonal light pattern assem- bly in mirror images. 8 doi:10.1371/journal.pone.0001282.g009 December 2007 \| Issue 12 \| e1282 PLoS ONE \| www.plosone.org 8 8 Geometric Biological Order Figure 10. Morphodynamics sequential images of electro-optical dynamic system. Final Momentum. Panel A–B–C. Triangular light clusters experiences displacement inside the interaction region. Higher magnification. Panel D–E. Interleaving of sub patterns of indefinite light clusters and defined left handed and right handed spirals are structured in interaction area. Panel F. Finally triangular geometric expression twisted 360u at the boundaries of the system generatting mirror images (Chirality). doi:10.1371/journal.pone.0001282.g010 Figure 10. Morphodynamics sequential images of electro-optical dynamic system. Final Momentum. Panel A–B–C. Triangular light clusters experiences displacement inside the interaction region. Higher magnification. Panel D–E. Interleaving of sub patterns of indefinite light clusters and defined left handed and right handed spirals are structured in interaction area. Panel F. Finally triangular geometric expression twisted 360u at the boundaries of the system generatting mirror images (Chirality). doi:10.1371/journal.pone.0001282.g010 This research suggests that molecular crystals represented by triangular chiral hexagons derived from collision events against collagen type I fibrils emerge at microscopic and macroscopic scales in lateral assembly of each side of hypertrophy of helicoid fibers that represent flow energy in cooperative hierarchically chiral electro- magnetic interaction in pathological tissues, and arises as a geometry of the equilibrium in perturbed biological systems . disorder. GTCHC complex brings polarity to the disorder system and is represented in triangular mirror images that conform to hexagons. The research’ images demonstrate a narrow relation- ship, at least morphologically, between the microscopic and macroscopic level. The link between macroscopic microscopic phenomena and molecular chirality proves that crystals are necessary for the direct assignment of absolute configuration of chiral molecules [16]. ACKNOWLEDGMENTS The authors thank the Pathology Department Medicine School of the Cooperative University of Colombia; specially their dean Dr Eduardo Rozo and directives of the Clinical Carlos Hugo Estrada, state social company, Policarpa Salavarrieta–Villavicencio, Colombia for their logis- tical and economic support. Under deductive analysis it is possible to affirm that in malignant tumors left handed and right handed order exists. Authors discern two oppossite diferent types of morphological subtrates, dark cromatine condensation, proliferative status and authofagic vacuoles apoptosis degenerative cystic changes, depending on magnetic spiral sub patterns direction of the tringular basic units of the GTCHC complexes. (Figures 7C, 8A, 8B, 8C, 8D). Author Contributions Conceived and designed the experiments: JD NJ MM. Performed the experiments: JD NJ MM. Analyzed the data: JD NJ MM. Contributed reagents/materials/analysis tools: JD NJ. Wrote the paper: JD NJ MM. Conceived and designed the experiments: JD NJ MM. Performed the experiments: JD NJ MM. Analyzed the data: JD NJ MM. Contributed reagents/materials/analysis tools: JD NJ. Wrote the paper: JD NJ MM. How did the authors interpret these images? The authors believe that the complexes GTCHC have a biological meaning in conformity with the microscopic and macroscopic finds. In tumour injuries, micro- scopically focal authofagic vacuolization and apoptotic changes are observed without elements of inflammatory or necrotic visible partners to these structures (Figure 5B, 5C). The interrelation between these changes represent probably the cystic formations that are visualized at macroscopic level inside these complexes (Figure 2E, 2F), which would inhibit the pathological progression of the tumor and its expansion. Geometries would be a transcisional dynamic step toward the known process of malignant tissues cystic degeneration disorder. GTCHC complex brings polarity to the disorder system and is represented in triangular mirror images that conform to hexagons. The research’ images demonstrate a narrow relation- ship, at least morphologically, between the microscopic and macroscopic level. The link between macroscopic microscopic phenomena and molecular chirality proves that crystals are necessary for the direct assignment of absolute configuration of chiral molecules [16]. The authors believe that the complexes GTCHC have a biological meaning in conformity with the microscopic and macroscopic finds. In tumour injuries, micro- scopically focal authofagic vacuolization and apoptotic changes are observed without elements of inflammatory or necrotic visible partners to these structures (Figure 5B, 5C). The interrelation between these changes represent probably the cystic formations that are visualized at macroscopic level inside these complexes (Figure 2E, 2F), which would inhibit the pathological progression of the tumor and its expansion. Geometries would be a transcisional dynamic step toward the known process of malignant tissues cystic degeneration The authors recommend that further interdisciplinary studies be carried out to investigate these geometric complexes to know them better and in the future, to be able to reproduce, manipulate, and amplify their activity and maybe use them as a base to develop new therapeutic strategies in cancer. It is thinkable reorganize a perturbed system through the spatio-temporal assemby of mirror image molecules. 2. Tamura S, Furuya Y, Tadakoro T, Higashidani Y, Yokoyama Y, et al. (2002) Pit pattern and three-dimensional configuration of isolated crypts from the patients with colorectal neoplasm. J Gastroenterol 37(10): 798–806. 1. Pokorna E, Zicha D, Chaloupkova A, Matouskova E, Versely P (2003) Two dynamic morphotypes of sarcoma cells, asymmetric stellate and triangle with leading lamella, are related to malignancy. Folia Biol (Praha) 49(1): 33–39. 2. Tamura S, Furuya Y, Tadakoro T, Higashidani Y, Yokoyama Y, et al. (2002) Pit pattern and three-dimensional configuration of isolated crypts from the patients with colorectal neoplasm. J Gastroenterol 37(10): 798–806. REFERENCES 1. Pokorna E, Zicha D, Chaloupkova A, Matouskova E, Versely P (2003) Two dynamic morphotypes of sarcoma cells, asymmetric stellate and triangle with leading lamella, are related to malignancy. Folia Biol (Praha) 49(1): 33–39. PLoS ONE \| www.plosone.org December 2007 \| Issue 12 \| e1282 9 Geometric Biological Order 3. Tadakoro T, Tamura S, Furuya Y, Araki K, Onishi S (2004) Evaluation of tumor development from the view point of the three dimensional configuration of isolated crypts in colorectal adenomas with a type IIIL pit pattern. JGastroenterol 39(7): 635–639. 11. Oda R, Lequeux E, Mendez (1996) Evidence for local orientation order in Salt free wormlike micelles a transient electric Birefringence study. J.Phys II 6: 1429–1439. 12. Prosser RS, Volkov VB, Shiyanovskaya IV (1998) Novel chelate-induced magnetic alignment of biological membranes. Biophys J 75(5): 2163–2169. J ( ) 4. Lanyi M (1982) An analysis of 136 groups of micro calcifications of benign origin. How specific is the ‘‘triangle principle’’. Rofo 136(2): 182–8. 13. Brizard A, Oda R, Huc I (2005) Chirality effects in self assembled fibrillar networks. Top Curr Chem 256: 167–268. 5. Cherel P, Becette V, Hagay C (2005) Stellate images: anatomic and radiologic correlations. Eur J Radiol 54(1): 37–54. correlations. Eur J Radiol 54(1): 37–54. 14. Hullinger J, Bebie H, Kluge S, Quintel A (2002) Growth-in 14. Hullinger J, Bebie H, Kluge S, Quintel A (2002) Growth-induced evolution of polarity in organic crystals. Chem. Mater 14: 1523–1529. 6. He HJ, Kanemura S, Yuan CP (2002) Determining the chirality of yucawa couplings via simple charged higgs boson photo collisions. Phys Rev lett 89 (10): 101803. polarity in organic crystals. Chem. Mater 14: 1523–152 15. Lifang Dong, Zengian Yin, Xuechen Li, Zhifang Chai, Yafeng He (2006) Spatio-temporal patterns in dielectric discharge in air at atmospheric pressure. Plasma Sources Sci Technol 15: 840–844. 7. Barron, Laurence D (2000) Chirality magnetism and light. Nature 405 (6789): 895–896. 16. Addadi L, Berkovitch-Yellin Z, Weissbuch I, Lahav M, Leiserowitz L (2007) Link Between Macroscopic Phenomena and Molecular Chirality: Crystal as probes for the Direct Assignment of absolute Configuration of Chiral Molecules. In Ernest L Eliel, Samuel H Wilen, Norman L Allinger, eds. Topics in Stereochemistry Volume 17. 8. Prockop DJ, Fertala A (1998) The Collagen Fibril: the almost crystalline structure. J. Struct Biol 122: 110–118. structure. J. Struct Biol 122: 110–118. 9. Shamos MH, Lavine LS (1967) Piezoelectricity as a fundamental property of biological tissues. REFERENCES Nature 213(5073): 267–269. 9. Shamos MH, Lavine LS (1967) Piezoelectricity biological tissues. Nature 213(5073): 267–269. biological tissues. Nature 213(5073): 267–269. 10. Hulmes DJ, Miller A (1979) Quasi-Hexagonal molecular packing in collagen fibrils. Nature 282(5741): 878–880. PLoS ONE \| www.plosone.org December 2007 \| Issue 12 \| e1282 10
https://openalex.org/W4200104293	https://covid19-preprints.microbe.ru/files/375	Russian	null	Анализ динамики эпидемического процесса COVID-19 в мире за неделю с 20.11.2021 г. по 26.11.2021 г.	null	2,021	cc-by	3,784	А.А. Зимирова, М.В. Поспелов, А.В. Иванова, Л.Н. Дмитриева, Е.А. Чу- мачкова, В.А. Сафронов, И. Г. Карнаухов, С. А. Щербакова, В. В. Кутырев Анализ динамики эпидемического процесса COVID-19 в мире за неделю с 20.11.2021 г. по 26.11.2021 г. ФКУЗ Российский научно-исследовательский противочумный институт «Микроб» Роспотребнадзора, Саратов, Российская Федерация В обзоре представлен анализ эпидемиологической ситуации в странах мира по COVID-19, за неделю с 20.11.2021 г. по 26.11.2021 г. Анализ проведен на основании данных следующих ресурсов: 1. www.worldometers.info/coronavirus/ 2. tass.ru/pandemiya-covid-19 3. www.skyscanner.ru/travel-restrictions 4. reopen.europa.eu/en/map/LVA/7001 5. intelyse.com/coronavirus-travel-restrictions/ 6.gisanddata.maps.arcgis.com/apps/dashboards/bda7594740fd40299423467b4 8e9ecf6 А.А. Зимирова, М.В. Поспелов, А.В. Иванова, Л.Н. Дмитриева, Е.А. Чу- мачкова, В.А. Сафронов, И. Г. Карнаухов, С. А. Щербакова, В. В. Кутырев Анализ динамики эпидемического процесса COVID-19 в мире за неделю с 20.11.2021 г. по 26.11.2021 г. ФКУЗ Российский научно-исследовательский противочумный институт «Микроб» Роспотребнадзора, Саратов, Российская Федерация В обзоре представлен анализ эпидемиологической ситуации в странах мира по COVID-19, за неделю с 20.11.2021 г. по 26.11.2021 г. Анализ проведен на основании данных следующих ресурсов: 1. www.worldometers.info/coronavirus/ 2. tass.ru/pandemiya-covid-19 3. www.skyscanner.ru/travel-restrictions 4. reopen.europa.eu/en/map/LVA/7001 5. intelyse.com/coronavirus-travel-restrictions/ 6.gisanddata.maps.arcgis.com/apps/dashboards/bda7594740fd40299423467b4 8e9ecf6 6.gisanddata.maps.arcgis.com/apps/dashboards/bda7594740fd40299423467b4 8e9ecf6 1 Всего в мире зарегистрировано 260 044 436 случаев заболевания (33992 на 1 млн. населения); прирост за неделю составил 4 475 143 случая (585,0 на 1 млн. населения) – 1,8%. Всего в мире зарегистрировано 5 182 911 летальных исходов (677,5 на 1 млн. населения); прирост за неделю составил 50 345 случаев (6,6 на 1 млн. населения) – 1%. За неделю с 20 по 26 ноября 2021 г. показатель прироста новых случаев в сравнении с предыдущей неделей (с 13 по 19 ноября 2021 г.) увеличился – с 1,5% до 1,8% (рис. 1; таб.1). Недельный прирост числа летальных исходов за анализируемую неделю не изменился - 1%. Анализ динамики эпидемического процесса COVID-19 в мире за неделю с 20.11.2021 г. по 26.11.2021 г. Рисунок 1 – Ежедневный прирост подтвержденных случаев в мире 100000 200000 300000 400000 500000 600000 700000 800000 900000 100000018.03.202018.04.202018.05.202018.06.202018.07.202018.08.202018.09.202018.10.202018.11.202018.12.202018.01.202118.02.202118.03.202118.04.202118.05.202118.06.202118.07.202118.08.202118.09.202118.10.202118.11.2021 Рисунок 1 – Ежедневный прирост подтвержденных случаев в мире 2 Таблица 1 – Еженедельный прирост случаев за последние полгода Неделя Недель- ный при- рост (случаев) Недель- ный при- рост (%) Неделя Недель- ный при- рост (случаев) Недель- ный при- рост (%) 29 мая – 4 июня 3 254 304 1,9 28 августа – 3 сентября 4 457 642 2,1 5 – 11 июня 2 745 583 1,6 4 – 10 сентября 4 108 660 1,9 12 – 18 июня 2 587 203 1,5 11 – 17 сентября 3 881 581 1,7 19 – 25 июня 2 576 278 1,5 18 – 24 сентября 3 544 821 1,6 26 июня – 2 июля 2 650 167 1,5 25 сентября – 1 октября 3 179 984 1,4 3 - 9 июля 2 927 889 1,6 2 – 8 октября 2 991 475 1,3 10 – 16 июля 3 363 018 1,8 9 – 15 октября 2 829 038 1,2 17 – 23 июля 3 656 571 1,9 16 – 22 октября 2 885 362 1,2 24 – 30 июля 3 978 967 2,1 23 – 29 октября 3 022 350 1,2 31 июля – 6 ав- густа 4 295 399 2,2 30 октября – 5 ноября 3 064 395 1,2 7 – 13 августа 4 386 968 2,3 6 – 12 ноября 3 322 096 1,3 14 – 20 августа 4 416 659 2,2 13 – 19 ноября 3 665 348 1,5 21 – 27 августа 4 617 376 2,2 20 – 26 ноября 4 475 143 1,8 3 3 Рисунок 2 – Количество зарегистрированных случаев с 20.11.2021 г. по 26.11.2021 г. Рисунок 2 – Количество зарегистрированных случаев с 20.11.2021 г. по 26.11.2021 г. За последнюю неделю в мире каждый день число новых случаев увеличива- лось на 500 – 700 тысяч. В общей структуре заболеваемости за неделю с 20 по 26 ноября 2021 г. наибольшая доля новых случаев приходится на Европейский регион (69,7%, показатель увеличился на 4,8%). Доля случаев в Американ- ском регионе за неделю снизилась на 3,8% и составила 19,4%. Доля Западно- Тихоокеанского региона уменьшилась на 0,2% в сравнении с предыдущей неделей – 4,8%. За анализируемый период доля Юго-Восточной Азии снизи- лась на 0,7% и составила 3,3%. Доля Восточно-Средиземноморского региона снизилась на 0,2% в сравнении с предыдущей неделей и составила 2,4%. Анализ динамики эпидемического процесса COVID-19 в мире за неделю с 20.11.2021 г. по 26.11.2021 г. Наименьший удельный вес новых случаев заболевания приходится на страны Африканского региона – 0,4%, доля увеличилась на 0,1% по сравнению с предыдущей неделей. 4 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 23.01.2020 23.02.2020 23.03.2020 23.04.2020 23.05.2020 23.06.2020 23.07.2020 23.08.2020 23.09.2020 23.10.2020 23.11.2020 23.12.2020 23.01.2021 23.02.2021 23.03.2021 23.04.2021 23.05.2021 23.06.2021 23.07.2021 23.08.2021 23.09.2021 23.10.2021 23.11.2021 Западно-Тихоокеанский регион Европейский регион Американский регион Восточно-Средиземноморский регион Юго-Восточная Азия Африканский регион 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 23.01.2020 23.02.2020 23.03.2020 23.04.2020 23.05.2020 23.06.2020 23.07.2020 23.08.2020 23.09.2020 23.10.2020 23.11.2020 23.12.2020 23.01.2021 23.02.2021 23.03.2021 23.04.2021 23.05.2021 23.06.2021 23.07.2021 23.08.2021 23.09.2021 23.10.2021 23.11.2021 Западно-Тихоокеанский регион Европейский регион Американский регион Восточно-Средиземноморский регион Юго-Восточная Азия Африканский регион Рисунок 3 – Доля регионов мира в ежедневном приросте случаев заболевания 5 Таблица 2 – Средняя доля региона от ежедневного прироста в мире Регионы ВОЗ 18 – 24 сен- тября 25 сен- тября – 1 ок- тября 2 – 8 ок- тября 9 – 15 ок- тября 16 – 22 ок- тября 23 – 29 ок- тября 30 ок- тября – 5 нояб- ря 6 – 12 нояб- ря 13 – 19 нояб- ря 20 – 26 нояб- ря Западно- Тихоокеан- ский регион 11,4 11,6 9,3 8,2 6,6 5,9 5,8 5,2 5 4,8 Европейский регион 32,3 36,9 42,2 48,7 55,4 59,4 62,6 65,2 64,9 69,7 Американский регион 37,9 34,4 32,7 28,9 25,5 23,7 21,8 21,7 23,2 19,4 Восточно- Средиземно- морский реги- он 6,1 5,6 5,4 4,9 4,6 3,9 3,7 3,3 2,6 2,4 Юго- Восточная Азия 10,5 9,8 9,2 8,4 7,1 6,4 5,6 4,7 4 3,3 Африканский регион 1,8 1,7 1,2 0,9 0,8 0,7 0,5 0,6 0,3 0,4 6 Американский регион В регионе на анализируемой неделе отмечено снижение числа новых слу- чаев заболевания на 9,3% в сравнении с предыдущей неделей (рис. 4). Рисунок 4 – Динамика ежедневного прироста новых случаев в Американском регионе Рисунок 4 – Динамика ежедневного прироста новых случаев в Американском регионе Количество стран региона, в которых зафиксирован рост числа новых случаев за неделю составило 12; наибольший по сравнению с предыдущей неделей – в Гватемале и Мексике (таб. 3). Таблица 3 – Страны с максимальным увеличением количества случаев, зарегистрированных за неделю (при сравнении недели с 13 по 19 ноября и не- дели с 20 по 26 ноября) Страна Зарегистрировано Увели- чение в/на с 13 по 19 ноября с 20 по 26 ноября абс. число на 1 млн абс. число на 1 млн Гватемала 3067 173,5 5180 293 68,9% Мексика 16686 130,6 21397 167 28,2% 7 На анализируемой неделе среди стран региона по числу еженедельно вы- являемых случаев доминируют Бразилия и США. Удельный вес новых случа- ев в общей заболеваемости в Американском регионе в этих двух странах на не- деле составил 83% (рис. 5). Рисунок 5 – Доля Бразилии, США и других стран в количестве случаев, зареги- стрированных за отчетную неделю Рисунок 5 – Доля Бразилии, США и других стран в количестве случаев, зареги- стрированных за отчетную неделю Наибольшее число заболевших в регионе на 1 млн населения за послед- нюю неделю зарегистрировано на Барбадосе (4380), в Доминике (3736) и Тринидаде и Тобаго (2786). Наибольшее число летальных исходов на 1 млн населения за последнюю неделю зарегистрировано в Тринидаде и Тобаго (95), Антигуа и Барбуде (93) и на Барбадосе (45). В настоящее время наблюдается ухудшение эпидситуации с COVID-19 в Боливии (рис. 6). Страна переживает четвертую волну эпидемии, в сутки реги- стрируется более 1000 новых случаев заражения. Самый высокий уровень забо- леваемости зафиксирован в департаментах «центральной оси»: Санта-Крус, за ним следуют Кочабамба, Ла-Пас и Тариха. Американский регион 8 8 Рисунок 6 – Динамика ежесуточного прироста новых случаев заболевания и летальности в Боливии (как отношения средних показателей прироста смер- тей и случаев за 7-дневный период) 0% 1% 2% 3% 4% 5% 6% 0 1000 2000 3000 4000 5000 6000 7000 800001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост 0% 1% 2% 3% 4% 5% 6% 0 1000 2000 3000 4000 5000 6000 7000 800001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост Рисунок 6 – Динамика ежесуточного прироста новых случаев заболевания и летальности в Боливии (как отношения средних показателей прироста смер- тей и случаев за 7-дневный период) Рисунок 6 – Динамика ежесуточного прироста новых случаев заболевания и летальности в Боливии (как отношения средних показателей прироста смер- тей и случаев за 7-дневный период) В США в настоящее время наблюдается рост уровня заболеваемости COVID-19 (рис. 7). Количество ежедневных случаев заражения в ноябре вырос- ло на 46% по сравнению с показателями на конец октября. За последние две не- дели число госпитализаций выросло на 15%. Наиболее тяжелая эпидситуация наблюдается в штатах Коннектикут, Мэн, Массачусетс, Нью-Гэмпшир, Род- Айленд и Вермонт. 9 0% 1% 2% 3% 4% 0 50000 100000 150000 200000 250000 300000 35000001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост 9 0% 1% 2% 3% 4% 0 50000 100000 150000 200000 250000 300000 35000001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост 9 Рисунок 7 – Динамика ежесуточного прироста новых случаев в США (как отношения средних показателей прироста смертей и случаев за 7-дневный пе- риод) Европейский регион В Европейском регионе на анализируемой неделе отмечен рост числа новых случаев заражения на 17,3% в сравнении с предыдущей неделей (рис. 8). Рисунок 8 – Динамика ежедневного прироста новых случаев в Европейском ре- гионе Рисунок 8 – Динамика ежедневного прироста новых случаев в Европейском ре- гионе Рост заболеваемости за неделю отмечен в 29 из 56 стран региона, наибольший – в Андорре, Франции, Бельгии и Лихтенштейне (таб. 4). 10 Таблица 4 – Страны с максимальным увеличением количества случаев, зарегистрированных за неделю (при сравнении недели с 13 по 19 ноября и не- дели с 20 по 26 ноября) Страна Зарегистрировано Увели- чение на с 13 по 19 ноября с 20 по 26 ноября абс. число на 1 млн абс. число на 1 млн Андорра 291 3820,1 531 6971 82,5% Франция 95021 1379,9 165715 2407 74,4% Бельгия 79751 6949,2 136562 11900 71,2% Лихтенштейн 237 6175,4 383 9980 61,6% Наибольшее число инфицированных на 1 млн населения в регионе за не- делю зафиксировано в Словакии (14335), Бельгии (11900) и Чехии (11003). Наибольшее число летальных исходов на 1 млн населения за последнюю неде- лю отмечено в Болгарии (130), Грузии (120) и Хорватии (112). Неблагоприятная эпидситуация с COVID-19 в настоящее время регистри- руется во Франции (рис. 10). Французские власти сообщили об очередном молниеносном росте заболеваемости. В течение последней недели уровень за- болеваемости в стране вырос почти на 80%. На фоне сложившейся ситуации государственные органы планируют более жестко задействовать систему COVID-сертификатов и другие ограничительные меры. В стране c 27 ноября всем жителям старше 18 лет начнут предлагать дополнительную бустерную до- зу вакцины, без которой в дальнейшем не будут действовать COVID- сертификаты. 11 11 Рисунок 9 – Динамика ежесуточного прироста новых случаев заболе- вания и летальности во Франции (как отношения средних показателей прироста смертей и случаев за 7-дневный период) 0% 1% 2% 3% 4% 5% 0 20000 40000 60000 80000 100000 120000 14000001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост 0% 1% 2% 3% 4% 5% 0 20000 40000 60000 80000 100000 120000 14000001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост Суточный прирост Рисунок 9 – Динамика ежесуточного прироста новых случаев заболе- вания и летальности во Франции (как отношения средних показателей прироста смертей и случаев за 7-дневный период) Рисунок 9 – Динамика ежесуточного прироста новых случаев заболе- вания и летальности во Франции (как отношения средних показателей прироста смертей и случаев за 7-дневный период) В настоящее время рост заболеваемости COVID-19 наблюдается в Чехии (рис. 9). Европейский регион С 26 ноября правительство страны ввело режим чрезвычайной ситуа- ции из-за роста заболеваемости. В течение 30 дней рождественские ярмарки, бары, рестораны и ночные клубы будут закрыты с 22:00 до 5:00. Заведения об- щепита будут работать только на вынос. Посещать культурные и спортивные мероприятия смогут максимум 1 тыс. человек. 12 0% 1% 2% 3% 4% 5% 6% 7% 0 5000 10000 15000 20000 25000 3000001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост 12 Рисунок 10 – Динамика ежесуточного прироста новых случаев заболева- ния и летальности в Чехии (как отношения средних показателей прироста смер- тей и случаев за 7-дневный период) Доля выявленных случаев в странах СНГ от всех зарегистрированных в Евро- пейском регионе составила 14,4% - снижение на 4,5% по сравнению с прошлой неделей (рис. 11). Рисунок 11 – Доля стран СНГ и других стран в количестве случаев, заре- гистрированных за отчетную неделю в Европейском регионе Рисунок 11 – Доля стран СНГ и других стран в количестве случаев, заре- гистрированных за отчетную неделю в Европейском регионе Западно-Тихоокеанский регион 15 Рисунок 14 – Динамика ежесуточного прироста новых случаев и леталь- ности во Вьетнаме (как отношения средних показателей прироста смертей и случаев за 7-дневный период) 0% 1% 2% 3% 4% 5% 0 5000 10000 15000 20000 25000 30000 35000 40000 4500001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост Рисунок 14 – Динамика ежесуточного прироста новых случаев и леталь- ности во Вьетнаме (как отношения средних показателей прироста смертей и 0% 1% 2% 3% 4% 5% 0 5000 10000 15000 20000 25000 30000 35000 40000 4500001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост Рисунок 14 – Динамика ежесуточного прироста новых случаев и леталь- ности во Вьетнаме (как отношения средних показателей прироста смертей и случаев за 7-дневный период) Рисунок 14 – Динамика ежесуточного прироста новых случаев и леталь- ности во Вьетнаме (как отношения средних показателей прироста смертей и случаев за 7-дневный период) Западно-Тихоокеанский регион На анализируемой неделе в Западно-Тихоокеанском регионе отмечен рост числа решистрируемых случаев на 3,5% в сравнении с прошлой неделей (рис. 12). 13 Рисунок 12 – Динамика ежедневного прироста новых случаев в Западно-Тихоокеанском регионе Рисунок 12 – Динамика ежедневного прироста новых случаев в Западно-Тихоокеанском регионе Рисунок 12 – Динамика ежедневного прироста новых случаев в Западно-Тихоокеанском регионе В 6 странах региона зафиксирован рост числа новых случаев за неделю, наибольший - во Вьетнаме и Папуа-Новой Гвинее (таб. 5). Таблица 5 – Страны с максимальным увеличением количества случаев, зарегистрированных за неделю (при сравнении недели с 13 по 19 ноября и не- дели с 20 по 26 ноября) Страна Зарегистрировано Увели- чение в/на с 13 по 19 ноября с 20 по 26 ноября абс. число на 1 млн абс. число на 1 млн Вьетнам 64572 671 102759 1068 59,1% Папуа-Новая Гвинея 959 109,3 1509 172 57,4% На Малайзию и Вьетнам приходится 67% от всех выявленных случаев за неделю в Западно-Тихоокеанском регионе (рис. 13). 14 Рисунок 13 – Доля Вьетнама, Малайзии и других стран в количестве слу- чаев, зарегистрированных за отчетную неделю. Рисунок 13 – Доля Вьетнама, Малайзии и других стран в количестве слу- чаев, зарегистрированных за отчетную неделю. Рисунок 13 – Доля Вьетнама, Малайзии и других стран в количестве слу- чаев, зарегистрированных за отчетную неделю. В регионе наибольшее число новых случаев заражения на 1 млн населе- ния за неделю зарегистрировано в Сингапуре (2092) и Лаосе (1197). Наиболь- шее число летальных исходов на 1 млн населения зафиксировано в Монголии (17) и на Филиппинах (13). В настоящее время продолжает ухудшаться эпидситуация во Вьетнаме (рис. 14). Тем не менее, Вьетнам возобновил международное авиасообщение для привлечения туристов. Пока местные власти открывают только несколько курортов, среди них остров Фукуок. Путешественникам понадобится сертифи- кат о полной вакцинации от COVID-19 или отрицательный результат ПЦР- теста, сделанного не более, чем за 72 часа до визита в страну, а также медицин- ская страховка. Проходить обязательный карантин туристом не нужно. Восточно-Средиземноморский регион В регионе на анализируемой неделе наблюдается снижение числа новых случаев заболевания на 0,1% в сравнении с прошлой неделей (рис. 15). 16 Рисунок 15 – Динамика ежедневного прироста новых случаев в Восточно- Средиземноморском регионе Рисунок 15 – Динамика ежедневного прироста новых случаев в Восточно- Средиземноморском регионе 16 За неделю с 20 по 26 ноября в 10 из 22 стран региона отмечен рост числа новых случаев. Максимальное увеличение недельного прироста зафиксировано в Тунисе, Бахрейне и Афганистане (таб. 7). Таблица 6 – Страны с максимальным увеличением количества случаев, зареги- стрированных за неделю (при сравнении недели с 13 по 19 ноября и недели с 20 по 26 ноября) Страна Зарегистрировано Увеличение в/на с 13 по 19 ноября с 20 по 26 ноября абс. число на 1 млн абс. число на 1 млн Тунис 499 42,6 873 75 74,9% Бахрейн 139 79,0 206 117 48,2% Афганистан 283 8,8 405 13 43,1% На Иран и Иорданию приходится 66,7% от всех выявленных случаев за неделю с 20 по 26 ноября в Восточно-Средиземноморском регионе (рис. 16). 17 Рисунок 16 – Удельный вес Ирана, Иордании и других стран в структуре случа- ев в Восточно-Средиземноморском регионе Рисунок 16 – Удельный вес Ирана, Иордании и других стран в структуре случа- ев в Восточно-Средиземноморском регионе 17 Наибольшее число заболевших на 1 млн населения за последнюю неделю зарегистрировано в Иордании (2494) и Ливане (1107). Наибольшее число ле- тальных исходов на 1 млн населения за последнюю неделю зарегистрировано в Иордании (14) и Ливии (12). В Иордании в настоящее время наблюдается неблагоприятная эпидситу- ация с COVID-19 (рис.17). В стране продолжается третья волна эпидемии. Чис- ло новых случаев заражения COVID-19, фиксируемых еженедельно в Иорда- нии, резко возросло примерно до 20 тыс, следует из данных Министерства здравоохранения. Увеличение доли положительных результатов тестов до 8% вызывает у властей обеспокоенность. Возможности больниц по лечению паци- ентов по-прежнему находятся на приемлемом уровне, так как заполняемость отделений интенсивной терапии и процент занятых аппаратов ИВЛ пока не превышает 30%. Рисунок 17 – Динамика ежесуточного прироста новых случаев заболева- ния и летальности в Иордании (как отношения средних показателей прироста смертей и случаев за 7-дневный период). Восточно-Средиземноморский регион 0% 1% 2% 3% 4% 5% 0 2000 4000 6000 8000 10000 1200001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост 0% 1% 2% 3% 4% 5% 0 2000 4000 6000 8000 10000 1200001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост Летальность (%) Рисунок 17 – Динамика ежесуточного прироста новых случаев заболева- ния и летальности в Иордании (как отношения средних показателей прироста смертей и случаев за 7-дневный период). Рисунок 17 – Динамика ежесуточного прироста новых случаев заболева- ния и летальности в Иордании (как отношения средних показателей прироста смертей и случаев за 7-дневный период). 18 Юго-Восточная Азия В регионе на анализируемой неделе наблюдается снижение числа новых случаев на 10,9% в сравнении с прошлой неделей (рис.18). Рисунок 18 – Динамика ежедневного прироста новых случаев в Юго-Восточной Азии Рисунок 18 – Динамика ежедневного прироста новых случаев в Юго-Восточной Азии Рост заболеваемости за последнюю неделю отмечен в одной стране реги- она – Бангладеш. Рост заболеваемости за последнюю неделю отмечен в одной стране реги- она – Бангладеш. она – Бангладеш. Таблица 7 - Страны с максимальным увеличением количества случаев, зарегистрированных за неделю (при сравнении недели с 13 по 19 ноября и не- дели с 20 по 26 ноября) Страна Зарегистрировано Увеличение в/на с 13 по 19 ноября с 20 по 26 ноября абс. число на 1 млн абс. число на 1 млн Бангладеш 1552 9,0 1727 10 11,3% Таблица 7 - Страны с максимальным увеличением количества случаев, зарегистрированных за неделю (при сравнении недели с 13 по 19 ноября и не- дели с 20 по 26 ноября) Страна Зарегистрировано Увеличение в/на с 13 по 19 ноября с 20 по 26 ноября абс. число на 1 млн абс. число на 1 млн Бангладеш 1552 9,0 1727 10 11,3% 19 На Таиланд и Индию приходится 86,7% от всех выявленных за неделю случаев в Юго-Восточной Азии в период с 20 по 26 ноября (рис.19). Рисунок 19 – Доля Индии, Таиланда и других стран региона в количестве слу- чаев, зарегистрированных за отчетную неделю Рисунок 19 – Доля Индии, Таиланда и других стран региона в количестве слу- чаев, зарегистрированных за отчетную неделю Наибольшее число заболевших на 1 млн населения за последнюю неделю зарегистрировано на Мальдивах (1422) и в Таиланде (659). Наибольшее число летальных исходов на 1 млн населения за последнюю неделю в регионе зареги- стрировано на Шри-Ланке (7) и Таиланде (5). В настоящее время стабилизируется эпидситуация на Шри-Ланке (рис. 20). C 25 ноября третью прививку от COVID-19 смогут сделать все жители Шри-Ланки старше 20 лет. Кроме того, власти намерены на следующей неделе частично смягчить действующие ограничения. Так, гражданам страны разрешат посещать кинотеатры и рестораны, проводить массовые мероприятия и засе- ляться в гостиницы при соблюдении строгих санитарных правил. 20 Рисунок 20 – Динамика ежесуточного прироста новых случаев заболева- ния и летальности в Шри-Ланке (как отношения средних показателей прироста смертей и случаев за 7-дневный период) 0% 1% 2% 3% 4% 5% 6% 7% 0 2000 4000 6000 8000 10000 1200001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост 0% 1% 2% 3% 4% 5% 6% 7% 0 2000 4000 6000 8000 10000 1200001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост Рисунок 20 – Динамика ежесуточного прироста новых случаев заболева- ния и летальности в Шри-Ланке (как отношения средних показателей прироста смертей и случаев за 7-дневный период) Рисунок 20 – Динамика ежесуточного прироста новых случаев заболева- ния и летальности в Шри-Ланке (как отношения средних показателей прироста смертей и случаев за 7-дневный период) Африканский регион В регионе на анализируемой неделе наблюдается рост числа новых слу- чаев заболевания на 56,9% в сравнении с прошлой неделей (рис. 21). 21 21 Рисунок 21 – Динамика ежедневного прироста новых случаев в Африкан- ском регионе Рисунок 21 – Динамика ежедневного прироста новых случаев в Африкан- ском регионе В 15 из 48 стран Африканского региона зарегистрирован рост числа но- вых случаев. Максимальное увеличение еженедельного прироста новых случа- ев по сравнению с предшествующей неделей демонстрируют ЮАР, Демокра- тическая Республика Конго и Мали (таб. 9). Таблица 8 – Страны с максимальным увеличением количества случаев, зареги- стрированных за неделю (при сравнении недели с 13 по 19 ноября и недели с 20 по 26 ноября). Страна Зарегистрировано Увеличе- ние в/на с 13 по 19 ноября с 20 по 26 ноября абс. число на 1 млн абс. число на 1 млн ЮАР 2142 39,0 7299 133 в 3,4 раза ДР Конго 141 1,4 257 3 82,3% Мали 234 11,9 348 18 48,7% На ЮАР и Маврикий приходится 52% от всех выявленных за неделю случаев в Африканском регионе в период с 20 по 26 ноября (рис. 22). 22 22 Рисунок 22 – Удельный вес ЮАР, Маврикия и других стран в структуре случа- ев, зарегистрированных за отчетную неделю в Африканском регионе Рисунок 22 – Удельный вес ЮАР, Маврикия и других стран в структуре случа- ев, зарегистрированных за отчетную неделю в Африканском регионе Наибольшее число заболевших на 1 млн населения за последнюю неделю зарегистрировано на Сейшелах (3735) и Маврикии (1534). Наибольшее число летальных исходов на 1 млн населения за последнюю неделю зарегистрировано в Мавритании (4) и ЮАР (4). Рост заболеваемости COVID-19 в настоящее время отмечается в Мали (рис.23). Рост заболеваемости COVID-19 в настоящее время отмечается в Мали (рис.23). Рисунок 23 – Динамика ежесуточного прироста новых случаев заболевания и летальности в Мали (как отношения средних показателей прироста смертей и случаев за 7-дневный период) 0% 2% 4% 6% 8% 10% 12% 14% 16% 18% 0 50 100 150 200 250 300 350 400 45001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост 0% 2% 4% 6% 8% 10% 12% 14% 16% 18% 0 50 100 150 200 250 300 350 400 45001.12.202001.01.202101.02.202101.03.202101.04.202101.05.202101.06.202101.07.202101.08.202101.09.202101.10.202101.11.2021 Летальность (%) Суточный прирост Рисунок 23 – Динамика ежесуточного прироста новых случаев заболевания и летальности в Мали (как отношения средних показателей прироста смертей и случаев за 7-дневный период) 23
https://openalex.org/W3125276211	https://www.chebsbornik.ru/jour/article/download/910/738	Russian	null	Рассеяние наклонно падающей плоской звуковой волны упругим цилиндром с неоднородным покрытием, находящимся вблизи плоской поверхности	Čebyševskij sbornik	2,020	cc-by	6,164	DOI 10.22405/2226-8383-2020-21-4-369-381 DOI 10.22405/2226-8383-2020-21-4-369-381 DOI 10.22405/2226-8383-2020-21-4-369-381 Л. А. Толоконников, Д. Ю. Ефимов Лев Алексеевич Толоконников — доктор физико-математических наук, Тульский госу- дарственный университет (г. Тула). e-mail: tolokonnikovla@mail.ru Ефимов Дмитрий Юрьевич — магистрант, Тульский государственный университет (г. Ту- ла). Лев Алексеевич Толоконников — доктор физико-математических наук, Тульский госу- дарственный университет (г. Тула). il t l k ik l @ il e-mail: tolokonnikovla@mail.ru e mail: tolokonnikovla@mail.ru Ефимов Дмитрий Юрьевич — магистрант, Тульский государственный университет (г. Ту- ла). Ефимов Дмитрий Юрьевич — магистрант, Тульский государственный унив ла). e-mail: bogart.efimov@yandex.ru 369 369 Рассеяние наклонно падающей плоской звуковой волны . . . 1Исследование выполнено за счет гранта Российского научного фонда (проект 18-11-00199). Аннотация В статье рассматривается задача о рассеянии плоской монохроматической звуковой волны, падающей произвольным образом на упругий круговой цилиндр с радиально- неоднородным покрытием в присутствии плоской поверхности (абсолютно жесткой и аку- стически мягкой). Методом мнимых источников с применением теорем сложения для ци- линдрических волновых функций получено аналитическое решение задачи. Волновые по- ля в содержащей среде и однородном упругом цилиндре находятся в виде разложений по волновым цилиндрическим функциям, а для нахождения полей смещений в неоднород- ных покрытиях построена краевая задача для системы обыкновенных дифференциальных уравнений второго порядка. Проведены численные расчеты частотных и угловых характеристик рассеянного поля для упругих однородных цилиндров с покрытием и без него, находящихся вблизи подсти- лающей плоскости. Выявлено существенное влияние непрерывно-неоднородных упругих покрытий на звукоотражающие свойства упругих цилиндрических тел. Ключевые слова: рассеяние, звуковые волны, упругий цилиндр, неоднородное упругое покрытие, плоская поврхность. Библиография: 15 названий. Рассеяние наклонно падающей плоской звуковой волны упругим цилиндром с неоднородным покрытием, находящимся вблизи плоской поверхности1 Л. А. Толоконников, Д. Ю. Ефимов Л. А. Толоконников, Д. Ю. Ефимов. Рассеяние наклонно падающей плоской звуковой волны упругим цилиндром с неоднородным покрытием, находящимся вблизи плоской поверхности // Чебышевcкий сборник, 2020, т. 21, вып. 4, с. 369–381. For citation: L. A. Tolokonnikov, D. Yu. Efimov, 2020, “Scattering of a plane sound waves by an elastic cylinder with an non-uniform coating situated near to a flat surface”, Chebyshevskii sbornik, vol. 21, no. 4, pp. 369–381. UDC 539.3:534.26 UDC 539.3:534.26 UDC 539.3:534.26 Для цитирования: Л. А. Толоконников, Д. Ю. Ефимов. Рассеяние наклонно падающей плоской звуковой волны упругим цилиндром с неоднородным покрытием, находящимся вблизи плоской поверхности // Чебышевcкий сборник, 2020, т. 21, вып. 4, с. 369–381. 370 Л. А. Толоконников, Д. Ю. Ефимов Lev Alexeevich Tolokonnikov — doctor of physical and mathematical Sciences, Tula State University (Tula). Lev Alexeevich Tolokonnikov — doctor of physical and mathematical Sciences, Tula State University (Tula). e-mail: tolokonnikovla@mail.ru Efimov Dmitrii Yurevich — undergraduate, Tula State University (Tula). Efimov Dmitrii Yurevich — undergraduate, Tula State University (Tula). e-mail: bogart.efimov@yandex.ru Efimov Dmitrii Yurevich — undergraduate, Tula State University (Tula). il b t fi @ d 2This Research was performed by a grant of Russian Science Foundation (project 18-11-00199). Abstract In article the problem of the scattering of an obliquely incident plane monochromatic sound wave by an elastic cylinder with a radially non-uniform elastic coating in presence of a flat surface (absolutely rigid and acoustically soft) is considered. The analytical solution of the problem by the method of imaginary sources using addition theorems for cylindrical wave functions is received. Wave fields in a containing medium and homogeneous elastic cylinder are found in the form of expansions in wave cylindrical functions. The boundary-value problem for the system of ordinary second order differential equations is constructed for determination of the displacement fields in inhomogeneous coatings. Numerical calculations of frequency and angular characteristics of the scattered field for elastic homogeneous cylinders with and without coating located near the underlying plane are performed. Influence of continuously inhomogeneous elastic coatings on sound-reflecting properties of elastic cylindrical bodies are revealed. Keywords: scattering, sound waves, elastic cylinder, non-uniform elastic coating, flat surfac Bibliography: 15 titles. Scattering of a plane sound waves by an elastic cylinder with an non-uniform coating situated near to a flat surface2 Scattering of a plane sound waves by an elastic cylinder with an non-uniform coating situated near to a flat surface2 DOI 10.22405/2226-8383-2020-21-4-369-381 DOI 10.22405/2226-8383-2020-21-4-369-381 Рассеяние наклонно падающей плоской звуковой волны . . . 371 решены как прямая задача дифракции, так и обратная задача об определении квадратичных законов неоднородности материала покрытия, обеспечивающих наименьшее звукоотражение в определенном угловом секторе и в заданном диапазоне частот. Моделирование непрерывно- неоднородного покрытия упругого цилиндра с заданными звукоотражающими свойствами осуществлено в [6, 7]. Зависимости плотности и модулей упругости материала покрытия от ра- диальной координаты аппроксимированы многочленами первой степени в [6] и второй степени в [7]. В работах, приведенных выше, полагалось, что цилиндрические тела находятся в безгра- ничном пространстве. В [8] решена задача дифракции плоской звуковой волны на упругом цилиндре с радиально-неоднородным упругим покрытием, находящемся вблизи плоской иде- альной поверхности (абсолютно жесткой и акустически мягкой), когда плоская волна пада- ет перпендикулярно оси цилиндра. В настоящей работе рассматривается задача о рассеянии плоской монохроматической звуковой волны, падающей произвольным образом на упругий круговой цилиндр, покрытый радиально-неоднородным упругим слоем, в присутствии под- стилающей плоскости (абсолютно жесткой и акустически мягкой). 2. Постановка задачи Рассмотрим бесконечный упругий цилиндр радиуса 𝑟0, материал которого характеризуется плотностью 𝜌0 и упругими постоянными 𝜆0 и 𝜇0. Цилиндр имеет покрытие в виде радиально- неоднородного изотропного упругого слоя с внешним радиусом 𝑟1. Полагаем, что модули упру- гости 𝜆и 𝜇материала неоднородного покрытия описываются дифференцируемыми функци- ями цилиндрической радиальной координаты, а плотность 𝜌— непрерывной функцией этой координаты. Окружающая цилиндрическое тело жидкость является идеальной и характери- зуется плотностью 𝜌1 и скоростью звука 𝑐. Цилиндр с покрытием находится вблизи плоской поверхности Γ, которая является абсолютно жесткой или акустически мягкой. Ось цилиндра параллельна плоскости Γ и отстоит от нее на расстоянии 𝑑. Пусть из внешнего полупространства на тело под произвольным углом падает плоская гармоническая звуковая волна с временной зависимостью 𝑒−𝑖𝜔𝑡, где 𝜔— круговая частота; 𝑡 — время. В дальнейшем временной множитель 𝑒−𝑖𝜔𝑡будем опускать. При рассеянии звука цилиндром, находящимся вблизи звукоотражающей границы, возни- кают многократные переотражения между телом и плоскостью, так что близко расположенная подстилающая поверхность оказывает существенное влияние на рассеяние звука цилиндром. Определим акустическое поле, рассеянное цилиндром с покрытием в присутствии плоскости. 1. Введение Возможность изменения звукоотражающих свойств цилиндрических тел с помощью по- крытий из функционально-градиентных материалов обсуждалась в ряде работ. В [1, 2] иссле- довано рассеяние плоских и цилиндрических звуковых волн жестким цилиндром с радиально- неоднородным упругим покрытием. При этом в [1] рассматривался случай нормального паде- ния плоской волны, а в [2] полагалось, что ось цилиндрического источника параллельна оси цилиндра. Задачи о рассеянии наклонно падающей плоской звуковой волны упругим цилин- дром с неоднородным покрытием решены в [3, 4]. В [3] неоднородное покрытие полагалось непрерывно-неоднородным, а в [4] — дискретно-слоистым. Дифракция плоской звуковой вол- ны на термоупругом цилиндре с радиально-неоднородным покрытием изучена в [5]. При этом Рассеяние наклонно падающей плоской звуковой волны . . . 3. Аналитическое решение задачи В рассматриваемой постановке задача является трехмерной. Так как подстилающая плос- кость полагается идеальной (абсолютно жесткой или акустически мягкой), то решение постав- ленной задачи можно найти методом мнимых источников. Решение задачи проведем, исполь- зуя подход, примененный в [8, 9]. Согласно методу мнимых источников исключим плоскость Γ, вводя в рассмотрение вто- рой цилиндр, являющийся зеркальным отражением исходного рассеивателя, и вторую плос- кую волну, распространяющуюся в направлении волнового вектора k2 [8]. Причем вектор k2 является зеркальным отражением волнового вектора k1 падающей плоской звуковой волны относительно плоскости Γ. В результате исходная задача сводится к задаче рассеяния двух плоских волн на двух идентичных цилиндрах, находящихся в безграничном пространстве, заполненном идеальной жидкостью [9]. 372 Л. А. Толоконников, Д. Ю. Ефимов Введём три одинаково ориентированные прямоугольные декартовы системы координат: основную (𝑥, 𝑦, 𝑧), связанную с плоскостью Γ, и две локальных (𝑥+1, 𝑦+1, 𝑧+1) и (𝑥−1, 𝑦−1, 𝑧−1), связанные с действительным и фиктивным цилиндрами соответственно (рис. 1). Плоскость 𝑥𝑧 совмещена с плоскостью Γ, координатные оси 𝑧+1 и 𝑧−1 совпадают с осями вращения первого и второго цилиндров соответственно, координатные оси 𝑦+1 и 𝑦−1 совпадают с осью 𝑦. Свяжем прямоугольные системы координат (𝑥, 𝑦, 𝑧), (𝑥+1, 𝑦+1, 𝑧+1), (𝑥−1, 𝑦−1, 𝑧−1) с цилин- дрическими системами координат (𝑟, 𝜙, 𝑧), (𝑟+1, 𝜙+1, 𝑧+1), (𝑟−1, 𝜙−1, 𝑧−1). Отметим, что 𝑧= 𝑧𝑙 (𝑙= ±1). В локальных цилиндрических координатах уравнения внешней и внутренней поверхностей покрытия 𝑙– го цилиндра имеют вид 𝑟𝑙= 𝑟1 и 𝑟𝑙= 𝑟0 (𝑙= ±1). Р 1 К Рис. 1: К задаче рассеяния звука на двух упругих цилиндрах с неоднородными покрытиями Потенциал скорости падающей волны, распространяющейся в направлении волнового век- тора k1, в основной системе координат имеет вид Потенциал скорости падающей волны, распространяющейся в направлении волнового век- тора k1, в основной системе координат имеет вид Ψ01 = 𝐴exp[𝑖(k1 · R)], где 𝐴— амплитуда волны; k1 = (𝑘sin 𝜃0 cos 𝜙0; 𝑘sin 𝜃0 sin 𝜙0; 𝑘cos 𝜃0); 𝑘= 𝜔/𝑐— волновое число жидкости; R = (𝑥, 𝑦, 𝑧) — радиус-вектор; 𝜃0 и 𝜙0 — полярный и азимутальный углы падения плоской волны. Чтобы граничные условия на плоскости Γ (при 𝑦= 0) удовлетворялись автоматически, потенциал скорости второй падающей плоской волны должен быть равен [8] Ψ02 = 𝐴exp[𝑖(k2 · R)], Ψ02 = 𝐴exp[𝑖(k2 · R)], если плоскость Γ жесткая, и Ψ02 = −𝐴exp[𝑖(k2 · R)], Ψ02 = −𝐴exp[𝑖(k2 · R)], если плоскость Γ мягкая. При этом k2 = (𝑘sin 𝜃0 cos 𝜙0; −𝑘sin 𝜃0 sin 𝜙0; 𝑘cos 𝜃0). Рассеяние наклонно падающей плоской звуковой волны . . . 3. Аналитическое решение задачи 373 373 С учетом того, что R = R𝑙+ R𝑂𝑙(𝑙= ±1), где R𝑙— радиус-вектор точки наблюдения в 𝑙– ой локальной системе координат; R𝑂𝑙= (0, 𝑑𝑙, 0) — радиус-вектор, соединяющий точку 𝑂с точкой 𝑂𝑙, потенциалы скорости падающих плоских волн в локальных цилиндрических координатах представляются в виде разложений [11] Ψ01(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝐴𝑒𝑖𝑑𝑙𝛽sin 𝜙0𝑒𝑖𝛼𝑧𝑙 ∞ ∑︁ 𝑛=−∞ 𝑖𝑛𝐽𝑛(𝛽𝑟𝑙)𝑒𝑖𝑛(𝜙𝑙−𝜙0), (1) Ψ02(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = ±𝐴𝑒−𝑖𝑑𝑙𝛽sin 𝜙0𝑒𝑖𝛼𝑧𝑙 ∞ ∑︁ 𝑛=−∞ 𝑖𝑛𝐽𝑛(𝛽𝑟𝑙)𝑒𝑖𝑛(𝜙𝑙−𝜙0), (2) (1) ︁ Ψ02(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = ±𝐴𝑒−𝑖𝑑𝑙𝛽sin 𝜙0𝑒𝑖𝛼𝑧𝑙 ∞ ∑︁ 𝑛=−∞ 𝑖𝑛𝐽𝑛(𝛽𝑟𝑙)𝑒𝑖𝑛(𝜙𝑙−𝜙0), (2) (2) ︁ где 𝐽𝑛— цилиндрическая функция Бесселя порядка 𝑛; 𝛼= 𝑘cos 𝜃0; 𝛽= 𝑘sin 𝜃0. Распространение гармонических звуковых волн в идеальной жидкости описывается урав- ︁ где 𝐽𝑛— цилиндрическая функция Бесселя порядка 𝑛; 𝛼= 𝑘cos 𝜃0; 𝛽= 𝑘sin 𝜃0. Распространение гармонических звуковых волн в идеальной жидкости описывается урав- нением Гельмгольца [10] ︁ где 𝐽𝑛— цилиндрическая функция Бесселя порядка 𝑛; 𝛼= 𝑘cos 𝜃0; 𝛽= 𝑘sin 𝜃0. нение гармонических звуковых волн в идеальной жидкости описывается урав- ольца [10] ΔΨ + 𝑘2Ψ = 0, ΔΨ + 𝑘2Ψ = 0, где Ψ — потенциал скорости полного акустического поля. где Ψ — потенциал скорости полного акустического поля. При этом скорость частиц v и акустическое давление 𝑝в жидкости определяются по фор- мулам При этом скорость частиц v и акустическое давление 𝑝в жидкости определяются по фор- мулам При этом скорость частиц v и акустическое давление 𝑝в жидкости определяются по фор- мулам v = gradΨ, 𝑝= 𝑖𝜌1𝜔Ψ. v = gradΨ, 𝑝= 𝑖𝜌1𝜔Ψ. v = gradΨ, 𝑝= 𝑖𝜌1𝜔Ψ. В силу линейной постановки задачи потенциал скорости полного акустического поля будет равен [8] В силу линейной постановки задачи потенциал скорости полного акустического поля будет равен [8] р [ ] Ψ = Ψ01 + Ψ02 + Ψ𝑠1 + Ψ𝑠2, (3) Ψ = Ψ01 + Ψ02 + Ψ𝑠1 + Ψ𝑠2, (3) Ψ = Ψ01 + Ψ02 + Ψ𝑠1 + Ψ𝑠2, (3) где Ψ𝑠1 и Ψ𝑠2 — потенциалы скорости рассеянных двумя цилиндрами первой и второй плоских волн соответственно. где Ψ𝑠1 и Ψ𝑠2 — потенциалы скорости рассеянных двумя цилиндрами первой и второй плоских волн соответственно. Потенциал скорости волны, рассеянной цилиндром в присутствии подстилающей плоской поверхности определяется выражением Ψ𝑠= Ψ02 + Ψ𝑠1 + Ψ𝑠2. (4) (4) Потенциалы Ψ𝑠𝑗представим в виде суммы двух слагаемых︁ Потенциалы Ψ𝑠𝑗представим в виде суммы двух слагаемых︁ Потенциалы Ψ𝑠𝑗представим в виде суммы двух слагаемых︁ Ψ𝑠𝑗= ∑︁ 𝑙=±1 Ψ(𝑙) 𝑠𝑗 (𝑗= 1, 2), (5) (5) ︁ каждое из которых представляет собой потенциал скорости волны, рассеянной 𝑙– ым цилин- дром. 3. Аналитическое решение задачи С учетом условия ограниченности функции 𝐹(𝑙) 𝑗, 𝐿(𝑙) 𝑗 и 𝑀(𝑙) 𝑗 будем искать в виде С учетом условия ограниченности функции 𝐹(𝑙) 𝑗, 𝐿(𝑙) 𝑗 и 𝑀(𝑙) 𝑗 будем искать в виде 𝐹(𝑙) 𝑗(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︁ 𝑛=−∞ 𝐵(𝑙) 𝑗𝑛𝐽𝑛(𝑘1𝑟𝑙)𝑒𝑖𝑛(𝜙−𝜙0) (𝑙= ±1, 𝑗= 1, 2), (7) 𝐹(𝑙) 𝑗(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︁ 𝑛=−∞ 𝐵(𝑙) 𝑗𝑛𝐽𝑛(𝑘1𝑟𝑙)𝑒𝑖𝑛(𝜙−𝜙0) (𝑙= ±1, 𝑗= 1, 2), (7) 𝐿(𝑙) 𝑗(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︁ 𝑛=−∞ 𝐶(𝑙) 𝑗𝑛𝐽𝑛(𝑘2𝑟𝑙)𝑒𝑖𝑛(𝜙−𝜙0) (𝑙= ±1, 𝑗= 1, 2), (8) 𝑀(𝑙) 𝑗(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︁ 𝑛=−∞ 𝐷(𝑙) 𝑗𝑛𝐽𝑛(𝑘2𝑟𝑙)𝑒𝑖𝑛(𝜙−𝜙0) (𝑙= ±1, 𝑗= 1, 2), (9)︁︀ (7) ︁ 𝐿(𝑙) 𝑗(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︁ 𝑛=−∞ 𝐶(𝑙) 𝑗𝑛𝐽𝑛(𝑘2𝑟𝑙)𝑒𝑖𝑛(𝜙−𝜙0) (𝑙= ±1, 𝑗= 1, 2), (8) 𝑀(𝑙) 𝑗(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︁ 𝑛=−∞ 𝐷(𝑙) 𝑗𝑛𝐽𝑛(𝑘2𝑟𝑙)𝑒𝑖𝑛(𝜙−𝜙0) (𝑙= ±1, 𝑗= 1, 2), (9)︁︀ (8) (9) ︁ де 𝑘1 = √︁ 𝑘2𝜂−𝛼2; 𝑘2 = √︀ 𝑘2𝜏−𝛼2. ︁ где 𝑘1 = √︁ 𝑘2𝜂−𝛼2; 𝑘2 = √︀ 𝑘2𝜏−𝛼2. ︁ где 𝑘1 = √︁ 𝑘2𝜂−𝛼2; 𝑘2 = √︀ 𝑘2𝜏−𝛼2. ︁ где 𝑘1 = √︁ 𝑘2𝜂−𝛼2; 𝑘2 = √︀ 𝑘2𝜏−𝛼2. √︁︀ Волновые поля в неоднородных упругих покрытиях цилиндров описываются общими урав- нениями движения упругой среды [13], которые в локальных цилиндрических координатах имеют вид √︁︀ Волновые поля в неоднородных упругих покрытиях цилиндров описываются общими урав- нениями движения упругой среды [13], которые в локальных цилиндрических координатах имеют вид (𝑙) (𝑙) (𝑙) (𝑙) (𝑙) 𝜕𝜎(𝑙) 𝑟𝑟 𝜕𝑟𝑙 + 1 𝑟𝑙 𝜕𝜎(𝑙) 𝑟𝜙 𝜕𝜙𝑙 + 𝜕𝜎(𝑙) 𝑟𝑧 𝜕𝑧𝑙 + 𝜎(𝑙) 𝑟𝑟−𝜎(𝑙) 𝜙𝜙 𝑟𝑙 = −𝜔2𝜌(𝑟𝑙)𝑢(𝑙) 𝑟, 𝜕𝜎(𝑙) 𝑟𝜙 𝜕𝑟𝑙 + 1 𝑟𝑙 𝜕𝜎(𝑙) 𝜙𝜙 𝜕𝜙𝑙 + 𝜕𝜎(𝑙) 𝜙𝑧 𝜕𝑧𝑙 + 2 𝑟𝑙 𝜎(𝑙) 𝑟𝜙= −𝜔2𝜌(𝑟𝑙)𝑢(𝑙) 𝜙, 𝜕𝜎(𝑙) 𝑟𝑧 𝜕𝑟𝑙 + 1 𝑟𝑙 𝜕𝜎(𝑙) 𝜙𝑧 𝜕𝜙𝑙 + 𝜕𝜎(𝑙) 𝑧𝑧 𝜕𝑧𝑙 + 1 𝑟𝑙 𝜎(𝑙) 𝑟𝑧= −𝜔2𝜌(𝑟𝑙)𝑢(𝑙) 𝑧, (10) (10) где 𝑢(𝑙) 𝑟, 𝑢(𝑙) 𝜙, 𝑢(𝑙) 𝑧и 𝜎(𝑙) 𝑝𝑞— компоненты вектора смещения u(𝑙) и тензора напряжений в покрытии 𝑙– го цилиндра, у которых здесь и далее индекс 𝑗(𝑗= 1, 2), показывающий под воздействием какой плоской волны происходит деформация в покрытии, опущен для упрощения записи. где 𝑢(𝑙) 𝑟, 𝑢(𝑙) 𝜙, 𝑢(𝑙) 𝑧и 𝜎(𝑙) 𝑝𝑞— компоненты вектора смещения u(𝑙) и тензора напряжений в покрытии 𝑙– го цилиндра, у которых здесь и далее индекс 𝑗(𝑗= 1, 2), показывающий под воздействием какой плоской волны происходит деформация в покрытии, опущен для упрощения записи. 3. Аналитическое решение задачи ︁ каждое из которых представляет собой потенциал скорости волны, рассеянной 𝑙– ым цилин- дром. ( ) Функции Ψ(𝑙) 𝑠𝑗являются решениями уравнений Гельмгольца в локальных цилиндрических координатах и должны удовлетворять условиям излучения на бесконечности. Эти функции будем искать в виде Ψ(𝑙) 𝑠𝑗(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︁ 𝑛=−∞ 𝐴(𝑙) 𝑗𝑛𝐻𝑛(𝛽𝑟𝑙)𝑒𝑖𝑛(𝜙𝑙−𝜙0) (𝑙= ±1, 𝑗= 1, 2), (6) (6) где 𝐻𝑛— цилиндрическая функция Ганкеля первого рода порядка 𝑛; 𝐴(𝑙) 𝑗𝑛— коэффициенты, подлежащие определению из граничных условий. где 𝐻𝑛— цилиндрическая функция Ганкеля первого рода порядка 𝑛; 𝐴(𝑙) 𝑗𝑛— коэффициенты, подлежащие определению из граничных условий. Рассмотрим теперь уравнения, описывающие распространение упругих волн в однородных упругих цилиндрах (действительном и мнимом). (𝑙) Представим вектор смещения u(𝑙) 0𝑗частиц упругого изотропного однородного 𝑙– го цилин- дра при воздействии 𝑗– ой плоской волны (𝑗= 1, 2) в виде u(𝑙) 0𝑗= grad𝐹(𝑙) 𝑗 + rotΦ(𝑙) 𝑗, divΦ(𝑙) 𝑗 = 0, Л. А. Толоконников, Д. Ю. Ефимов 374 где 𝐿(𝑙) 𝑗 и Φ(𝑙) 𝑗 — скалярный и векторный потенциалы смещения, которые в случае установивше- гося режима колебаний являются решениями скалярного и векторного уравнений Гельмгольца [12] где 𝐿(𝑙) 𝑗 и Φ(𝑙) 𝑗 — скалярный и векторный потенциалы смещения, которые в случае установивше- гося режима колебаний являются решениями скалярного и векторного уравнений Гельмгольца [12] Δ𝐹(𝑙) 𝑗 + 𝑘2 𝜂𝐹(𝑙) 𝑗 = 0, ΔΦ𝑗(𝑙) + 𝑘2 𝜏Φ𝑗(𝑙) = 0 (𝑙= ±1, 𝑗= 1, 2), где 𝑘𝜂= 𝜔/𝑐𝜂и 𝑘𝜏= 𝜔/𝑐𝜏 — волновые числа продольных и поперечных упругих волн; 𝑐𝜂= √︀ (𝜆0 + 2𝜇0)/𝜌0 и 𝑐𝜏= √︀ 𝜇0/𝜌0 — скорости продольных и поперечных волн. Представим вектор Φ(𝑙) 𝑗 в виде где 𝑘𝜂= 𝜔/𝑐𝜂и 𝑘𝜏= 𝜔/𝑐𝜏 — волновые числа продольных и поперечных упругих волн; 𝑐𝜂= √︀ (𝜆0 + 2𝜇0)/𝜌0 и 𝑐𝜏= √︀ 𝜇0/𝜌0 — скорости продольных и поперечных волн. Представим вектор Φ(𝑙) 𝑗 в виде ︀ Представим вектор Φ(𝑙) 𝑗 Φ𝑗(𝑙) = rot(𝐿(𝑙) 𝑗𝑒𝑧𝑙) + 𝑘𝜏𝑀(𝑙) 𝑗𝑒𝑧𝑙, где 𝐿(𝑙) 𝑗 и 𝑀(𝑙) 𝑗 — скалярные функции пространственных координат 𝑟𝑙, 𝜙𝑙, 𝑧𝑙; 𝑒𝑧𝑙— единичный вектор оси 𝑧𝑙. где 𝐿(𝑙) 𝑗 и 𝑀(𝑙) 𝑗 — скалярные функции пространственных координат 𝑟𝑙, 𝜙𝑙, 𝑧𝑙; 𝑒𝑧𝑙— единичный вектор оси 𝑧𝑙. Тогда векторное уравнение относительно функции Φ𝑗сведется к двум скалярным уравне- ниям Гельмгольца относительно функций 𝐿(𝑙) 𝑗 и 𝑀(𝑙) 𝑗 Δ𝐿(𝑙) 𝑗+ 𝑘2 𝜏𝐿(𝑙) 𝑗 = 0, Δ𝑀(𝑙) 𝑗 + 𝑘2 𝜏𝑀(𝑙) 𝑗 = 0. Δ𝐿(𝑙) 𝑗+ 𝑘2 𝜏𝐿(𝑙) 𝑗 = 0, Δ𝑀(𝑙) 𝑗 + 𝑘2 𝜏𝑀(𝑙) 𝑗 = 0. 3. Аналитическое решение задачи ︁ Подставляя разложения (11) в уравнения (10), записанные через функции 𝑢(𝑙) 𝑟(𝑟𝑙, 𝜙𝑙, 𝑧𝑙), 𝑢(𝑙) 𝜙(𝑟𝑙, 𝜙𝑙, 𝑧𝑙), 𝑢(𝑙) 𝑧(𝑟𝑙, 𝜙𝑙, 𝑧𝑙), получим следующую систему линейных обыкновенных дифферен- циальных уравнений второго порядка относительно неизвестных функций 𝑈(𝑙) 𝑖𝑛(𝑟𝑙) (𝑖= 1, 2, 3) для каждого 𝑛: ^A(𝑙) 𝑛U(𝑙)′′ 𝑛 + ^B(𝑙) 𝑛U(𝑙)′ 𝑛 + ^C(𝑙) 𝑛U(𝑙) 𝑛= 0 (𝑙= ±1, 𝑗= 1, 2), (12) где U(𝑙) 𝑛= (︁ 𝑈(𝑙) 1𝑛(𝑟𝑙), 𝑈(𝑙) 2𝑛(𝑟𝑙), 𝑈(𝑙) 3𝑛(𝑟𝑙) )︁𝑇 ; ^A(𝑙) 𝑛= (𝑎𝑛𝑝𝑞)3×3, ^B(𝑙) 𝑛= (𝑏𝑛𝑝𝑞)3×3, ^C(𝑙) 𝑛= (𝑐𝑛𝑝𝑞)3×3 — матрицы третьего порядка с элементами (12) 𝑛 𝑛 𝑛 𝑛 𝑛 𝑛 ( , 𝑗 , ), ( ) где U(𝑙) 𝑛= (︁ 𝑈(𝑙) 1𝑛(𝑟𝑙), 𝑈(𝑙) 2𝑛(𝑟𝑙), 𝑈(𝑙) 3𝑛(𝑟𝑙) )︁𝑇 ; ^A(𝑙) 𝑛= (𝑎𝑛𝑝𝑞)3×3, ^B(𝑙) 𝑛= (𝑏𝑛𝑝𝑞)3×3, ^C(𝑙) 𝑛= (𝑐𝑛𝑝𝑞)3×3 — матрицы третьего порядка с элементами 𝑎(𝑙) 𝑛11 = (𝜆+ 2𝜇) 𝑟2 𝑙, 𝑎(𝑙) 𝑛22 = 𝑎(𝑙) 𝑛33 = 𝜇𝑟2 𝑙, 𝑎(𝑙) 𝑛𝑝𝑞= 0 (𝑝̸= 𝑞) , 𝑏(𝑙) 𝑛11 = (︀ 𝜆′ + 2𝜇′)︀ 𝑟2 𝑙+ (𝜆+ 2𝜇) 𝑟𝑙, 𝑏(𝑙) 𝑛12 = 𝑖𝑛(𝜆+ 𝜇) 𝑟𝑙, 𝑏(𝑙) 𝑛13 = 𝑖𝛼(𝜆+ 𝜇) 𝑟2 𝑙, 𝑏(𝑙) 𝑛21 = 𝑖𝑛(𝜆+ 𝜇) 𝑟𝑙, 𝑏(𝑙) 𝑛22 = 𝜇′𝑟2 𝑙+ 𝜇𝑟𝑙, 𝑏(𝑙) 𝑛23 = 0, 𝑏(𝑙) 𝑛31 = 𝑖𝛼(𝜆+ 𝜇) 𝑟2 𝑙, 𝑏(𝑙) 𝑛32 = 0, 𝑏(𝑙) 𝑛33 = 𝜇′𝑟2 𝑙+ 𝜇𝑟𝑙, 𝑐(𝑙) 𝑛11 = 𝜆′𝑟𝑙−𝜆− (︀ 𝑛2 + 2 + 𝛼2𝑟2 𝑙 )︀ 𝜇+ 𝜔𝜌𝑟2 𝑙, 𝑐(𝑙) 𝑛12 = 𝑖𝑛 (︀ 𝜆′𝑟𝑙−𝜆−3𝜇 )︀ , 𝑐(𝑙) 𝑛13 = 𝑖𝛼𝜆′𝑟2 𝑙, 𝑐(𝑙) 𝑛22 = −𝜇′𝑟𝑙−𝑛2𝜆− (︀ 2𝑛2 + 𝛼2𝑟2 𝑙+ 1 )︀ 𝜇+ 𝜔2𝜌𝑟2 𝑙, 𝑐(𝑙) 𝑛23 = −𝑛𝛼(𝜆+ 𝜇) 𝑟𝑙, 𝑐(𝑙) 𝑛31 = 𝑖𝛼 [︀ 𝜇′𝑟2 𝑙+ (𝜆+ 𝜇) 𝑟𝑙 ]︀ , 𝑐(𝑙) 𝑛32 = −𝑛𝛼(𝜆+ 𝜇) 𝑟𝑙, 𝑐(𝑙) 𝑛33 = − (︀ 𝑛2 + 2𝛼2𝑟2 𝑙 )︀ 𝜇−𝛼2𝜆𝑟2 𝑙+ 𝜔2𝜌𝑟2 𝑙, 𝜆= 𝜆(𝑟𝑙), 𝜇= 𝜇(𝑟𝑙). 𝑎(𝑙) 𝑛11 = (𝜆+ 2𝜇) 𝑟2 𝑙, 𝑎(𝑙) 𝑛22 = 𝑎(𝑙) 𝑛33 = 𝜇𝑟2 𝑙, 𝑎(𝑙) 𝑛𝑝𝑞= 0 (𝑝̸= 𝑞) , ︀︀ 𝜆= 𝜆(𝑟𝑙), 𝜇= 𝜇(𝑟𝑙). Искомые функции Ψ(𝑙) 𝑠𝑗, 𝐹(𝑙) 𝑗, 𝐿(𝑙) 𝑗, 𝑀(𝑙) 𝑗, 𝑢(𝑙) 𝑟, 𝑢(𝑙) 𝜙, 𝑢(𝑙) 𝑧 (𝑙= ±1, 𝑗= 1, 2) должны удовлетворять граничным условиям. Граничные условия на внешней поверхности неоднородного покрытия 𝑙– го цилиндра заключаются в равенстве нормальных скоростей частиц упругой неоднородной среды и жид- кости, равенстве на ней нормального напряжения и акустического давления, отсутствии ка- сательных напряжений: при 𝑟𝑙= 𝑟1 −𝑖𝜔𝑢(𝑙) 𝑟 = 𝑣𝑟, 𝜎(𝑙) 𝑟𝑟= −𝑝, 𝜎(𝑙) 𝑟𝜙= 0, 𝜎(𝑙) 𝑟𝑧= 0. 3. Аналитическое решение задачи Используя соотношения, связывающие компоненты тензора напряжений с компонентами вектора смещения, запишем уравнения (10) через компоненты вектора смещения [3] в локаль- ных цилиндрических координатах. Рассеяние наклонно падающей плоской звуковой волны . . . 375 375 Компоненты вектора смещения u(𝑙) в неоднородном упругом покрытии 𝑙– го цилиндра будем искать в виде рядов Фурье 𝑢(𝑙) 𝑟(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︀ 𝑛=−∞ 𝑈(𝑙) 1𝑛(𝑟𝑙)𝑒𝑖𝑛(𝜙𝑙−𝜙0), 𝑢(𝑙) 𝜙(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︀ 𝑛=−∞ 𝑈(𝑙) 2𝑛(𝑟𝑙)𝑒𝑖𝑛(𝜙𝑙−𝜙0), (11) 𝑢(𝑙) 𝑟(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︀ 𝑛=−∞ 𝑈(𝑙) 1𝑛(𝑟𝑙)𝑒𝑖𝑛(𝜙𝑙−𝜙0), 𝑢(𝑙) 𝜙(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︀ 𝑛=−∞ 𝑈(𝑙) 2𝑛(𝑟𝑙)𝑒𝑖𝑛(𝜙𝑙−𝜙0), (11) (11) ︀︀ ( ) 𝑢(𝑙) 𝑧(𝑟𝑙, 𝜙𝑙, 𝑧𝑙) = 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︁ 𝑛=−∞ 𝑈(𝑙) 3𝑛(𝑟𝑙)𝑒𝑖𝑛(𝜙𝑙−𝜙0). 3. Аналитическое решение задачи (13) (13) На внутренней поверхности покрытия 𝑙– го цилиндра при переходе через границу раздела упругих сред должны быть непрерывны составляющие вектора смещения частиц, а также нормальные и тангенциальные напряжения: при 𝑟𝑙= 𝑟0 𝑢(𝑙) 𝑟 = 𝑢(𝑙) 0𝑟, 𝑢(𝑙) 𝜙= 𝑢(𝑙) 0𝜙, 𝑢(𝑙) 𝑧 = 𝑢(𝑙) 0𝑧, 𝜎(𝑙) 𝑟𝑟= 𝜎(𝑙) 0𝑟𝑟, 𝜎(𝑙) 𝑟𝜙= 𝜎(𝑙) 0𝑟𝜙, 𝜎(𝑙) 𝑟𝑧= 𝜎(𝑙) 0𝑟𝑧, (14) (14) где 𝜎(𝑙) 0𝑝𝑞— компоненты тензора напряжений в однородном 𝑙– ом цилиндре (индекс 𝑗опущен). где 𝜎(𝑙) 0𝑝𝑞— компоненты тензора напряжений в однородном 𝑙– ом цилиндре (индекс 𝑗опущен). Л. А. Толоконников, Д. Ю. Ефимов 376 Компоненты вектора смещения u(𝑙) 0𝑗и тензора напряжений 𝜎(𝑙) 0𝑝𝑞в однородном упругом 𝑙– ом цилиндре запишем через функции 𝐹(𝑙) 𝑗, 𝐿(𝑙) 𝑗 и 𝑀(𝑙) 𝑗, а компоненты тензора напряжений в неоднородном покрытии 𝑙– го цилиндра 𝜎(𝑙) 𝑝𝑞— через компоненты вектора смещения u(𝑙) [3]. С помощью теоремы сложения для волновых цилиндрических функций [9] из первых двух граничных условий (13) получаем две бесконечные системы линейных алгебраических урав- нений (при 𝑖= 1 и 𝑖= 2) относительно неизвестных коэффициентов 𝐴(𝑙) 𝑗𝑛(𝑙= ±1) 𝐴(𝑙) 𝑗𝑛+ ∞ ∑︁ 𝑚=−∞ 𝛼(−𝑙,𝑙) 𝑖𝑛𝑚𝐴(−𝑙) 𝑗𝑚= 𝑆(𝑙) 𝑖𝑛 (𝑖= 1, 2; 𝑛= 0, ±1, ±2, . . . ; 𝑙= ±1), (15) (15) где 𝛼(−𝑙,𝑙) 1𝑛𝑚= 𝐽′ 𝑛(𝛽𝑟1) 𝐻′𝑛(𝛽𝑟1)𝐻𝑚−𝑛(2𝛽𝑑)𝑒𝑖(𝑚−𝑛)(𝜙−𝑙,𝑙−𝜙0), 𝛼(−𝑙,𝑙) 2𝑛𝑚= 𝐽𝑛(𝛽𝑟1) 𝐻𝑛(𝛽𝑟1)𝐻𝑚−𝑛(2𝛽𝑑)𝑒𝑖(𝑚−𝑛)(𝜙−𝑙,𝑙−𝜙0), 𝑆(𝑙) 1𝑛= −𝐴𝑖𝑛𝐽′ 𝑛(𝛽𝑟1) 𝐻′𝑛(𝛽𝑟1)𝑒𝑖𝛽𝑑𝑙sin 𝜙0 −𝑖𝜔𝑈(𝑙) 1𝑛(𝑟1) 𝛽𝐻′𝑛(𝛽𝑟1) , 𝑆(𝑙) 2𝑛= −𝐴𝑖𝑛𝐽𝑛(𝛽𝑟1) 𝐻𝑛(𝛽𝑟1)𝑒𝑖𝛽𝑑𝑙sin 𝜙0+ + 𝑖 𝜌1𝜔𝐻𝑛(𝛽𝑟1) [︂ (𝜆(𝑟1) + 2𝜇(𝑟1)) 𝑈(𝑙)′ 1𝑛(𝑟1) + 𝜆(𝑟1) 𝑟1 (︁ 𝑈(𝑙) 1𝑛(𝑟1) + 𝑖𝑛𝑈(𝑙) 2𝑛(𝑟1) )︁ + 𝑖𝛼𝜆(𝑟𝑙)𝑈(𝑙) 3𝑛(𝑟𝑙) ]︂ , (2 𝑙)/2 𝛼(−𝑙,𝑙) 1𝑛𝑚= 𝐽′ 𝑛(𝛽𝑟1) 𝐻′𝑛(𝛽𝑟1)𝐻𝑚−𝑛(2𝛽𝑑)𝑒𝑖(𝑚−𝑛)(𝜙−𝑙,𝑙−𝜙0), 𝛼(−𝑙,𝑙) 2𝑛𝑚= 𝐽𝑛(𝛽𝑟1) 𝐻𝑛(𝛽𝑟1)𝐻𝑚−𝑛(2𝛽𝑑)𝑒𝑖(𝑚−𝑛)(𝜙−𝑙,𝑙−𝜙0), (𝑙) 𝐽′ (𝛽𝑟1) 𝑖𝜔𝑈(𝑙)(𝑟1) (𝑙) 𝐽(𝛽𝑟1) 𝛼(−𝑙,𝑙) 1𝑛𝑚= 𝐽′ 𝑛(𝛽𝑟1) 𝐻′𝑛(𝛽𝑟1)𝐻𝑚−𝑛(2𝛽𝑑)𝑒𝑖(𝑚−𝑛)(𝜙−𝑙,𝑙−𝜙0), 𝛼(−𝑙,𝑙) 2𝑛𝑚= 𝐽𝑛(𝛽𝑟1) 𝐻𝑛(𝛽𝑟1)𝐻𝑚−𝑛(2𝛽𝑑)𝑒𝑖(𝑚−𝑛)(𝜙−𝑙,𝑙−𝜙0), 𝑆(𝑙) 1𝑛= −𝐴𝑖𝑛𝐽′ 𝑛(𝛽𝑟1) 𝐻′𝑛(𝛽𝑟1)𝑒𝑖𝛽𝑑𝑙sin 𝜙0 −𝑖𝜔𝑈(𝑙) 1𝑛(𝑟1) 𝛽𝐻′𝑛(𝛽𝑟1) , 𝑆(𝑙) 2𝑛= −𝐴𝑖𝑛𝐽𝑛(𝛽𝑟1) 𝐻𝑛(𝛽𝑟1)𝑒𝑖𝛽𝑑𝑙sin 𝜙0+ + 𝑖 𝜌1𝜔𝐻𝑛(𝛽𝑟1) [︂ (𝜆(𝑟1) + 2𝜇(𝑟1)) 𝑈(𝑙)′ 1𝑛(𝑟1) + 𝜆(𝑟1) 𝑟1 (︁ 𝑈(𝑙) 1𝑛(𝑟1) + 𝑖𝑛𝑈(𝑙) 2𝑛(𝑟1) )︁ + 𝑖𝛼𝜆(𝑟𝑙)𝑈(𝑙) 3𝑛(𝑟𝑙) ]︂ , 𝜙−𝑙,𝑙= 𝜋(2 −𝑙)/2, штрихи означают дифференцирование по аргументу функций. штрихи означают дифференцирование по аргументу функций. ( ) Для решения бесконечных систем линейных уравнений (15) воспользуемся методом усече- ния [14]. Как и в [9] методом обратной матрицы найдем решения двух усеченных систем (15) при 𝑖= 1 и 𝑖= 2, выбрав порядок усечения 𝑁. Получим два выражения для коэффициентов 𝐴(𝑙) 𝑗𝑛, записанных через значения функций 𝑈(𝑙) 1𝑛(𝑟𝑙), 𝑈(𝑙) 2𝑛(𝑟𝑙), 𝑈(𝑙) 3𝑛(𝑟𝑙) на внешней поверхности покрытия (при 𝑟𝑙= 𝑟1). 3. Аналитическое решение задачи Приравнивая между собой эти выражения, получаем краевое условие при 𝑟𝑙= 𝑟1 для нахождения решения системы дифференциальных уравнений (12). Условия отсутствия касательных напряжений в (13) дают еще два краевых условия при 𝑟𝑙= 𝑟1 ( ) ( )′ ( ) ( ) ствия касательных напряжений в (13) дают еще два краевых условия при V(𝑙) 𝑛(𝑟1)U(𝑙)′ 𝑛 + W(𝑙) 𝑛(𝑟1)U(𝑙) 𝑛= 0, V(𝑙) 𝑛(𝑟1)U(𝑙)′ 𝑛 + W(𝑙) 𝑛(𝑟1)U(𝑙) 𝑛= 0, (16) 𝑙 1 V(𝑙) 𝑛(𝑟1)U(𝑙)′ 𝑛 + W(𝑙) 𝑛(𝑟1)U(𝑙) 𝑛= 0, (16) где где V(𝑙) 𝑛(𝑟1) = (︁ 𝑣(𝑙) 𝑛𝑝𝑞 )︁ 2×3 , W(𝑙) 𝑛(𝑟1) = (︁ 𝑤(𝑙) 𝑛𝑝𝑞 )︁ 2×3 ; 𝑣(𝑙) 𝑛11 = 𝑣(𝑙) 𝑛13 = 𝑣(𝑙) 𝑛21 = 𝑣(𝑙) 𝑛22 = 0, 𝑣(𝑙) 𝑛12 = 𝑣(𝑙) 𝑛23 = 𝜇(𝑟1) 𝑤(𝑙) 𝑛11 = 𝑖𝑛𝜇(𝑟1)/𝑟1, 𝑤(𝑙) 𝑛12 = −𝜇(𝑟1)/𝑟1, 𝑤(𝑙) 𝑛21 = 𝑖𝛼𝜇(𝑟1), 𝑤(𝑙) 𝑛13 = 𝑤(𝑙) 𝑛22 = 𝑤(𝑙) 𝑛23 = 0. Из первых трех граничных условий (14) выразим неизвестные коэффициенты 𝐵(𝑙) 𝑗𝑛, 𝐶(𝑙) 𝑗𝑛, 𝐷(𝑙) 𝑗𝑛через значения функций 𝑈(𝑙) 1𝑛(𝑟𝑙), 𝑈(𝑙) 2𝑛(𝑟𝑙), 𝑈(𝑙) 3𝑛(𝑟𝑙) на внутренней поверхности покрытия (при 𝑟𝑙= 𝑟0). Получаем K(𝑙) 𝑛= [︁ T(𝑙) 𝑛(𝑟0) ]︁−1 U(𝑙) 𝑛 (𝑙= ±1), (17) (17) где где где ︁︁ K(𝑙) 𝑛= (︁ 𝐵(𝑙) 𝑗𝑛, 𝐶(𝑙) 𝑗𝑛, 𝐷(𝑙) 𝑗𝑛 )︁𝑇 , T(𝑙) 𝑛(𝑟0) = (︁ 𝑡(𝑙) 𝑛𝑝𝑞 )︁ 3×3 ; Рассеяние наклонно падающей плоской звуковой волны . . . 377 Рассеяние наклонно падающей плоской звуковой волны . . . 377 𝑡(𝑙) 𝑛11 = 𝑘1𝐽′ 𝑛(𝑘1𝑟0), 𝑡(𝑙) 𝑛12 = 𝑖𝛼𝑘2𝐽′ 𝑛(𝑘2𝑟0), 𝑡(𝑙) 𝑛13 = 𝑖𝑛𝑘𝜏𝐽𝑛(𝑘2𝑟0)/𝑟0, 𝑡(𝑙) 𝑛21 = 𝑖𝑛𝐽𝑛(𝑘1𝑟0)/𝑟0, 𝑡(𝑙) 𝑛22 = −𝛼𝑛𝐽𝑛(𝑘2𝑟0)/𝑟0, 𝑡(𝑙) 𝑛23 = −𝑘𝜏𝑘2𝐽′ 𝑛(𝑘2𝑟0), 𝑡(𝑙) 𝑛31 = 𝑖𝛼𝐽𝑛(𝑘1𝑟0), 𝑡(𝑙) 𝑛32 = (𝑘2 𝜏−𝛼2)𝐽𝑛(𝑘2𝑟0), 𝑡(𝑙) 𝑛33 = 0. 𝑡(𝑙) 𝑛11 = 𝑘1𝐽′ 𝑛(𝑘1𝑟0), 𝑡(𝑙) 𝑛12 = 𝑖𝛼𝑘2𝐽′ 𝑛(𝑘2𝑟0), 𝑡(𝑙) 𝑛13 = 𝑖𝑛𝑘𝜏𝐽𝑛(𝑘2𝑟0)/𝑟0, 𝑡(𝑙) 𝑛21 = 𝑖𝑛𝐽𝑛(𝑘1𝑟0)/𝑟0, 𝑡(𝑙) 𝑛22 = −𝛼𝑛𝐽𝑛(𝑘2𝑟0)/𝑟0, 𝑡(𝑙) 𝑛23 = −𝑘𝜏𝑘2𝐽′ 𝑛(𝑘2𝑟0), 𝑡(𝑙) 𝑛31 = 𝑖𝛼𝐽𝑛(𝑘1𝑟0), 𝑡(𝑙) 𝑛32 = (𝑘2 𝜏−𝛼2)𝐽𝑛(𝑘2𝑟0), 𝑡(𝑙) 𝑛33 = 0. 3. Аналитическое решение задачи Из последних трех граничных условий (14) находим Из последних трех граничных условий (14) находим Из последних трех граничных условий (14) находим E(𝑙) 𝑛(𝑟0)U(𝑙)′ 𝑛(𝑟0) + H(𝑙) 𝑛(𝑟0)U(𝑙) 𝑛(𝑟0) = G(𝑙) 𝑛(𝑟0)K(𝑙) 𝑛 (𝑙= ±1), (18) E(𝑙) 𝑛(𝑟0)U(𝑙)′ 𝑛(𝑟0) + H(𝑙) 𝑛(𝑟0)U(𝑙) 𝑛(𝑟0) = G(𝑙) 𝑛(𝑟0)K(𝑙) 𝑛 (𝑙= ±1), (18) (18) где где E(𝑙) 𝑛(𝑟0) = (︁ 𝑒(𝑙) 𝑛𝑝𝑞 )︁ 3×3 , H(𝑙) 𝑛(𝑟0) = (︁ ℎ(𝑙) 𝑛𝑝𝑞 )︁ 3×3 , G(𝑙) 𝑛(𝑟𝑙) = (︁ 𝑔(𝑙) 𝑛𝑝𝑞 )︁ 3×3 ; 𝑒(𝑙) 𝑛11 = 𝜆(𝑟0) + 2𝜇(𝑟0) , 𝑒(𝑙) 𝑛22 = 𝑒(𝑙) 𝑛33 = 𝜇(𝑟0) , 𝑒(𝑙) 𝑛𝑖𝑗= 0 (𝑝̸= 𝑞), ℎ(𝑙) 𝑛11 = 𝜆(𝑟0)/𝑟0, ℎ(𝑙) 𝑛12 = 𝑖𝑛𝜆(𝑟0)/𝑟0, ℎ(𝑙) 𝑛13 = 𝑖𝛼𝜆(𝑟0), ℎ(𝑙) 𝑛21 = 𝑖𝑛𝜇(𝑟0)/𝑟0, ℎ(𝑙) 𝑛22 = −𝜇(𝑟0)/𝑟0, ℎ(𝑙) 𝑛23 = ℎ(𝑙) 𝑛32 = ℎ(𝑙) 𝑛33 = 0, ℎ(𝑙) 𝑛31 = 𝑖𝛼𝜇(𝑟0), 𝑔(𝑙) 𝑛11 = −𝜆0𝑘2 𝜂𝐽𝑛(𝑘1𝑟0) + 2𝜇0𝑘2 1𝐽′′ 𝑛(𝑘1𝑟0) , 𝑔(𝑙) 𝑛12 = 2𝑖𝛼𝜇0𝑘2 2𝐽′′ 𝑛(𝑘2𝑟0) , 𝑔(𝑙) 𝑛13 = 2𝑖𝑛𝜇0𝑘𝜏 1 𝑟0 [︂ 𝑘2𝐽′ 𝑛(𝑘2𝑟0) −1 𝑟0 𝐽𝑛(𝑘2𝑟0) ]︂ , 𝑔(𝑙) 𝑛21 = 2𝑖𝑛𝜇0 1 𝑟0 [︂ 𝑘1𝐽′ 𝑛(𝑘1𝑟0) −1 𝑟0 𝐽𝑛(𝑘1𝑟0) ]︂ , 𝑔(𝑙) 𝑛22 = −2𝛼𝑛𝜇0 1 𝑟0 [︂ 𝑘2𝐽′ 𝑛(𝑘2𝑟0) −1 𝑟0 𝐽𝑛(𝑘2𝑟0) ]︂ , 𝑔(𝑙) 𝑛23 = −𝜇0𝑘𝜏 [︂ 𝑘2 2𝐽′′ 𝑛(𝑘2𝑟0) −𝑘2 1 𝑟0 𝐽′ 𝑛(𝑘2𝑟0) + 𝑛2 1 𝑟2 0 𝐽𝑛(𝑘2𝑟0) ]︂ , 𝑔(𝑙) 2𝑖𝛼𝜇𝑘𝐽′ (𝑘𝑟) 𝑔(𝑙) 𝜇𝑘 (︀ 𝑘2 2𝛼2)︀ 𝐽′ (𝑘𝑟) 𝑔(𝑙) 𝛼𝑛𝜇𝑘𝐽(𝑘𝑟)/𝑟 де E(𝑙) 𝑛(𝑟0) = (︁ 𝑒(𝑙) 𝑛𝑝𝑞 )︁ 3×3 , H(𝑙) 𝑛(𝑟0) = (︁ ℎ(𝑙) 𝑛𝑝𝑞 )︁ 3×3 , G(𝑙) 𝑛(𝑟𝑙) = (︁ 𝑔(𝑙) 𝑛𝑝𝑞 )︁ 3×3 ; 𝑒(𝑙) 𝑛11 = 𝜆(𝑟0) + 2𝜇(𝑟0) , 𝑒(𝑙) 𝑛22 = 𝑒(𝑙) 𝑛33 = 𝜇(𝑟0) , 𝑒(𝑙) 𝑛𝑖𝑗= 0 (𝑝̸= 𝑞), ℎ(𝑙) 𝑛11 = 𝜆(𝑟0)/𝑟0, ℎ(𝑙) 𝑛12 = 𝑖𝑛𝜆(𝑟0)/𝑟0, ℎ(𝑙) 𝑛13 = 𝑖𝛼𝜆(𝑟0), (𝑙) (𝑙) (𝑙) (𝑙) (𝑙) (𝑙) E(𝑙) 𝑛(𝑟0) = (︁ 𝑒(𝑙) 𝑛𝑝𝑞 )︁ 3×3 , H(𝑙) 𝑛(𝑟0) = (︁ ℎ(𝑙) 𝑛𝑝𝑞 )︁ 3×3 , G(𝑙) 𝑛(𝑟𝑙) = (︁ 𝑔(𝑙) 𝑛𝑝𝑞 )︁ 3×3 ; 𝑔𝑛11 0 𝜂 ( 0) 𝜇0 1 𝑛( 0) , 𝑔𝑛12 𝜇0 2 𝑛( 0) , 𝑔(𝑙) 𝑛13 = 2𝑖𝑛𝜇0𝑘𝜏 1 𝑟0 [︂ 𝑘2𝐽′ 𝑛(𝑘2𝑟0) −1 𝑟0 𝐽𝑛(𝑘2𝑟0) ]︂ , 𝑔(𝑙) 𝑛21 = 2𝑖𝑛𝜇0 1 𝑟0 [︂ 𝑘1𝐽′ 𝑛(𝑘1𝑟0) −1 𝑟0 𝐽𝑛(𝑘1𝑟0) ]︂ , 𝑔(𝑙) 𝑛22 = −2𝛼𝑛𝜇0 1 𝑟0 [︂ 𝑘2𝐽′ 𝑛(𝑘2𝑟0) −1 𝑟0 𝐽𝑛(𝑘2𝑟0) ]︂ , 𝑔(𝑙) 𝑛23 = −𝜇0𝑘𝜏 [︂ 𝑘2 2𝐽′′ 𝑛(𝑘2𝑟0) −𝑘2 1 𝑟0 𝐽′ 𝑛(𝑘2𝑟0) + 𝑛2 1 𝑟2 0 𝐽𝑛(𝑘2𝑟0) ]︂ ,︀︀ ︂︂ 𝑔(𝑙) 𝑛31 = 2𝑖𝛼𝜇0𝑘1𝐽′ 𝑛(𝑘1𝑟0) , 𝑔(𝑙) 𝑛32 = 𝜇0𝑘2 (︀ 𝑘2 𝜏−2𝛼2)︀ 𝐽′ 𝑛(𝑘2𝑟0) , 𝑔(𝑙) 𝑛33 = −𝛼𝑛𝜇0𝑘𝜏𝐽𝑛(𝑘2𝑟0)/𝑟0. 3. Аналитическое решение задачи Подставляя (17) в (18), получаем три краевых условия для системы (12) при 𝑟𝑙= 𝑟0 ︂︂ 𝑔(𝑙) 𝑛31 = 2𝑖𝛼𝜇0𝑘1𝐽′ 𝑛(𝑘1𝑟0) , 𝑔(𝑙) 𝑛32 = 𝜇0𝑘2 (︀ 𝑘2 𝜏−2𝛼2)︀ 𝐽′ 𝑛(𝑘2𝑟0) , 𝑔(𝑙) 𝑛33 = −𝛼𝑛𝜇0𝑘𝜏𝐽𝑛(𝑘2𝑟0)/𝑟0. Подставляя (17) в (18), получаем три краевых условия для системы (12) при 𝑟𝑙= 𝑟0 E(𝑙) 𝑛(𝑟0)𝑈(𝑙)′ 𝑛(𝑟0) + Y(𝑙) 𝑛(𝑟0)𝑈(𝑙) 𝑛(𝑟0) = 0 (𝑙= ±1), E(𝑙) 𝑛(𝑟0)𝑈(𝑙)′ 𝑛(𝑟0) + Y(𝑙) 𝑛(𝑟0)𝑈(𝑙) 𝑛(𝑟0) = 0 (𝑙= ±1), (19) (19) где Y(𝑙) 𝑛(𝑟0) = H(𝑙) 𝑛(𝑟0) −G(𝑙) 𝑛(𝑟0) [︁ T(𝑙) 𝑛(𝑟0) ]︁−1 . ︁︁ Таким образом, для нахождения функций 𝑈(𝑙) 1𝑛(𝑟𝑙), 𝑈(𝑙) 2𝑛(𝑟𝑙) и 𝑈(𝑙) 3𝑛(𝑟𝑙) (𝑙= ±1) для всех 𝑛 (𝑛= 0, ±1, ±2, . . . , ±𝑁) необходимо найти решение системы 6(2𝑁+ 1) обыкновенных диф- ференциальных уравнений (12), удовлетворяющих полученным выше краевым условиям. По- строенная краевая задача может быть решена каким-либо численным или аналитическим методом. д Затем находим неизвестные коэффициенты 𝐴(𝑙) 𝑗𝑛, 𝐵(𝑙) 𝑗𝑛, 𝐶(𝑙) 𝑗𝑛и 𝐷(𝑙) 𝑗𝑛разложений (6) – (9) (𝑙= ±1; 𝑗= 1, 2; 𝑛= 0, ±1, ±2, . . . , ±𝑁). В результате на основании (4) – (6) получаем аналитическое описание акустического поля, рассеянного цилиндром с покрытием в присутствии плоскости Ψ𝑠= 2 ∑︁ 𝑗=1 ∑︁ 𝑙=±1 𝑒𝑖𝛼𝑧𝑙 ∞ ∑︁ 𝑛=−∞ [︁ ±𝐴𝑒−𝑖𝑑𝑙𝛽sin 𝜙0𝑖𝑛𝐽𝑛(𝛽𝑟𝑙) + 𝐴(𝑙) 𝑗𝑛𝐻𝑛(𝛽𝑟𝑙) ]︁ 𝑒𝑖𝑛(𝜙𝑙−𝜙0). (20) (20) ︁︁︁︁︁ Отметим, что, используя полученное решение Ψ𝑠1 дифракционной задачи для случая, ко- гда падающая плоская волна имеет потенциал скорости Ψ01, легко записать решение задачи дифракции плоской волны с потенциалом скорости Ψ02. Для этого достаточно заменить ком- поненты волнового вектора k1 на компоненты вектора k2, если подстилающая плоскость яв- ляется акустически жесткой, и дополнительно заменить амплитуду 𝐴на −𝐴, если плоскость — акустически мягкая. 378 Л. А. Толоконников, Д. Ю. Ефимов 4. Численные исследования На основе полученного аналитического решения задачи были проведены численные рас- четы частотных и угловых характеристик рассеянного поля в дальней зоне. Краевая задача для системы (12) решалась методом сплайн-коллокации [15]. Каждая функция 𝑈(𝑙) 1𝑛(𝑟𝑙), 𝑈(𝑙) 2𝑛(𝑟𝑙), 𝑈(𝑙) 3𝑛(𝑟𝑙) представлялась в виде разложения по базисным 𝐵– сплай- нам. На большом удалении от цилиндра (𝑘𝑟𝑙⩾1) справедливы соотношения 𝑟𝑙≈𝑟−𝑙𝑑sin 𝜙, 𝜙𝑙≈𝜙 (𝑟>> 𝑑; 𝑙= ±1). (21) (21) Расчеты проводились по формуле (20) в основной системе координат (𝑟, 𝜙, 𝑧) с учетом (21) при 𝑟= 100 м Расчеты проводились по формуле (20) в основной системе координат (𝑟, 𝜙, 𝑧) с учетом (21) при 𝑟= 100 м. Рассматривался алюминиевый цилиндр (𝜌0 = 2, 7 · 103 кг/м3, 𝜆0 = 5, 3 · 1010 Н/м2, 𝜇0 = 2, 6 · 1010 Н/м2) радиуса 𝑟0 = 0, 8 м с покрытием толщиной 0,2 м, находящийся в по- лупространстве, заполненном водой (𝜌1 = 1000 кг/м3, 𝑐= 1485 м/с). Покрытие цилиндра вы- полнено на основе полимерного материала, неоднородного по плотности, с характерной плот- ностью ˜𝜌= 1070 кг/м3 и характерными модулями упругости ˜𝜆= 3, 9 · 109 Н/м2, ˜𝜇= 9, 8 · 108 Н/м2 (поливинилбутираль). Расстояние от оси цилиндра до плоскости 𝑑= 2 м. Полагалось, что плоская звуковая волна единичной амплитуды падает на тело под углами 𝜃0 = 𝜋/4 и 𝜙0 = −𝜋/6. Механические характеристики материала неоднородного покрытия менялись по толщине слоя по законам толщине слоя по законам 𝜌= ˜𝜌𝑓(𝑟+1), 𝜆= ˜𝜆, 𝜇= ˜𝜇, 𝑓(𝑟+1) = 0, 75 [︃(︂𝑟+1 −𝑟0 𝑟1 −𝑟0 )︂2 + 1 ]︃ , 𝑟0 ⩽𝑟+1 ⩽𝑟1. 𝜌= ˜𝜌𝑓(𝑟+1), 𝜆= ˜𝜆, 𝜇= ˜𝜇, 𝜌= ˜𝜌𝑓(𝑟+1), 𝜆= ˜𝜆, 𝜇= ˜𝜇, где 𝑓(𝑟+1) = 0, 75 [︃(︂𝑟+1 −𝑟0 𝑟1 −𝑟0 )︂2 + 1 ]︃ , 𝑟0 ⩽𝑟+1 ⩽𝑟1. На рис. 2 представлены частотные зависимости амплитуды обратного рассеяния звука ⃒⃒⃒⃒ Ψ𝑠 𝐴 ⃒⃒⃒⃒ от волнового размера тела 𝑘𝑟1 в интервале 0 < 𝑘𝑟1 ⩽10 (𝜙= 5/6𝜋) в случае абсолютно жест- кой подстилающей поверхности. Сплошная линия соответствует случаю упругого цилиндра с неоднородным упругим покрытием, пунктирная — упругому цилиндру без покрытия. На рис. 2 представлены частотные зависимости амплитуды обратного рассеяния звука ⃒⃒⃒⃒ Ψ𝑠 𝐴 ⃒⃒⃒⃒ от волнового размера тела 𝑘𝑟1 в интервале 0 < 𝑘𝑟1 ⩽10 (𝜙= 5/6𝜋) в случае абсолютно жест- кой подстилающей поверхности. Сплошная линия соответствует случаю упругого цилиндра с неоднородным упругим покрытием, пунктирная — упругому цилиндру без покрытия. С На рис. 2 представлены частотные зависимости амплитуды обратного рассеяния звука ⃒⃒⃒⃒ Ψ𝑠 𝐴 ⃒⃒⃒⃒ На рис. 4. Численные исследования 2 представлены частотные зависимости амплитуды обратного рассеяния звука ⃒⃒⃒⃒ Ψ𝑠 𝐴 ⃒⃒⃒⃒ от волнового размера тела 𝑘𝑟1 в интервале 0 < 𝑘𝑟1 ⩽10 (𝜙= 5/6𝜋) в случае абсолютно жест- кой подстилающей поверхности. Сплошная линия соответствует случаю упругого цилиндра с На рис. 2 представлены частотные зависимости амплитуды обратного рассея На рис. 2 представлены частотные зависимости амплитуды обратного рассеяния звука ⃒⃒⃒⃒ Ψ𝑠 𝐴 ⃒⃒⃒⃒ ⃒ ⃒ от волнового размера тела 𝑘𝑟1 в интервале 0 < 𝑘𝑟1 ⩽10 (𝜙= 5/6𝜋) в случае абсолютно жест- кой подстилающей поверхности. Сплошная линия соответствует случаю упругого цилиндра с неоднородным упругим покрытием, пунктирная — упругому цилиндру без покрытия. Сравнение частотных зависимостей показывает существенное влияние неоднородного по- крытия на звукоотражающие свойства цилиндра. Это проявляется в смещении максимумов и минимумов коэффициента обратного отражения и изменении их уровней. Отличие частотных характеристик для тела с покрытием и без него возрастает с увеличением частоты падающей звуковой волны. Рассеяние наклонно падающей плоской звуковой волны . . . 379 Рис. 2: Зависимость амплитуды обратного рассеяния звука от волнового размера цилиндра Рис. 2: Зависимость амплитуды обратного рассеяния звука от волнового размера цилиндра Рис. 3: Диаграммы направленности рассеянного поля Рис. 3: Диаграммы направленности рассеянного поля На рис. 3 представлены диаграммы направленности поля, рассеянного упругим цилиндром с однородным покрытием (𝜌= ˜𝜌, 𝜆= ˜𝜆, 𝜇= ˜𝜇) при 𝑘𝑟1 = 3. Зависимости ⃒⃒⃒⃒ Ψ𝑠 𝐴 ⃒⃒⃒⃒от 𝜙рассчитаны в плоскости 𝑥𝑦. Сплошной линией изображена диаграмма для случая наклонного падения плоской волны (𝜃0 = 𝜋/4), а пунктирная линия соответствует случаю нормального падения (𝜃0 = 𝜋/2). Стрелкой показано направление падения плоской звуковой волны (𝜙0 = −𝜋/6). Из рисунка видно, что диаграмма направленности при наклонном падении плоской волны на цилиндр существенно отличается от диаграммы, соответствующей нормальному падению. Из рисунка видно, что диаграмма направленности при наклонном падении плоской волны на цилиндр существенно отличается от диаграммы, соответствующей нормальному падению. 5. Заключение Выявлено существенное влияние непрерывно-неоднородных упругих покрытий на звуко- отражающие свойства упругих цилиндрических тел. С помощью непрерывно-неоднородных покрытий можно эффективно изменять характеристики рассеяния тел в определенных на- правлениях, если подобрать соответствующие законы неоднородности для механических пара- метров покрытия. Такое покрытие можно реализовать с помощью дискретной системы тонких Л. А. Толоконников, Д. Ю. Ефимов 380 однородных упругих слоев, имеющих различные значения механических параметров (плотно- сти и упругих постоянных). REFERENCES 1. Romanov, A. G. & Tolokonnikov, L. A. 2011, “The scattering of acoustic waves by a cylinder with a non-uniform elastic coating”, J. Appl. Math. Mech., vol. 75, no. 5, pp. 595-600. 2. Tolokonnikov, L. A. 2013, “Diffraction of cylindrical sound waves by an cylinder with a non- uniform elastic coating”, Izv. Tul. Gos. Univ., Ser. Estestv. Nauki, no. 3, pp. 202-208, [in Russian]. 3. Tolokonnikov, L. A. 2013, “Scattering of an obliquely incident plane sound wave by an elastic cylinder with a non-uniform covering”, Izv. Tul. Gos. Univ., Ser. Estestv. Nauki, no. 2-2, pp. 265-274, [in Russian]. 4. Larin, N. V. & Tolokonnikov, L. A. 2015, “The scattering of a plane sound wave by an elastic cylinder with a discrete-layered covering”, J. Appl. Math. Mech., vol. 79. no 2, pp. 164-169. 5. Larin, N. V. 2017, “Diffraction of a plane acoustic wave on the thermoelastic cylinder with the continuously inhomogeneous covering”, Izv. Tul. Gos. Univ., Ser. Tekh. Nauki, no 6, pp. 154-173, [in Russian]. 6. Tolokonnikov, L. A., Larin, N. V. & Skobel’tsyn, S. A. 2014, “About definition of linear laws of heterogeneity of the cylindrical elastic layer having the least reflexion in the set direction at sound scattering”, Izv. Tul. Gos. Univ., Ser. Estestv. Nauki, no. 4, pp. 54-62, [in Russian]. 7. Tolokonnikov, L. A., Larin, N. V. & Skobel’tsyn, S. A. 2017, “Modeling of inhomogeneous coating of an elastic cylinder with given sound-reflecting properties”, J. Appl. Mech. and Techn. Physics, vol. 58, no 4, pp. 733-742. 8. Tolokonnikov, L. A. 2018, “Diffraction of a plane sound waves by an elastic cylinder with an non-uniform coating situated near to a flat surface”, Izv. Tul. Gos. Univ., Ser. Tekh. Nauki , no 9, pp. 276-289, [in Russian]. 9. Tolokonnikov, L. A. 2018, “Diffraction of a plane sound waves on two elastic cylinders with non-uniform coatings”, Chebyshevskii sbornik, vol. 19, no 1, pp. 238-254, [in Russian]. 10. Shenderov, E. L. 1972, “Wave problems of underwater acoustics”, Sudostroenie, Leningrad, 352 p, [in Russian]. 11. Ivanov, E. 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https://openalex.org/W3158167347	https://www.researchsquare.com/article/rs-147853/latest.pdf	English	null	HOXB7 Overexpression Leads Triple-Negative Breast Cancer Cells to a Less Aggressive Phenotype	Biomedicines	2,021	cc-by	14,567	HOXB7 overexpression leads triple-negative breast cancer cells to a less aggressive phenotype Simone Aparecida de Bessa Garcia I3S: Universidade do Porto Instituto de Investigacao e Inovacao em Saude Mafalda Araújo I3S: Universidade do Porto Instituto de Investigacao e Inovacao em Saude Tiago Pereira I3S: Universidade do Porto Instituto de Investigacao e Inovacao em Saude Renata Freitas (  renata.freitas@ibmc.up.pt ) I3S: Universidade do Porto Instituto de Investigacao e Inovacao em Saude https://orcid.org/0000- 0002-0123-7232 Research Keywords: Breast Cancer, HOXB7, Invasion, Metastasis, MDA-MB-231 Posted Date: January 19th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-147853/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License HOXB7 overexpression leads triple-negative breast cancer cells to a less aggressive phenotype Simone Aparecida de Bessa Garcia I3S: Universidade do Porto Instituto de Investigacao e Inovacao em Saude Mafalda Araújo I3S: Universidade do Porto Instituto de Investigacao e Inovacao em Saude Tiago Pereira I3S: Universidade do Porto Instituto de Investigacao e Inovacao em Saude Renata Freitas (  renata.freitas@ibmc.up.pt ) I3S: Universidade do Porto Instituto de Investigacao e Inovacao em Saude https://orcid.org/0000- 0002-0123-7232 Research Keywords: Breast Cancer, HOXB7, Invasion, Metastasis, MDA-MB-231 Posted Date: January 19th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-147853/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License HOXB7 overexpression leads triple-negative breast cancer cells to a less aggressive phenotype Simone Aparecida de Bessa Garcia I3S: Universidade do Porto Instituto de Investigacao e Inovacao em Saude Mafalda Araújo I3S: Universidade do Porto Instituto de Investigacao e Inovacao em Saude Tiago Pereira I3S: Universidade do Porto Instituto de Investigacao e Inovacao em Saude Renata Freitas (  renata.freitas@ibmc.up.pt ) I3S: Universidade do Porto Instituto de Investigacao e Inovacao em Saude https://orcid.org/0000- 0002-0123-7232 Research Result During the phenotypic characterization of the HOXB7-overexpressing cells, we found consistently a less aggressive behavior represented by lower cell viability, inhibition of cell migration, invasion and attachment-independent colony formation capacities added to the more compact and organized spheroids growth in 3D culture. In addition, we detected that these phenotypic changes may relate to the direct or indirect interaction of the HOXB7 protein with CTNNB1, EGFR, FGF2, CDH1, DNMT3B and COMMD7 genes. Research Keywords: Breast Cancer, HOXB7, Invasion, Metastasis, MDA-MB-231 Posted Date: January 19th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-147853/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/34 Page 1/34 Page 1/34 Conclusion Taken together, these results highlight the plasticity of the HOXB7 function in breast cancer, according to the cellular genetic background and expression levels and provide evidence that in triple-negative breast cancer cells, HOXB7 overexpression has the potential to promote less aggressive phenotypes. Background Breast cancer is a serious public health issue worldwide and, despite the advances in the understanding of this disease, its great complexity and heterogeneity still represent a major hurdle for accurate diagnosis and therapy decision-making. In addition to the biomarkers found to be useful in the prognosis a treatment of breast cancer, HOX genes have been proposed to be involved in the progression of this disease. For example, HOXB7 alterations in the expression and methylation patterns have been reported to promote breast cancer progression, most likely in a molecular subtype dependent way. Methods Here we induced HOXB7 overexpression in MDA-MB-231 cells, cellular model of Triple-Negative Breast Cancer, and evaluated the phenotypic changes in cell viability, morphogenesis, migration, invasion and formation of colonies. We also evaluated the expression of putative downstream targets and their direct binding to HOXB7 by Chip-qPCR in HOXB7-overexpressing cells and controls, namely CTNNB1, EGFR, FGF2, CDH1, DNMT3B and COMMD7. Background Page 2/34 Breast cancer is the most common cancer in women and a leading cause of cancer death among women worldwide 9. In spite of the fact that a number of molecular biomarkers and gene signatures are being used in standard clinical practice, and a growing body of others are being studied and tested, the great complexity and heterogeneity of breast cancer still limits accurate diagnosis and therapy decision- Page 2/34 making 44. In this context, HOX gene aberrant expression has been proposed as a breast cancer hallmark that is worth further investigation in order to improve prognosis and even develop novel targeted therapies 4, 10, 22. The HOX genes are organized in four clusters in the human genome: HOXA (7p15), HOXB (17q21.2), HOXC (12q13) and HOXD (2q31). They are highly conserved from Drosophila to Homo sapiens, namely due to a characteristic feature: the homeobox sequence. This 180 base pair sequence encodes a terminal or sub-terminal tri-helical domain, the homeodomain 28, 40, which is responsible for the recognition and binding of the HOX proteins to specific DNA motifs (ATTA/TAAT) 12. Apart from their role as transcription factors, the HOX proteins can also interact with other proteins to regulate cell or tissue-specific gene expression. Moreover, the genomic regions in which HOX genes are embedded produce numerous non-coding RNAs with important roles in gene regulation 23. These “HOX products” have central roles during embryonic development 57 and are also required for maintenance of cellular homeostasis during adulthood 35. In the breast, a structure that continues to form and remodel throughout a woman’s life, HOX genes assume a fundamental role in normal development and disease conditions 17, as their aberrant expression is frequently associated with breast tumorigenesis 22. This is the case of HOXB7, a gene expressed during the branching of the ductal tree and alveolar bud differentiation and also during the involution process after lactation 22. This HOX gene has a tendency to be overexpressed in primary breast cancer tumours with a more prominent overexpression in metastasis 67 and has also shown to be overexpressed in a variety of cell lines representative of distinct molecular subtypes 22, 27. Among breast cancer cell lines representing four molecular subtypes, the highest HOXB7 expression was detected in Luminal A and B models (MCF7 and BT474, respectively), and the lowest was observed in Triple Negative (MDA-MB-468 and MDA-MB-231) and HER2 + models (SKBR3) 27. Cell Culture The human breast cancer line MDA-MB-231 (MDA231) was authenticated by the Genomics Scientific Platform at i3S using the PowerPlex®16 HS System (Promega Corporation). Detection of the amplified fragments was made with automated capillary electrophoresis using the 3130 Genetic Analyzer (Applied Biosystems) and the assignment of genotypes was performed in GeneMapper software v5.0 (Applied Biosystems). The cells were cultured in Dulbecco’s modified Eagle’s medium, DMEM 1X (GIBCO, Paisley, UK) supplemented with 10% (V/V) heat-inactivated fetal bovine serum (FBS, Biowest, South American Origin) and 1X antibiotic solution penicillin-streptomycin, pen-strep (Gibco, Grand Island, USA), and maintained at 37 °C in a 5% CO2 humidified atmosphere. Cells were used in experiments upon reaching 70–80% of confluence. The cells generated after the transfection assays were cultured in the same medium as MDA231 cells adding 700 µg/mL geneticin (Gibco, Thailand) for the maintenance of cell selection pressure. Note that, for the assays described bellow, the transfected cells were kept in complete medium without geneticin. Background These subtype-specific expression profiles suggest that HOXB7 may have distinct roles in different breast cancer contexts 22, 31. In vivo and in vitro breast cancer models have associated the increased levels of HOXB7 with the activation of TGFB signalling 47) and with the expression levels of HER2 33 and EGFR 32. Studies performed in transgenic mice suggest that HOXB7 promotes breast cancer progression and metastisation by activating the TGFB signalling pathway 47. It was suggested that activation of TGFB/SMAD3 signaling is activated by HOXB7, because SMAD3 phosphorylation seems to be higher in primary mammary tumours from Hoxb7/Her2 double-transgenic mice than from Her2/neu single-transgenic mice. These authors also provide evidence that HOXB7 may bind to the TGFB2 promoter in double-transgenic tumours, in which TGFB2 expression is frequently higher than in single-transgenic mice. Moreover, TGFB2 expression was found to be higher in two breast cancer cell lines transfected with HOXB7 (SKBR3, MDA- MB-231) and the knockdown of TGFB2 in MDA-MB-231 cells overexpressing HOXB7 seems to inhibit lung metastasis in mice. To further study the impact of HOXB7 modulation on Triple Negative Breast Cancer (TNBC), having the MDA-MB-231 cells as cellular model, we generated HOXB7-overexpressing clones in order to analyse the associated phenotypic changes. TNBC is a group of tumours characterized by the absence of the ER Page 3/34 (Estrogen Receptor) / PR (Progesterone Receptor) / HER2 expression, which represents for up to 15% of the cases, and have the worse overall survival and breast cancer cause-specific survival time, in every stage and sub-stage, when compared to non-TNBC 41, 45. This high aggressiveness is represented by early relapses, few and non-targeted treatment options, and low response durability 54, 66. We found that in a HOXB7-overexpressing clone cell viability was lower, cell migration, invasion and colony formation in soft agar were inhibited and sphere formation in 3D cultures appeared more organized. In addition, we detected that in the MDA-MB-231 cell model, the HOXB7 protein interacts with the promoter regions of CTNNB1, FGF2, and CDH1 genes as HOXB7-overexpressing cells show an increment in CDH1 interaction and EGFR, DNMT3B and COMMD7 appear as potential new targets. (Estrogen Receptor) / PR (Progesterone Receptor) / HER2 expression, which represents for up to 15% of the cases, and have the worse overall survival and breast cancer cause-specific survival time, in every stage and sub-stage, when compared to non-TNBC 41, 45. Background This high aggressiveness is represented by early relapses, few and non-targeted treatment options, and low response durability 54, 66. We found that in a HOXB7-overexpressing clone cell viability was lower, cell migration, invasion and colony formation in soft agar were inhibited and sphere formation in 3D cultures appeared more organized. In addition, we detected that in the MDA-MB-231 cell model, the HOXB7 protein interacts with the promoter regions of CTNNB1, FGF2, and CDH1 genes as HOXB7-overexpressing cells show an increment in CDH1 interaction and EGFR, DNMT3B and COMMD7 appear as potential new targets. Overall, our results suggest that particular levels of HOXB7 overexpression can inhibit the aggressive behavior of TNBC cells possible through the direct or indirect regulation of CTNNB1, FGF2, CDH1, EGFR, DNMT3B and COMMD7. This adds important information to the current understanding of the HOXB7- dependent signalling pathways in TNBC cells for which the discovery of reliable predictive biomarkers is imperative for improving patient prognosis. RNA expression analyses The total RNA extraction was performed using TRIzol™ reagent (Ambion, Carlsbad, USA) according to the manufacturer’s instructions and adding one more wash with ethanol 75%. After assessment of RNA 260/280 nm ratio and concentration using NanoDrop 1000 (Thermo Scientific), 800 ng of RNA was subjected to reverse transcription, using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Vilnius, Lithuania), following the manufacturer’s instructions. The qPCRs reactions were performed in duplicates and carried out in the CFX96™ Real-Time PCR Detection System (Bio-Rad). Each 10µL amplification reaction contained 2µL of the respective cDNA diluted 1:4; the indicated quantity of forward and reverse 10 µM primers; 5µL of 2X iTaq™ Universal SYBR Green Supermix (Bio-Rad, USA) and 2µL of DNase/RNase free H2O. The run conditions were: 95˚C for 3 minutes (min.), 40 cycles of 95˚C for 10 seconds (sec.) and 60˚C for 30sec. followed by the default dissociation curve capture. The qPCR result analyses were performed using the method described by Schmittgen and Livak 58 using the formula: RATIO = E target – (CT sample target gene) /E GAPDH – (CT sample GAPDH), in which “E” is the primer pair efficiency previously calculated and GAPDH (glyceraldehyde-3-phosphate dehydrogenate) is the reference gene. The primer sequences used for qPCR were synthetized by Sigma Aldrich (Darmstadt, Germany) and are described in Supplementary Table 1. Stable Transfection Assay To generate stable HOXB7-overexpressing and control cells, MDA231 were transfected with the pCMV6- AC-GFP-HOXB7 (Origene, RG204495) or pCMV6-AC-GFP (Origene, PS100010) constructs. Transfections were performed using TurboFectin 8.0 (Origene) reagent according to manufacturer’s instructions. Briefly, MDA231 cells were seeded in a 6-well plate on the previous day in order to get 50–70% confluence on the following day. The complexes formation were made in two different ratios, 1 µg DNA: 3 µl TurboFectin 8.0 and 1.5 µg DNA: 6 µl TurboFectin 8.0, and the transfected cells were incubated at 37 °C in a 5% CO2 Page 4/34 Page 4/34 Page 4/34 humidified atmosphere for 24 hours post-plating (h). After incubation, the cells medium was changed and geneticin (1.2 mg/mL) was added for the selection of the stably transfected cells. The GFP- positive/geneticin-resistant cells were then sorted in the FACSAria™ II cell sorter (BD Biosciences). Cells transfected with pCMV6-AC-GFP-HOXB7 plasmid vector were seeded in 96-well plate (1 cell/well) containing 20% FBS in order to achieve the clonal expansion of one cell and the remaining cells were collected in a T25 flask. The cells transfected with the empty vector (pCMV6-AC-GFP) were collected in a T25 flask with the conventional complete medium. Once cells restarted proliferation both in the T25 flask and in the 96-well plate they were kept in the conventional medium containing 700 µg/mL geneticin. Three different cell transfectants were obtained and named as Empty Vector (EV), for the cells transfected with the control empty vector (pCMV6-AC-GFP); B7, for the pool of cells transfected with pCMV6-AC-GFP-HOXB7 vector and overexpressing variable levels of HOXB7; D3, for the clone obtained from the 96-well plate sorted cells transfected with pCMV6-AC-GFP-HOXB7 vector. ChIP-qPCR assay The cells were seeded in cell culture dishes with 15 cm in diameter in conventional medium and grown until reaching 80–90% confluence. Then, the DNA / proteins crosslink was performed using 1% formaldehyde solution incubation under agitation during 10 min. at room temperature. Next, to stop crosslink, 0.15M glycine was added directly onto the shaking plates and incubated for 5 min. at room temperature followed by two washes with cold 1X PBS (Gibco, Paisley, UK) and the cells were collected in 1.5 mL tubes after cell scrape. The obtained pellets, after 5 min. centrifugation at 1000 g at 4 °C, were suspended in Cell lysis buffer (5 mM Pipes, 85 mM KCl, 0.5% Igepal, 1X Roche cOmplete™ Protease Inhibitor Cocktail, 1 µL/mL Trypsin, 10 µL/mL PMSF 200 mM, 1 µL/mL DTT 50 mM in MilliQ H2O) and incubated on ice for 10 min. followed by a centrifugation of 1000 g for 5 min. at 4 °C. The cell pellet was Page 5/34 suspended in Nuclear lysis buffer (50 mM Tris, 10 mM EDTA, 1% SDS 1X Roche cOmplete™ Protease Inhibitor Cocktail, 1 µL/mL Trypsin, 10 µL/mL PMSF 200 mM, 1 µL/mL DTT 50 mM in MilliQ H2O), incubated for 10 min. on ice and immediately sonicated (30 cycles, 30 sec. ON/30 sec. OFF, high mode) in the Bioruptor Plus (Diagenode). The sonicated DNA (1 µg) fragment sizes were analysed in a 1% agarose gel with an expected smear under 1000 bp with a strong band at 500 bp. The DNA quantification was made in Nanodrop 1000 (Thermo Scientific) after the following preparation: 20 µl of sonicated samples was mixture in 20 µl of 10% Chelex® 100 resin (Bio-Rad, USA), incubated for 5 min. at 90 °C and centrifuged 15000 g for 1 min. The blank sample was prepared adding 20 µl of nuclear lysis buffer to 20 µl of 10% Chelex® 100 resin and following the same steps mentioned for the sample preparation. After DNA quantification and gel analyses, the sonicated DNA samples were heated for 5 min. at 65 °C and centrifuged 15000 g for 30 sec. Forty micrograms of DNA per Immunoprecipitation (IP) was diluted 1:20 in Dilution buffer (0.01% SDS, 1,1% Triton X-100, 1.2 mM EDTA pH 8.0, 16.7 mM Tris pH 8.1, 167 mM NaCl in MilliQ H2O) and 4 µg of the desired antibody was added. ChIP-qPCR assay The IPs were incubated overnight at 4 °C under rotation. An input control was prepared for each sample that was used diluting, also in a 1:20 ratio, 4 µg of each sample (10% of the IP) in Dilution buffer followed by the addition of 3 parts absolute ethanol and an overnight precipitation at -80 °C. On the day after, considering that 40 µl of Dynabeads A/G were used per IP, a mixture was prepared of 1 volume Dynabeads A and 1 volume Dynabeads G, followed by the beads separation with a magnetic stand, aspiration of the buffer and the addition of the same initial volume of IP buffer (10% Nuclear lysis buffer, 90% Dilution buffer). Next, the samples with antibodies were centrifuged for 10 min. at 12000 g, the supernatants were transferred to a new tube and 40 µl of Dynabeads preparation were added followed by an incubation for 2 h at 4 °C with rotation. The beads were, then, separated with a magnetic stand and washed (15 min. rotation at room temperature followed by beads separation) twice with 1 mL Dialysis buffer (2 mM EDTA pH 8.0, 50 mM Tris pH 8.1, 0.2% Sarkosyl in MilliQ H2O) and four times with 1 mL Wash buffer (0.5 M LiCl, 1% Igepal, 1% Na- deoxycholate, 33.2 mM Tris pH 8.1 in MilliQ H2O). After the last wash, beads were eluted in 150 µl Elution buffer (50 mM NaHCo3, 1% SDS), heated 15 min. at 65 °C with vortex each 5 min. and the supernatant transferred to a new tube. This step was repeated yielding 300 µl of supernatant per sample. The input controls were centrifuged 12000 g for 10 min. after the precipitation step and the pellets were washed twice with 70% ETOH (1 mL 70% ETOH and centrifugation), eluted with 300 µl Elution buffer and heated 15 min. at 65 °C with vortex every 5 min. To IPs and Input samples 2.4 µl Proteinase K (20 mg/mL) were added followed by 1 h incubation at 55 °C at 90 g and purification with QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Samples were suspended in DNase/RNase free H2O and stored at -20 °C. The ChIP-qPCR reactions were performed using 2µL of the purified sample without dilution and the run conditions were: 95˚C for 3 min., 40 cycles of 95˚C for 15 sec., 55˚C for 30 sec. ChIP-qPCR assay and 72˚C for 30 sec. followed by the default dissociation curve capture. The assays analyses were made using the “percent input” method according to Lacazette 42 The primers sequences suspended in Nuclear lysis buffer (50 mM Tris, 10 mM EDTA, 1% SDS 1X Roche cOmplete™ Protease Inhibitor Cocktail, 1 µL/mL Trypsin, 10 µL/mL PMSF 200 mM, 1 µL/mL DTT 50 mM in MilliQ H2O), incubated for 10 min. on ice and immediately sonicated (30 cycles, 30 sec. ON/30 sec. OFF, high mode) in the Bioruptor Plus (Diagenode). The sonicated DNA (1 µg) fragment sizes were analysed in a 1% agarose gel with an expected smear under 1000 bp with a strong band at 500 bp. The DNA quantification was made in Nanodrop 1000 (Thermo Scientific) after the following preparation: 20 µl of sonicated samples was mixture in 20 µl of 10% Chelex® 100 resin (Bio-Rad, USA), incubated for 5 min. at 90 °C and centrifuged 15000 g for 1 min. The blank sample was prepared adding 20 µl of nuclear lysis buffer to 20 µl of 10% Chelex® 100 resin and following the same steps mentioned for the sample preparation. After DNA quantification and gel analyses, the sonicated DNA samples were heated for 5 min. at 65 °C and centrifuged 15000 g for 30 sec. Forty micrograms of DNA per Immunoprecipitation (IP) was diluted 1:20 in Dilution buffer (0.01% SDS, 1,1% Triton X-100, 1.2 mM EDTA pH 8.0, 16.7 mM Tris pH 8.1, 167 mM NaCl in MilliQ H2O) and 4 µg of the desired antibody was added. The IPs were incubated overnight at 4 °C under rotation. An input control was prepared for each sample that was used diluting, also in a 1:20 ratio, 4 µg of each sample (10% of the IP) in Dilution buffer followed by the addition of 3 parts absolute ethanol and an overnight precipitation at -80 °C. On the day after, considering that 40 µl of Dynabeads A/G were used per IP, a mixture was prepared of 1 volume Dynabeads A and 1 volume Dynabeads G, followed by the beads separation with a magnetic stand, aspiration of the buffer and the addition of the same initial volume of IP buffer (10% Nuclear lysis buffer, 90% Dilution buffer). Next, the samples with antibodies were centrifuged for 10 min. ChIP-qPCR assay at 12000 g, the supernatants were transferred to a new tube and 40 µl of Dynabeads preparation were added followed by an incubation for 2 h at 4 °C with rotation. The beads were, then, separated with a magnetic stand and washed (15 min. rotation at room temperature followed by beads separation) twice with 1 mL Dialysis buffer (2 mM EDTA pH 8.0, 50 mM Tris pH 8.1, 0.2% Sarkosyl in MilliQ H2O) and four times with 1 mL Wash buffer (0.5 M LiCl, 1% Igepal, 1% Na- deoxycholate, 33.2 mM Tris pH 8.1 in MilliQ H2O). After the last wash, beads were eluted in 150 µl Elution buffer (50 mM NaHCo3, 1% SDS), heated 15 min. at 65 °C with vortex each 5 min. and the supernatant transferred to a new tube. This step was repeated yielding 300 µl of supernatant per sample. The input controls were centrifuged 12000 g for 10 min. after the precipitation step and the pellets were washed twice with 70% ETOH (1 mL 70% ETOH and centrifugation), eluted with 300 µl Elution buffer and heated 15 min. at 65 °C with vortex every 5 min. To IPs and Input samples 2.4 µl Proteinase K (20 mg/mL) were added followed by 1 h incubation at 55 °C at 90 g and purification with QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Samples were suspended in DNase/RNase free H2O and stored at -20 °C. The ChIP-qPCR reactions were performed using 2µL of the purified sample without dilution and the run conditions were: 95˚C for 3 min., 40 cycles of 95˚C for 15 sec., 55˚C for 30 sec. and 72˚C for 30 sec. followed by the default dissociation curve capture. The assays analyses were made using the “percent input” method according to Lacazette 42. The primers sequences used for ChIP-qPCR were synthetized by Sigma Aldrich (Darmstadt, Germany) and are described in Protein expression assays Protein expression assays Page 6/34 Total protein extraction was obtained collecting the cells pellets after dissociation, suspending them in the lysis solution (0.1% Triton X-100, 0.1% Igepal, 1X cOmplete™ Protease Inhibitor Cocktail [Roche, Germany], PBS 1X [Gibco, Paisley, UK]) and incubating on ice for 20 min. with vortex every 5 min. After incubation, the tubes were centrifuged 9000 g for 10 min. at 4 °C and the supernatant transferred to a new tube. An aliquot of the protein extracts was separated for posterior quantification. Samples were stored at -80 °C. Total protein extraction was obtained collecting the cells pellets after dissociation, suspending them in the lysis solution (0.1% Triton X-100, 0.1% Igepal, 1X cOmplete™ Protease Inhibitor Cocktail [Roche, Germany], PBS 1X [Gibco, Paisley, UK]) and incubating on ice for 20 min. with vortex every 5 min. After incubation, the tubes were centrifuged 9000 g for 10 min. at 4 °C and the supernatant transferred to a new tube. An aliquot of the protein extracts was separated for posterior quantification. Samples were stored at -80 °C. Cytoplasmic and nuclear proteins fractions extractions were performed using the ab113474 Nuclear Extraction Kit (Abcam) according to the manufacturer's recommendations. The nuclear proteins fractions were sonicated in Bioruptor Plus (Diagenode) for 3 cycles, 10 seconds ON / 10 sec. OFF at high mode to increase extraction yield. Aliquots of both fractions were separated for posterior quantification. Samples were stored at -80 °C. Protein concentrations were determined using the Pierce Detergent Compatible Bradford Assay Kit (Thermo Scientifc, Rockford, USA) following the product data sheet instructions. The albumin standards were prepared in a work range of 100–1500 µg/mL and the samples were quantified in a 1:10 dilution. Both, samples dilutions and albumin standards were prepared in RNase/DNase free H2O. Twenty to thirty micrograms of protein lysates were separated on a 12% SDS-PAGE gel prepared with 40% Acrylamide – Bisacrylamide 29:1 (Invitrogen, Carlsbad, USA) and 4X separating buffer (Alfa Aesar, Ward Hill, USA) for the separating gel or 4X stacking buffer (Alfa Aesar, Karlsruhe, Germany) for the stacking gel. After run the proteins were blotted onto a nitrocellulose membrane of the iBlot® gel transfer stacks (Kiryat Shmona, Israel) using the iBlot™ dry transfer system (Life Technologies, Israel). Protein expression assays Blots were blocked for 1 h at room temperature in 3% BSA/TBS-T (20 mM Tris, 137 mM NaCl, 0.1% Tween-20) solution or 5% skim milk/TBS-T solution and incubated overnight at 4 °C with the primary antibodies, diluted in the respective blocking solutions. Then, membranes were washed four times in TBS-T for 5 min., two times in TBS (20 mM Tris, 137 mM NaCl) for 5 min. and incubated with the appropriate horseradish peroxidase- conjugated secondary IgG antibody for 1.30 min. at room temperature. The bolts were washed again, as already described, followed by the detection of the immunoreactive proteins using the Clarity™ Western ECL Substrate (Bio-Rad, USA) in the ChemiDoc Gel Image System (Bio-Rad). The primary and secondary antibodies used in this work are listed on Supplementary Table 3. The assessment of the bands density was made in the ImageLab software (BioRad) using the measures of Tubulin (for total and cytoplasmic protein fractions) and HDAC1/Lamin B1 (for nuclear protein fraction) as loading controls. Wound Healing assay Cells were seeded in 24-well plates at a density of 2 × 105 cells/well in 500 µl of Dulbecco’s modified Eagle’s medium, DMEM 1X (GIBCO, Paisley, UK) supplemented with 5% (V/V) Charcoal Stripped Fetal Bovine Serum Qualified One Shot™ (Gibco, Mexico), for cells mitosis synchronization, and 1X antibiotic solution penicillin-streptomycin (pen-strep, Gibco™). At least 48 h post-seed, to ensure cell synchronization and growth to 90%-100% confluence, a single scratch wound was made in each well using a 200 µl disposable pipette tip. The cells were then incubated for 15 h in the InCell Analyser 2000 Automated fluorescence widefield HCS microscope for the capture of wound images every 3 h. The extent of wound closure was measured using the MRI wound healing tool from ImageJ software 59. On-top 3D cell culture For the morphogenesis assay, MDA231 wild-type (WT), EV and D3 cells were dissociated to obtain a suspension containing single cells. The cells (2 × 103), suspended in 400 µL of completed media containing 3% of Matrigel® Matrix Basement Membrane growth factor reduced (Corning, Bedford, USA), were seeded in 8-well glass chamber slides containing a pre-prepared bed of 30 µl Matrigel® Matrix Basement Membrane growth factor reduced, as described by Debnath et al. 24 and incubated at 37 °C in 5% CO2 humidified atmosphere for 8 days. The cells spheroids morphologies were analysed and registered every two-days in Axiovert 200M inverted fluorescent microscope (Carl Zeiss). On day-4, 100 µl of media containing 3% Matrigel® Matrix Basement Membrane growth factor reduced were added to the wells to prevent the effects of medium evaporation and nutrient scarcity. MTT assay with docetaxel treatments Cell viability was determined using MTT assay [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] (EMD Millipore, China) following the manufacturer's instructions. Cells were seeded in 96-well plates (1 × 104 cells/well) and maintained at 37 °C in 5% CO2 humidified atmosphere in complete media supplemented with 5% FBS for 24 h. In this time point (24 h), the Docetaxel (Sigma-Aldrich, China) treatments with 5 nM and 50 nM started. The cells viability measures were made by absorbance read at 570 nm using the Synergy™ 2 Plate reader (BioTek) at 24 h, 48 h, 72 h and 96 h post-seed. The medium Page 7/34 Page 7/34 containing Docetaxel or vehicle (ETOH) was changed every 24 h to ensure that cells were exposed to the same drug concentration along of time points analysed. The control cells were treated with the drug vehicle (ETOH) in the volume corresponding to the biggest drug concentration used. An additional assay was made for the measure of the cell viability in standard medium without ETOH. Invasion assay The invasion assay was made using the 24-well plate growth factor reduced Corning® Matrigel® Invasion chambers 8 µm pore size (Corning, Bedford, USA) and 24-well Control Inserts 8 µm pore size (Corning, Bedford, USA) according to manufacturer’s instructions. Briefly, cells (1 × 105) in 500 µl serum- free medium were plated into the upper chamber and the bottom wells were filled with 750 µl complete medium. The cells were incubated at 37 °C in 5% CO2 humidified atmosphere for 16 h. Then, cells in the upper chamber were removed using cotton swabs and the cells invading the bottom of the membrane were fixed with 4% paraformaldehyde for 20 min. The nuclei were stained with DAPI (Sigma-Aldrich, Darmstadt, Germany) 1 µg/mL plus 1% Triton X-100 in 1X PBS (Gibco, Paisley, UK) for 15 min. followed by two washes in Milli-Q H2O. Ten random fields from each membrane were photographed using the Zoe fluorescent cell imager (Bio-Rad) and the cells nuclei were counted using the Analyze Particles tool from ImageJ software 59. Clonogenic assay Clonogenic assay Page 8/34 Clonogenic assay is a versatile tool for in vitro screening of the capacity of a single-cell suspension to form a colony of 50 or more cells under different circumstances. The assay was made according to Franken et al. 26. A total of 100 cells were seeded into 6-well plates containing standard culture media. After 10 days, colonies were stained and the well images captured in the InCell Analyser 2000 Automated fluorescence widefield HCS microscope. Colony number and respective areas were measured using the colony count plugin from ImageJ software 59. Clonogenic assay is a versatile tool for in vitro screening of the capacity of a single-cell suspension to form a colony of 50 or more cells under different circumstances. The assay was made according to Franken et al. 26. A total of 100 cells were seeded into 6-well plates containing standard culture media. After 10 days, colonies were stained and the well images captured in the InCell Analyser 2000 Automated fluorescence widefield HCS microscope. Colony number and respective areas were measured using the colony count plugin from ImageJ software 59. Soft agar colony formation assay This assay was made as described by Borowicz and colleagues 7. The cells (1.5 × 104/well) were seeded in 6-well plates and incubated for four weeks at 37 °C in a 5% CO2 humidified atmosphere. The colonies, without staining, of nine random fields were counted using the Zoe fluorescent cell imager (Bio-Rad). MCF7 cells cultured in the same medium as MDA231 cell linewere used as a parameter for Luminal behavior analyses. Slow Aggregation Assay This assay was performed according to the protocol described by Boterberg et al 8. Briefly, 2 × 104 cells per well were seeded over an agar layer in a 96-well plate with and without the addition of MB2, an anti- CDH1 antibody, as a way to show if the formed aggregates are dependent on the CDH1/CTNNB1 complex functionality. The cells were photographed after 24 h, 48 h, 72 h and 96 h in an Axiovert 200M inverted fluorescent microscope (Carl Zeiss). The MDA231-WT and MCF7 cells were used, respectively, as negative and positive controls of the aggregation capacity. MDA231-WT cells do not express CDH1 and, therefore, they do not aggregate while MCF7 cells, which express high levels of CDH1, form well-defined aggregates when CDH1/CTNNB1 complex is active. Statistical analyses The statistical differences were determined by unpaired T test with Welch’s correction or by Brown- Forsythe and Welch ANOVA tests using the Prism8 software (GraphPad Software, La Jolla, USA). P - values were considered statistically significant when P ≤ 0.05. Data were reported as the mean ± SD of at least three independent experiments. 2.1 More compact spheroid organizations in 3D culture The three-dimensional (3D) culture on a reconstituted basement membrane results in formation of spheroids that recapitulate several aspects of glandular architecture in vivo 24. The morphology of B7 and D3 cell growth in the conventional 2D culture did not show impacting differences in comparison to the morphology observed in WT and EV cells (Supplementary Fig. 1). However, we hypothesized that 3D cellular organization could be different in the HOXB7 overexpressing cells. To address this question, the cells were grown on-top in Matrigel® for 8 days (Fig. 2), which allowed us to see that D3 cells form spheroids with a more compact growth without the formation of protrusions. In contrast, WT, EV and B7 cells showed a more spread growth with numerous protrusion formations. Interestingly, the spheroids growth pattern of D3 cells resembles that observed for MCF7 cells (Luminal A) as shown by Tasdemir et al. 64 and Kai et al. 34. 1. HOXB7 overexpression induced in MDA231 cells HOXB7 is typically overexpressed in breast cancer cell lines in comparison with normal cells [15]. However, the level of HOXB7 overexpression is lower in MDA231 cells (triple-negative, claudin-low) in comparison to MCF7 (Luminal A), BT474 (Luminal B), SKBR3 (HER2+) and MDA468 (Triple-negative, basal-like). Thus, we selected the MDA231 cell line to conduct functional assays aiming to further explore the mechanistic effect of HOXB7 up-regulation. To this end, we transfected these cells with pCMV6-AC- GFP plasmid vector as control generating the EV (Empty Vector) cells and with pCMV6-AC-GFP-HOXB7 plasmid vector generating the B7 and D3 cells. Page 9/34 Page 9/34 Subsequent mRNA expression analysis revealed that the levels of HOXB7 were identical in EV and WT cells, suggesting that the transfection procedure does not affect HOXB7 expression (Fig. 1A). In contrast, the B7 and D3 cells presented a significantly higher HOXB7 mRNA expression levels (P = 0.02 and P = 0.013, respectively) when compared to EV cells. In addition, the HOXB7 expression was significantly higher in the D3 than in the B7 cells (P = 0.015). Noteworthy, in terms of protein expression, we observed that HOXB7 levels did not change among WT, EV, B7 and D3 cells in the cytoplasmic fraction (Fig. 1B). However, in the nuclear fraction (Fig. 1C), HOXB7 expression was significantly higher only in D3 cells, in comparison with EV (P = 0.026), with a borderline significance in B7 (P = 0.049) cells. Thus, in summary, we confirmed that pCMV6-AC-GFP-HOXB7 transfection led to mRNA HOXB7 overexpression in B7 and D3 cells, and we further detected that only D3 cells present significant HOXB7 overexpression at nuclear protein level. Subsequent mRNA expression analysis revealed that the levels of HOXB7 were identical in EV and WT cells, suggesting that the transfection procedure does not affect HOXB7 expression (Fig. 1A). In contrast, the B7 and D3 cells presented a significantly higher HOXB7 mRNA expression levels (P = 0.02 and P = 0.013, respectively) when compared to EV cells. In addition, the HOXB7 expression was significantly higher in the D3 than in the B7 cells (P = 0.015). Noteworthy, in terms of protein expression, we observed that HOXB7 levels did not change among WT, EV, B7 and D3 cells in the cytoplasmic fraction (Fig. 1B). However, in the nuclear fraction (Fig. 1. HOXB7 overexpression induced in MDA231 cells 1C), HOXB7 expression was significantly higher only in D3 cells, in comparison with EV (P = 0.026), with a borderline significance in B7 (P = 0.049) cells. Thus, in summary, we confirmed that pCMV6-AC-GFP-HOXB7 transfection led to mRNA HOXB7 overexpression in B7 and D3 cells, and we further detected that only D3 cells present significant HOXB7 overexpression at nuclear protein level. 2. Phenotypic characterization of the HOXB7-overexpressing cells 2.4 Lower soft agar colony formation efficiency with no changes on clonogenic growth The ability of the transformed cells to grow and form colonies independently of a solid surface is a hallmark of carcinogenesis 7. This capacity was evaluated in the soft agar colony formation assay and it was observed that D3 cells formed significantly less colonies than WT (P = 0.041) and EV (P = 0.007) cells (Fig. 6). Moreover, when compared to a Luminal A cell line (MCF7), MDA231 WT and EV cells showed a higher number of colonies (P = 0.009 and P = 0.007, respectively), while D3 cells showed a colony number similar to MCF7 cells. In summary, D3 cells presented a colony formation efficiency that represents about 50% of the efficiencies observed in WT and EV cells, which is similar to what is observed in MCF7 cells. We also evaluated the capacity of a single cell to form progeny in an attachment- permissive environment by the clonogenic assay and it was observed that, in this condition, the clonogenic capacity of EV and D3 is similar both in the number and area of the colonies formed (Supplementary Fig. 2). 2.3 Lower migration and invasion capacities The cell migration capacity is essential for disease progression and is considered the first step for lymphovascular invasion and tumour metastasis 37. The data obtained by wound-healing assay showed that D3 cells had a significant delay in wound closure capacity in all analysed time points when compared to EV and B7 cells (Fig. 4). It is interesting to note that at time 0 h there are significant differences between EVxB7 (P < 0.0001) and B7xD3 (P = 0.0001) cells. This could be due to differences in the areas of the opened wounds. The EVxB7 differences decrease along the time (P = 0.0004 at 3 h) and disappear after 6 h, while the B7xD3 (P < 0.0001) as well as the EVxD3 differences were kept throughout the assay (P = 0.027 at 3 h and P < 0.0001 at 6 h, 9 h, 12 h and 15 h). The invasion capacity, which is another important skill developed by cancer cells to successfully spread locally and for distant sites, was analysed by Matrigel® invasion chamber assay. It was observed that D3 cells present lower capacity (P = 0.004) to invade the Matrigel® layer in comparison to EV cells with an invasion index of 0.4 (Fig. 5), which means that D3 cells invasion capacity is approximately 60% less than in EV cells. 2.2 Lower cell viability and no effect on sensitivity to Docetaxel treatments The effect of HOXB7 overexpression on cell viability and Docetaxel sensitivity was analysed in WT, EV, B7 and D3 cells by MTT assay at different time points (Fig. 3). At 48 h post-plating, the D3 cells exhibited lower viability in comparison to EV (P = 0.002) and B7 (P = 0.015) cells (Fig. 3A). The B7 (P = 0.001) and D3 (P = 0.0001) cells showed a lower viability in comparison to EV cells at 96hpost-plating. However, these differences in 96 h could lead to misinterpretations because at this time-point the cells were in a confluence near 100%. We then evaluated the sensitivity to Docetaxel, a chemotherapeutic drug that induces different types of cell death depending on the administrated concentration 30. We observed that, in the presence of Docetaxel (5 nM and 50 nM), the cells do not reach the exponential growth as observed in absence of the drug (Fig. 3B). The viability differences observed at 24 h post-plating (time 0 h for drug treatments) among the cells before and after Docetaxel treatment are probably related to cell loading and/or cell attachment issues. Despite the differences in 24 h, it is possible to observe the lower viability Page 10/34 Page 10/34 of the D3 cells kept with ETOH in 96 h compared to EV (P = 0.005) and B7 (P = 0.003) cells corroborating the findings of the curve in Fig. 3A. In the ETOH assay the cells were sub confluent at 96 h, possibly due to the differences observed at 24 h. Thus, no differences in response to Docetaxel treatments were observed over time. 3.1 Impact on CTNNB1 expression 3.1 Impact on CTNNB1 expression The current knowledge on the direct and/or indirect targets of HOXB7 is scarce; therefore, the search for these molecules is crucial to better understand HOXB7 functions in different tumours. Given the results Page 11/34 Page 11/34 obtained in 3D culture, showing a more compact and organised spheroid formation in D3 cells, we speculate that molecules involved in cell-cell adhesion and cytoskeletal organization (i.e. CDH1/CTNNB1 complex) could be influenced by HOXB7 overexpression. In this line of thought, we performed a slow aggregation assay in order to verify if the phenotypes mentioned above could be related to changes in CDH1 functionality, given that MDA231 cells do not express CDH1 and consequently do not form aggregates in this assay. However, no alterations were observed in the D3 cell aggregation profiles, in the presence and absence of MB2 antibody, in comparison to WT and EV cells (Supplementary Fig. 3), and the phenotypes observed in 3D cell culture were not directly linked to variations in CDH1 activity. Next, we analysed the expression of CTNNB1, a multi-functional molecule with key roles in normal and disease conditions 8. Interestingly, we found a significant downregulation of CTNNB1 mRNA (P = 0.021, Fig. 7A) and total protein (P = 0.007, Fig. 7B-C) in D3 cells. 3.2 HOXB7 interacts with CDH1, FGF2 and CTNNB1 promoter regions and, when overexpressed, EGFR, DNMT3B and COMMD7 genes become new targets 3.2 HOXB7 interacts with CDH1, FGF2 and CTNNB1 promoter regions and, when overexpressed, EGFR, DNMT3B and COMMD7 genes become new targets Based on previous studies, several putative targets of HOXB7 may explain its mechanistic role when overexpressed in breast cancer cells: EGFR, FGF2, CTNNB1, CDH1, DNMT3B and COMMD7. HOXB7 seems to establish direct interactions with EGFR in MCF7 32 and BT474 cells 29 and with FGF2 in BT474 cells 29. In addition, the knockdown of CTNNB1 in TNBC cell lines (MDA231 and HCC38) significantly impairs their ability to migrate and form anchorage-independent colonies 68 besides showing a down- regulation in our HOXB7-overexpressing cells (Fig. 7). Moreover, reduced expression of CDH1 is linked to the invasion capacity of cancer cells 19; however its interaction with HOXB7 has not been explored in breast cancer cells. Regarding DNMT3B, its function relates to de novo methylation, which has an important impact on epigenetic regulation of several genes 48 and TNBC prognosis 50. 3.1 Impact on CTNNB1 expression It was also shown that HOXB7 binds directly to TGFB2 promoter in MCF7 cells and upregulates this same molecule in MDA231 cells leading to increased cell migration and invasion [17]. Finally, the analysis of ChIP-Seq data provided by Heinonen et al. 29 suggests that COMMD7 might be a direct target of HOXB7 in BT474 cells (Supplementary Fig. 4A), as its function is explored in the progression of hepatocellular 70 and pancreatic carcinomas69. Here we analysed COMMD7 expression in a panel of breast cancer cells and found up- regulation in BT474 and MDA231 (Supplementary Fig. 4B). We then used ChIP-qPCR to explore the interaction of HOXB7 with all putative targets mentioned above. We detected three HOXB7 interaction patterns (Fig. 8): 1) interaction in EV and D3 cells with no increments in D3 cells for CTNNB1 and FGF2; 2) interaction only in D3 cells for EGFR, DNMT3B and COMMD7; and 3) interaction in EV and D3 cells with an increment in D3 cells for CDH1. No interactions between HOXB7 and TGFB2 were found in EV or D3 cells. The calculated P values are described in the legend of Fig. 8. Discussion Page 12/34 Triple-negative breast cancers (TNBC) corresponds to up to 15% of the total breast cancer cases and have the worse prognosis among breast cancer subtypes due to the absence of target-directed therapies and the increased rate of distant metastasis 41, 45, 54. As reviewed by de Bessa Garcia et al, HOXB7 has been frequently considered as an oncogene in breast cancer 22. However, our analyses of the HOXB7 basal mRNA expression profile 27 showed that the lowest levels were observed in SKBR3 (HER2+) and MDA231 (TNBC, claudin-low) cells, which represent the molecular subtypes with worse prognosis, while the highest expression levels were observed in the luminal A and B cells (MCF7 and BT474, respectively), representing tumour molecular sub-types with better prognosis 52. Surprisingly, the MDA231 cells overexpressing HOXB7 mRNA and nuclear protein showed less aggressive phenotypes, represented by more compact spheroid formation in 3D culture and lower cell viability, migration, invasion and anchorage-independent colony formation. Moreover, the search for potential downstream targets showed that the HOXB7-overexpressing cells, along with a decreased CTNNB1 expression, present enriched HOXB7 protein direct interaction with the promoter regions of EGFR, DNMT3B, COMMD7 and CDH1 genes. Triple-negative breast cancers (TNBC) corresponds to up to 15% of the total breast cancer cases and have the worse prognosis among breast cancer subtypes due to the absence of target-directed therapies and the increased rate of distant metastasis 41, 45, 54. As reviewed by de Bessa Garcia et al, HOXB7 has been frequently considered as an oncogene in breast cancer 22. However, our analyses of the HOXB7 basal mRNA expression profile 27 showed that the lowest levels were observed in SKBR3 (HER2+) and MDA231 (TNBC, claudin-low) cells, which represent the molecular subtypes with worse prognosis, while the highest expression levels were observed in the luminal A and B cells (MCF7 and BT474, respectively), representing tumour molecular sub-types with better prognosis 52. Surprisingly, the MDA231 cells overexpressing HOXB7 mRNA and nuclear protein showed less aggressive phenotypes, represented by more compact spheroid formation in 3D culture and lower cell viability, migration, invasion and anchorage-independent colony formation. Moreover, the search for potential downstream targets showed that the HOXB7-overexpressing cells, along with a decreased CTNNB1 expression, present enriched HOXB7 protein direct interaction with the promoter regions of EGFR, DNMT3B, COMMD7 and CDH1 genes. Discussion Therefore, this is a strong indication that the HOXB7 role in tumour progression is dependent on the cellular genetic background, especially concerning the HER2 profile and ECM interactions. The cell/cell and cell/ECM interactions are important for cellular architectural maintenance and growth control among other processes 56. To metastasize, breast cancer cells must detach from the tumour mass and resist to anoikis, a programmed cell death induced by lack of cell/ECM communication 5. The MDA231 cell line was established from cells recovered from a patient’s pleural effusion 14. Therefore, these cells had already accomplished the entire metastization process and, additionally, maintained its metastatic capability in in vivo mouse models. Here, we showed that HOXB7 overexpression leads to a decreased capacity of colony formation in an anchorage-independent environment. Moreover, HOXB7 overexpressing cells showed a capacity similar to that observed in MCF-7 cells, which is also a cell line established from a pleural effusion but non-metastatic in vivo. Akekawatchai et al 1, through proteomic analyses, identified 54 proteins that were expressed only in suspended and adherent MDA231 but not in MCF-7 cells in the same conditions. Could MDA231 HOXB7-overexpression have change the expression profiles of some proteins in order to have a genetic background more similar to the MCF7 cells? Could these proteins be related to the other phenotypic changes observed? These questions deserve attention. Controversially, the HOXB7-overexpression in SKBR3 cells (HER2+) increased their ability to form colonies in semisolid medium 16. Once again, the contradictory findings could be related to the different genetic profile of the cells and consequently to the pathways modulated in each particular condition. Thus, characterization of the HOXB7 targets is an urgent issue for better understanding of the HOXB7 functions in different cellular contexts. In this study, we investigate the physical interaction between HOXB7 protein and the promoter regions of the genes EGFR, DNMT3B, CDH1, CTNNB1, FGF2, TGFB2 and COMMD7. The EGFR (Epidermal Growth Factor Receptor) overexpression is recognized as a driver mechanism in the initiation, progression and therapy resistance of several carcinomas such as lung, breast and pancreatic cancers 3, being the interaction between HOXB7 and the EGFR promoter region already demonstrated 29. EGFR is also an important target in multiple chemotherapeutic regimens 13. It is a member of ERBB family, which is composed of ERBB-1 (HER1/EGFR), ERBB-2 (HER2), ERBB-3 (HER3) and ERBB-4 (HER4) receptors, and are activated when in dimers after ligand-binding 55. Discussion The culture of breast cells on a reconstituted basement membrane results in a 3D-growth that recapitulates several aspects of glandular architecture in vivo that are lost in 2D culture, as demonstrated with MCF10A cells 21, 24. Interestingly, MDA231 HOXB7-overexpressing cells (D3) formed compact spheroids compared to the spread and branched growth shown by MDA231 EV cells. When cultured in 2D conditions, the EV and D3 cells did not show impacting differences in their morphology. Moreover, the spheroids of MDA231 HOXB7-overexpressing cells resembled those generated by BT474 and MCF7 cells 34, 43, reinforcing the idea that HOXB7 up-regulation should be related to a more luminal phenotype. The plasma membrane protrusions are the result of the continuous synthesis and remodelling of the cytoskeleton actin filaments and are closely related to the promotion and driving of cell migration. The formation of protrusions occurs in both 2D and 3D cell migration. In 3D cell movement, these protrusions are collectively known as invadossomes that establish close contact with the ECM (Extracellular Matrix) and perform a proteolytic matrix degradation to invade the connective tissues 36. Thus, the decreased protrusions formation observed in 3D culture could explain the impaired capacity of HOXB7- overexpressing cells to migrate in a 2D culture and to invade through the Matrigel® matrix as will be discussed in detail ahead. The impact of HOXB7 overexpression on breast cancer cell proliferation has been demonstrated in different cellular models. Caré et al 16 showed that HOXB7 overexpression in SKBR3 cells (ER-/PR-/HER2+) leads to increased cell proliferation through the up-regulation of FGF2. Ma et al 49 corroborated these results in MCF7 cells (ER+/PR+/HER2-) in which HOXB7 down-regulation decreased the proliferation ratios. However, an in vivo study conducted by Chen et al 18 showed that the HOXB7 overexpression alone was insufficient to induce tumour formation and had a dual role when co- overexpressed with HER2. The Hoxb7/Her2 transgenic mice, compared to Her2 transgenic animals, showed a delayed tumour onset reflected in a decreased tumour multiplicity but, once the tumour was Page 13/34 Page 13/34 established, Hoxb7 promoted tumour progression leading to the formation of larger masses and to a higher index of pulmonary micrometastasis. Additionally, in silico analyses of public microarray data showed that high level of HOXB7 predict a poor outcome in HER2-positive, but not in HER2-negative breast cancers 18. Discussion Jin et al 32 showed that tamoxifen resistance in MCF7 cells is related to a progressive increasing in HOXB7 expression levels, along with the up-regulation of EGFR and its ligands. Making a parallel between the genetic backgrounds of MDA231 and MCF7 cells concerning HOXB7, EGFR and ER expression 20, 27, 61, we can infer that MDA231 is HOXB7-low, EGFR-high and ER-negative, while MCF7 is HOXB7-high, EGFR-low, and ER-positive. Therefore, in both cells, we observed an inverse correlation among the three molecules. Additionally, BT474 cells are HOXB7-high, EGFR-intermediary, ER-positive, but resistant to hormone therapy. Noteworthy, BT474 cells also express intermediary levels of HER2 for which EGFR is the preferred dimer- Page 14/34 Page 14/34 partner. Added to our finding that HOXB7/EGFR interaction occurred only in HOXB7-overexpressing MDA231 cells (HOXB7-high, EGFR-high, ER-negative) this is are supplementary evidence that HOXB7 action is dependent, not only on its own expression, but also on the molecules that are active in the cell, named ER, EGFR and HER2. Thus, different genetic profile combinations could lead to different responses for the same stimulus in the same tissue. partner. Added to our finding that HOXB7/EGFR interaction occurred only in HOXB7-overexpressing MDA231 cells (HOXB7-high, EGFR-high, ER-negative) this is are supplementary evidence that HOXB7 action is dependent, not only on its own expression, but also on the molecules that are active in the cell, named ER, EGFR and HER2. Thus, different genetic profile combinations could lead to different responses for the same stimulus in the same tissue. The interaction between HOXB7/DNMT3B was only observed in HOXB7-overexpressing cells as the case of EGFR. No studies exist correlating HOXB7 and DNMT3B molecules. However, it is widely known that the levels of DNMTs, especially of DNMT3B, DNMT3A, and DNMT3L, are often increased in various cancer tissues and cell lines, and may account for the hypermethylation of tumour suppressor genes in a variety of malignancies 62. As the HOXB7 methylation, which have already been described in several cancers and shown to be related to patients’ prognosis 53, the search for genes that are hypermethylated in response to HOXB7-binding to DNMT3B is also an important and interesting approach to pursue. CDH1 was another gene shown to interact with HOXB7 in MDA231 cells with an interaction increment in those overexpressing HOXB7. It was already demonstrated that HOXB7-overexpression in MCF10A cells, causes a reduction on CDH1 protein expression 67. Discussion This could be due to the fact that the signals released by CTNNB1 knockdown to promote changes in stemness are not the same as those provided by the HOXB7 overexpression effects on CTNNB1 expression. The same explanations could be applied to the absence of sensitivity changes to docetaxel treatments. The chemotherapeutic drugs have different action modes and different mechanisms of resistance. Cisplatin and doxorubicin are DNA-interfering drugs, while docetaxel acts on tubulin impairing the microtubules dynamics and consequently, inhibiting cell cycle progression 39, 60. Another gene that showed a direct interaction with HOXB7 in both EV and D3 cells was FGF2. The HOXB7/FGF2 interaction has already been described in melanoma cell lines 15 and in BT474 breast cancer cell line 29. Moreover, when the coordinated expression of HOXB7 and FGF2 was demonstrated in melanomas, glioblastomas and leukemias, it was also shown that SKBR3 (HER2+) cells are negative for HOXB7 and FGF2 expression 16. We found that FGF2 is expressed only in MCF10A (normal breast) and MDA468 (TNBC, basal) cells (Supplementary Fig. 6). Even in MCF7, BT474 and D3 cells, which show high levels of HOXB7, the FGF2 expression was undetectable (Supplementary Fig. 6). Despite the existence of HOXB7/FGF2 interaction in different cell models and the described relationship between the increased expression of FGF2 in breast cancer 6, further studies are needed to establish in which context HOXB7 may be involved in FGF2 expression regulation. Although the HOXB7 direct binding to the activation of TGFB2 promoter has already been described in MCF7 cells 47, this interaction was not observed in MDA231 EV and D3 cells. Liu and colleagues 47 showed that the knockdown of TGFB2 leads to a decrease in the migration and invasion capacities of MDA231 cells overexpressing HOXB7, along with a dramatic inhibition of lung metastasis formation in an in vivo model. The TGFB2 mRNA levels were also analysed in EV and D3 cells but no differences were detected (Supplementary Fig. 7). The role of TGFB2 in breast cancer progression is ambiguous, since it was shown to display tumour-suppressing and -enhancing effects. 2, 11, 72. Thus, a number of questions still need to be addressed in order to completely understand the regulation of TGFB2 and its interaction with pathways activated in the course of tumour progression. Finally, COMMD7, a gene that we found to be overexpressed in BT474 and MDA231 breast cancer cell lines (Supplementary Fig. Discussion In breast cancer, deregulation of the CDH1 function plays crucial roles in metastases and is related to worse prognosis and shorter overall survival of patients 19. Despite the higher HOXB7/CDH1 interaction found in MDA231 HOXB7-overexpressing cells, the CDH1 functionality did not change between EV and D3 cells, as accessed by the aggregation assay. However, CDH1 interacts with the actin cytoskeleton 19 and therefore the increment of the HOXB7/CDH1 interaction in MDA231 HOXB7-overexpressing cells could be related with the absence of protrusion formations in these cells. Additionally, CDH1 works in complex with CTNNB1 to ensure cell–cell adhesion between epithelial cells 8. The balance between CDH1 and CTNNB1 expression is responsible for the cell adherens- junction maintenance or for the CTNNB1 release from the complex followed by its degradation or nuclear translocation, where it forms complexes with members of the TCF/LEF family to activate the transcription of target genes involved in self-renewal, EMT (Epithelial to Mesenchymal Transition) and cell proliferation 51, 65. Moreover, in breast cancer patients, nuclear and cytosolic accumulation of CTNNB1, but not the membrane-associated form, is associated with reduced overall survival 38. To our surprise, the CTNNB1 promoter region showed to be a HOXB7 target in MDA231 EV and D3 cells, adding the fact that the CTNNB1 mRNA and protein levels were decreased in HOXB7-overexpressing cells. Xu et al 68 demonstrated that CTNNB1-knockdown impaired the ability of the MDA231 and HCC38 cells to migrate. Moreover, the HCC38 cells showed a decreased capacity to form anchorage-independent colonies in soft agar, a lower stemness potential and a reduced tumorigenic potential in vivo and an increased sensitivity to chemotherapeutic agents (cisplatin and doxorubicin). Another study by Lin and colleagues 46 showed that the CTNNB1 downregulation, promoted by luteolin treatment, effectively reverses EMT in the MDA231 and BT549 TNBC cells and suppresses the metastatic potential of MDA231 cells in vivo. These findings could be related to the phenotypes of migration and invasion observed in D3 cells. Concerning the stemness, we analysed the mRNA expression of the c-Myc, NANOG, OCT4, and Page 15/34 Page 15/34 SOX2 transcription factors, known as master regulators of pluripotency and stemness 63, and did not observe any differences between EV and D3 cells (Supplementary Fig. 5). Discussion 4) and that potentially presents enriched interactions with HOXB7 in BT474 29, was found to bind to a target of HOXB7 in D3 cells. The COMMD family is a group of proteins that act as scaffold proteins to facilitate the assembling of crucial molecules involved in the control of several biological processes such as the NF-κB signalling 25. No information exists on the specific role of COMMD7 in breast cancer but data exists revealing that COMMD7 expression levels are up-regulated in hepatocellular carcinoma tissues 71 and in pancreatic ductal adenocarcinoma (PDAC) tissues and cell lines [43]. Interestingly, HOXB7 expression is also high in liver and pancreatic tissues, as reviewed by de Bessa Garcia and colleagues 22. Therefore, it is possible to infer that HOXB7 and COMMD7 could have Page 16/34 Page 16/34 correlated and interaction-dependent functions not only in breast cancer, but also in other cancers. A summary of the main findings of this work is represented in the scheme shown in Fig. 9. correlated and interaction-dependent functions not only in breast cancer, but also in other cancers. A summary of the main findings of this work is represented in the scheme shown in Fig. 9. Conclusion Triple Negative breast cancer cells expressing high levels of HOXB7 mRNA and nuclear protein show a less aggressive phenotype represented by lower viability and decreased migration, invasion and anchorage-independent colony formation capacities along with a more compact spheroid formation in 3D culture. In MDA231 cells overexpressing HOXB7, the HOXB7 protein interacts with CTNNB1, FGF2, CDH1, EGFR, DNMT3B and COMMD7 promoter regions. Moreover, these cells present CTNNB1 down- regulation, which relates with the phenotypes observed. Thus, the present study raises important questions concerning, not only the downstream targets of HOXB7, but also the molecules that could be involved in HOXB7 regulation and nuclear translocation as well as the genes that are differentially methylated in response to HOXB7/DNMT3B interaction. In addition, our results suggest that the HOXB7 role in breast cancer is strictly dependent on the cellular genetic background, especially concerning ER, EGFR and HER2 expression, and modulated by the transcriptional levels of this transcription factor. Authors’ contributions SABG and RF designed the study, SABG executed the assays, performed data interpretation and wrote the manuscript, MA performed the COMMD7 qPCR and in silico analyses, TP performed the expression analyses of stemness markers, CTNNB1, FGF2 and TGFB2. RF provided financial support, discussed results, participated in the writing of the manuscript and supervised the work. Funding FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-030562 (PTDC/BTM-TEC/30562/2017). Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethical approval and consent to participate No applicable The authors declare no competing interests The authors declare no competing interests Consent for publication No applicable Abbreviations Abbreviations 2D- Two-dimensional 3D- Three-dimensional ECM- Extracellular Matrix ER- Estrogen receptor ETOH - Ethanol EV- Empty Vector FBS- Fetal Bovine Serum g- gravitational force equivalent h- hours IP- Immunoprecipitation min.- minutes PR- Progesterone receptor sec.- seconds Page 17/34 TNBC- Triple Negative Breast Cancer V/V- volume/volume WT- Wild-Type Acknowledgments We acknowledge Barbara Sousa for the assistance in the aggregation assay protocol, Joana Paredes for kindly provide the MDA-MB-231 cells and the MB2 antibody, Carmen Jerónimo for kindly provide the additional breast cancer cell lines. We are grateful to Ana Paço for participating in the preliminary data collection and Raul Guizzo for editing assistance and proofreading. We also acknowledge the i3S Page 18/34 scientific platforms (https://www.i3s.up.pt/scientific-platforms) for technical support and equipment provided: 1) Biochemical and Biophysical Technologies (b2Tech), NanoDrop 1000 (Thermo Scientific) and Bioruptor Plus (Diagenode); 2) Bioimaging (member of the national infrastructure PPBI - Portuguese Platform of Bioimaging, PPBI-POCI-01-0145-FEDER-022122), Axiovert 200M inverted fluorescent microscope (Carl Zeiss); 3) BioSciences Screening (member of the national infrastructure PPBI - Portuguese Platform of Bioimaging, PPBI-POCI-01-0145-FEDER-022122), Synergy™ 2 Plate reader (BioTek) and InCell Analyser 2000 Automated fluorescence widefield HCS microscope; 4) Cell Culture and Genotyping (CCGen), cell culture room, CFX96™ Real-Time PCR Detection System (Bio-Rad), ChemiDoc Gel Image System (Bio-Rad) and Zoe fluorescent cell imager (Bio-Rad); 5) Translational Cytometry Unit, TraCy (project MICROXXI - NORTE-07-0162-FEDER-000033 funded by ON2), FACSAria™ II cell sorter (BD Biosciences). 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COMMD7 Regulates NF-κB Signaling Pathway in Hepatocellular Carcinoma Stem-like Cells. Molecular therapy oncolytics12: 112-123. 72 Zugmaier G, Ennis BW, Deschauer B, Katz D, Knabbe C, Wilding G et al (1989). Transforming growth factors type beta 1 and beta 2 are equipotent growth inhibitors of human breast cancer cell lines. Journal of cellular physiology141: 353-361. Figures Page 24/34 Figure 1 Page 25/34 Figure 1 HOXB7 mRNA and protein expression characterization in MDA231 cells after transfection. A, HOXB7 mRNA expression in WT (wild-type cells), EV (empty-vector transfected cells), B7 (pool of cells transfected with HOXB7-expression vector), D3 (clone of cells transfected with HOXB7-expression vector). B, HOXB7 cytoplasmic protein expression in WT (wild type cells), EV (empty-vector transfected cells), B7 (pool of cells transfected with HOXB7-expression vector), D3 (clone of cells transfected with HOXB7-expression Figure 1 HOXB7 mRNA and protein expression characterization in MDA231 cells after transfection. A, HOXB7 mRNA expression in WT (wild-type cells), EV (empty-vector transfected cells), B7 (pool of cells transfected with HOXB7-expression vector), D3 (clone of cells transfected with HOXB7-expression vector). B, HOXB7 cytoplasmic protein expression in WT (wild type cells), EV (empty-vector transfected cells), B7 (pool of cells transfected with HOXB7-expression vector), D3 (clone of cells transfected with HOXB7-expression Page 25/34 Page 25/34 vector). C, HOXB7 nuclear protein expression in WT (wild type cells), EV (empty-vector transfected cells), B7 (pool of cells transfected with HOXB7-expression vector), D3 (clone of cells transfected with HOXB7- expression vector). The bars represent the mean ±SD obtained in at least three independent experiments. , Statistically significant differences, P≤0.05, obtained by Brown-Forsythe and Welch ANOVA tests (Multiple comparisons) with Games-Howell’s correction. (Multiple comparisons) with Games Howells correction. Figure 2 HOXB7 overexpression effect on MDA231 cells spheroids morphology in 3D cell culture. The cells were seeded in 8-well glass slides on-top in Matrigel® and after 8-days they were photographed in an inverted fluorescent microscope. The WT, EV and B7 cells show a similar pattern of 3D-growth with spread spheroids that form a number of protrusions. These patterns are clearly different from that observed in Figure 2 HOXB7 overexpression effect on MDA231 cells spheroids morphology in 3D cell culture. The cells were seeded in 8-well glass slides on-top in Matrigel® and after 8-days they were photographed in an inverted fluorescent microscope. The WT, EV and B7 cells show a similar pattern of 3D-growth with spread spheroids that form a number of protrusions. These patterns are clearly different from that observed in D3 cells, which grow in compact spheroids without protrusions formation. HOXB7 overexpression effect on MDA231 cells spheroids morphology in 3D cell culture. The cells were seeded in 8-well glass slides on-top in Matrigel® and after 8-days they were photographed in an inverted fluorescent microscope. The WT, EV and B7 cells show a similar pattern of 3D-growth with spread spheroids that form a number of protrusions. These patterns are clearly different from that observed in D3 cells, which grow in compact spheroids without protrusions formation. Page 26/34 Page 26/34 Figure 3 HOXB7 overexpression effect on viability and Docetaxel sensitivity of MDA231 cells. A, MTT cell-viability measure in EV, B7 and D3 cells in 24h, 48h, 72h and 96h post-plating. B, EV, B7 and D3 cell sensitivity to 5nM and 50nM Docetaxel treatment in 24h, 48h, 72h and 96h post-plating. The cells were kept in ETOH as a control of vehicle action. The graphs represent the mean ±SD obtained in at least three independent experiments. , Statistically significant differences, P≤0.05, obtained through 2way ANOVA test (Multiple HOXB7 overexpression effect on viability and Docetaxel sensitivity of MDA231 cells. A, MTT cell-viability measure in EV, B7 and D3 cells in 24h, 48h, 72h and 96h post-plating. B, EV, B7 and D3 cell sensitivity to 5nM and 50nM Docetaxel treatment in 24h, 48h, 72h and 96h post-plating. The cells were kept in ETOH as a control of vehicle action. The graphs represent the mean ±SD obtained in at least three independent experiments. , Statistically significant differences, P≤0.05, obtained through 2way ANOVA test (Multiple HOXB7 overexpression effect on viability and Docetaxel sensitivity of MDA231 cells. A, MTT cell-viability measure in EV, B7 and D3 cells in 24h, 48h, 72h and 96h post-plating. B, EV, B7 and D3 cell sensitivity to 5nM and 50nM Docetaxel treatment in 24h, 48h, 72h and 96h post-plating. The cells were kept in ETOH as a control of vehicle action. The graphs represent the mean ±SD obtained in at least three independent experiments. , Statistically significant differences, P≤0.05, obtained through 2way ANOVA test (Multiple Page 27/34 Page 27/34 comparisons) with Tukey’s correction. In B, the P values for the time-point of 24h are: ETOH, EVxB7 (P=0.003) and B7xD3 (P=<0.0001); Docetaxel 5nM EVxD3 (P=0.0002) and B7xD3 (P=0.04); and Docetaxel 50nM EVxD3 (P<0.0001) and B7xD3 (P=0.0022). comparisons) with Tukey’s correction. In B, the P values for the time-point of 24h are: ETOH, EVxB7 (P=0.003) and B7xD3 (P=<0.0001); Docetaxel 5nM EVxD3 (P=0.0002) and B7xD3 (P=0.04); and Docetaxel 50nM EVxD3 (P<0.0001) and B7xD3 (P=0.0022). Docetaxel 50nM EVxD3 (P<0.0001) and B7xD3 (P=0.0022). Figure 4 Figure 4 Figure 4 HOXB7 overexpression effect on MDA231 cells migration capacity. A, representative images of the wound healing assay in EV, B7 and D3 cells at 0h, 9h and 15h after the scratch wound. B, wound areas of the EV, Page 28/34 B7 and D3 cells along the time. The graph represents the mean ±SD obtained in at least three independent experiments. , Statistically significant differences, P≤0.05, obtained through 2way ANOVA test (Multiple comparisons) with Tukey’s correction. , Represents the comparison between EV cells and the cells of the respective rectangle, when not specified. B7 and D3 cells along the time. The graph represents the mean ±SD obtained in at least three independent experiments. , Statistically significant differences, P≤0.05, obtained through 2way ANOVA test (Multiple comparisons) with Tukey’s correction. , Represents the comparison between EV cells and the cells of the respective rectangle, when not specified. B7 and D3 cells along the time. The graph represents the mean ±SD obtained in at least three independent experiments. , Statistically significant differences, P≤0.05, obtained through 2way ANOVA test (Multiple comparisons) with Tukey’s correction. , Represents the comparison between EV cells and the cells of the respective rectangle, when not specified. Page 29/34 the cells of the respective rectangle, when not specified. Figure 5 Page 29/34 Figure 5 Figure 5 HOXB7 overexpression effect on MDA231 cells invasion capacity. A, representative images of the nuclei of the EV and D3 cells that invaded the Matrigel® layer of the invasion chamber and migrate through the control chambers. B, invasion percentage of EV and D3 cells with the respective invasion index. The graph represents the mean ±SD obtained in at least three independent experiments. * Statistically significant differences, P≤0.05, obtained by unpaired T test with Welch’s correction. Figure 6 HOXB7 overexpression effect on MDA231 cells anchorage-independent colony formation efficiency. Number of colonies formed by MCF7 (Luminal A) and MDA231 (Triple-negative; WT, EV and D3). The graph represents the mean ±SD obtained in at least three independent experiments. , Statistically significant differences, P≤0.05, obtained by Brown-Forsythe and Welch ANOVA tests (Multiple comparisons) with Games-Howell’s correction. Figure 6 HOXB7 overexpression effect on MDA231 cells anchorage-independent colony formation efficiency. Number of colonies formed by MCF7 (Luminal A) and MDA231 (Triple-negative; WT, EV and D3). The graph represents the mean ±SD obtained in at least three independent experiments. , Statistically significant differences, P≤0.05, obtained by Brown-Forsythe and Welch ANOVA tests (Multiple comparisons) with Games-Howell’s correction. Page 30/34 Page 30/34 Figure 7 HOXB7 overexpression effect on CTNNB1 mRNA and protein expression in MDA231 cells. A, CTNNB1 mRNA relative expression in EV and D3 cells. B, CTNNB1 total protein relative expression EV and D3 cells. C, western blot representative of the CTNNB1 total protein analyses in EV and D3 cells being the expression of Tubulin the protein loading control. The bars represent the mean ±SD obtained in at least Figure 7 HOXB7 overexpression effect on CTNNB1 mRNA and protein expression in MDA231 cells. A, CTNNB1 mRNA relative expression in EV and D3 cells. B, CTNNB1 total protein relative expression EV and D3 cells. C, western blot representative of the CTNNB1 total protein analyses in EV and D3 cells being the expression of Tubulin the protein loading control. The bars represent the mean ±SD obtained in at least Page 31/34 Page 31/34 three independent experiments. , Statistically significant differences, P≤0.05, obtained by unpaired T test with Welch’s correction. test with Welch’s correction. Page 32/34 Figure 8 HOXB7 protein direct interaction with the promoter regions of EGFR (A), FGF2 (A), CTNNB1 DNMT3B (D), TGFB2 (E) and COMMD7 (F). IgG, Immunoprecipitations with anti-IgG antibo negative control). HOXB7, Immunoprecipitations with anti-HOXB7 antibody. The bars repre Page 32/34 Figure 8 HOXB7 protein direct interaction with the promoter regions of EGFR (A), FGF2 (A), CTNNB1 (B), CDH1 (C), DNMT3B (D), TGFB2 (E) and COMMD7 (F). IgG, Immunoprecipitations with anti-IgG antibody (interaction negative control). HOXB7, Immunoprecipitations with anti-HOXB7 antibody. The bars represent the mean Figure 8 Page 32/34 HOXB7 protein direct interaction with the promoter regions of EGFR (A), FGF2 (A), CTNNB1 (B), CDH1 (C), DNMT3B (D), TGFB2 (E) and COMMD7 (F). IgG, Immunoprecipitations with anti-IgG antibody (interaction negative control). HOXB7, Immunoprecipitations with anti-HOXB7 antibody. The bars represent the mean Page 32/34 Page 32/34 ±SD obtained in at least three independent experiments. , Statistically significant differences, P≤0.05, obtained by 2way ANOVA test (Multiple comparisons) with Bonferroni’s correction (EGFR, FGF2, CTNNB1 DISTAL, CTNNB1 PROX, CDH1 CpG and CDH1 PROM) or by Brown-Forsythe and Welch ANOVA tests (Multiple comparisons) with Games-Howell’s correction (DNMT3B, TGFB2 and COMMD7). The calculated P values were: EGFR (IgG_D3xD3=0.01), FGF2 (IgG_EVxEV=0.035, IgG_D3xD3=0.036), CTNNB1 (DISTAL: IgG_EVxEV=0.032, IgG_D3xD3=0.011. PROX: IgG_EVxEV=0.022, IgG_D3xD3=0.017), CDH1 (CpG: IgG_EVxEV=0.018, IgG_D3xD3=0.002. PROM: IgG_EVxEV=0.034, IgG_D3xD3=0.0001, EVxD3=0.031), DNMT3B (IgG_D3xD3=0.033) and COMMD7 (IgG_D3xD3=0.013). ±SD obtained in at least three independent experiments. *, Statistically significant differences, P≤0.05, obtained by 2way ANOVA test (Multiple comparisons) with Bonferroni’s correction (EGFR, FGF2, CTNNB1 DISTAL, CTNNB1 PROX, CDH1 CpG and CDH1 PROM) or by Brown-Forsythe and Welch ANOVA tests (Multiple comparisons) with Games-Howell’s correction (DNMT3B, TGFB2 and COMMD7). The calculated P values were: EGFR (IgG_D3xD3=0.01), FGF2 (IgG_EVxEV=0.035, IgG_D3xD3=0.036), CTNNB1 (DISTAL: IgG_EVxEV=0.032, IgG_D3xD3=0.011. PROX: IgG_EVxEV=0.022, IgG_D3xD3=0.017), CDH1 (CpG: IgG_EVxEV=0.018, IgG_D3xD3=0.002. PROM: IgG_EVxEV=0.034, IgG_D3xD3=0.0001, EVxD3=0.031), DNMT3B (IgG_D3xD3=0.033) and COMMD7 (IgG_D3xD3=0.013). Figure 7 g _ , g _ g _ , g _ , ), DNMT3B (IgG_D3xD3=0.033) and COMMD7 (IgG_D3xD3=0.013). Figure 9 Summary of the HOXB7 nuclear overexpression effects in the TNBC cell line MDA-MB-231. The HOXB7 nuclear overexpression leads the MDA-MB-231 cells to a less aggressive phenotype represented by a more organized spheroid formation in 3D culture and reduced cell viability, migration, invasion and anchorage-independent colony formation. The observed phenotypes could be related to changes in CTNNB1 expression and to HOXB7 binding to genes with important roles in breast cancer progression. Exception made to COMMD7 whose function in cancer is still little studied. Figure 9 Summary of the HOXB7 nuclear overexpression effects in the TNBC cell line MDA-MB-231. The HOXB7 nuclear overexpression leads the MDA-MB-231 cells to a less aggressive phenotype represented by a more organized spheroid formation in 3D culture and reduced cell viability, migration, invasion and anchorage-independent colony formation. The observed phenotypes could be related to changes in CTNNB1 expression and to HOXB7 binding to genes with important roles in breast cancer progression. Exception made to COMMD7 whose function in cancer is still little studied. Summary of the HOXB7 nuclear overexpression effects in the TNBC cell line MDA-MB-231. The HOXB7 nuclear overexpression leads the MDA-MB-231 cells to a less aggressive phenotype represented by a more organized spheroid formation in 3D culture and reduced cell viability, migration, invasion and anchorage-independent colony formation. The observed phenotypes could be related to changes in CTNNB1 expression and to HOXB7 binding to genes with important roles in breast cancer progression. Exception made to COMMD7 whose function in cancer is still little studied. SupplementaryData.pdf Supplementary Files Page 33/34 This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. SupplementaryData.pdf SupplementaryData.pdf Page 34/34
https://openalex.org/W4205622891	https://periodicos2.uesb.br/index.php/reed/article/download/8854/6539	Portuguese	null	A DIMENSÃO DA SOCIALIZAÇÃO PROFISSIONAL DOS SABERES DOCENTES: EXPERIÊNCIAS SIGNIFICATIVAS PARA A DOCÊNCIA	Revista de Estudos em Educação e Diversidade	2,021	cc-by	6,673	Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 LA DIMENSIÓN DE LA SOCIALIZACIÓN PROFESIONAL DE LOS SABERES DOCENTES: EXPERIENCIAS SIGNIFICATIVAS PARA LA ENSEÑANZA Edmila Silva de Oliveira1 Denise Aparecida Brito Barreto2 Resumo: O presente artigo é o recorte de uma pesquisa de Mestrado que analisou as representações sociais de professores sobre a docência superior. O objetivo da discussão foi apresentar a dimensão da socialização profissional que está imbricada nos saberes docentes, articulando a mobilização desses saberes com questões que envolvem a experiência e os contextos significativos para o exercício profissional. A abordagem da pesquisa é qualitativa com a perspectiva interpretativa. Os participantes foram seis professores do curso de Pedagogia de uma universidade pública da Bahia. Os dados foram obtidos por meio de entrevista semiestruturada e a análise foi efetivada a partir da técnica de Análise de Conteúdo. Por meio do estudo, constatamos que o processo de socialização envolve uma construção de saberes ligados tanto a questões profissionais quanto as questões de ordem pessoal, as quais os docentes levarão consigo para o resto de suas vidas e que repercutirão no exercício docente. 1 Palavras-chave: Saberes docentes. Socialização profissional. Exercício docente. Abstract: The present article is an excerpt of a Master's research that analyzed the social representations of teachers about higher education teaching. One of the purposes of the discussion was to present the dimension of professional socialization that is embedded in teachers' knowledge, articulating the mobilization of this knowledge with issues involving experience and significant contexts for professional practice. The research approach is qualitative with an interpretative perspective. The participants were six professors from the Pedagogy course of a public university in Bahia. The data were obtained through semi-structured interviews and the analysis was carried out using the Content Analysis technique. Through the study, we found that the socialization process involves a construction of knowledge linked to both professional and personal issues, which teachers 1 Mestra em Educação. Universidade Estadual do Sudoeste da Bahia -UESB. Professora substituta da Universidade Federal do Recôncavo da Bahia/UFRB. Membro do Grupo de Pesquisa Estudos em Linguagem, Formação de Professores e Práticas Educativas (GELFORPE). E-mail: edmilaoliveira@ufrb.edu.br. Orcid: https://orcid.org/0000-0003-4357-0698. 1 Mestra em Educação. Universidade Estadual do Sudoeste da Bahia -UESB. Professora substituta da Universidade Federal do Recôncavo da Bahia/UFRB. Membro do Grupo de Pesquisa Estudos em Linguagem, Formação de Professores e Práticas Educativas (GELFORPE). E-mail: edmilaoliveira@ufrb.edu.br. Orcid: https://orcid.org/0000-0003-4357-0698. 2 Doutora em Educação. Universidade Federal da Bahia - UFBA. Professora plena da Universidade Estadual do Sudoeste da Bahia/UESB. LA DIMENSIÓN DE LA SOCIALIZACIÓN PROFESIONAL DE LOS SABERES DOCENTES: EXPERIENCIAS SIGNIFICATIVAS PARA LA ENSEÑANZA Líder do Grupo de Pesquisa Estudos em Linguagem, Formação de Professores e Práticas Educativas (GELFORPE). E-mail: deniseabrito@gmail.com. Orcid: https://orcid.org/0000-0003-3448- 5109. 2 Doutora em Educação. Universidade Federal da Bahia - UFBA. Professora plena da Universidade Estadual do Sudoeste da Bahia/UESB. Líder do Grupo de Pesquisa Estudos em Linguagem, Formação de Professores e Práticas Educativas (GELFORPE). E-mail: deniseabrito@gmail.com. Orcid: https://orcid.org/0000-0003-3448- 5109 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 will carry with them for the rest of their lives and which will have repercussions on the teaching practice. Keywords: Teaching knowledge. Professional socialization. Teaching practice. Resumen: Este artículo es el extracto de una investigación de maestría que analizó las representaciones sociales de los profesores sobre la educación superior. Uno de los propósitos de la discusión fue presentar la dimensión de la socialización profesional que está impregnada en el conocimiento docente, articulando la movilización de este conocimiento con cuestiones que involucran la experiencia y los contextos significativos para el ejercicio profesional. El enfoque de la investigación es cualitativo con una perspectiva interpretativa. Los participantes fueron seis profesores del curso de Pedagogía de una universidad pública de Bahía. Los datos se obtuvieron mediante entrevistas semiestructuradas y el análisis se realizó a partir de la técnica del Análisis de Contenido. A través del estudio, encontramos que el proceso de socialización implica una construcción de conocimiento vinculada tanto a cuestiones profesionales como personales, que los profesores llevarán consigo el resto de su vida y que repercutirá en el ejercicio de la docencia. Palabras-clave: Saberes docentes. Socialización profesional. Práctica docente. Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. do modo de pensar e agir dos docentes no exercício de suas práticas e estão atreladas com as questões que envolvem o processo constante e incansável de qualificação, a qual, de acordo com Pimenta e Anastasiou (2014, p. 39) é “um fator chave no fomento da qualidade em qualquer profissão, especialmente na educação, que experimenta constante mudança”. Este estudo considera o saber docente como um saber plural, como propõe Tardif (2014), e não um conjunto de conteúdos cognitivos definidos de uma vez por todas, mas um processo em construção ao longo de uma carreira profissional na qual o professor vai aprendendo a dominar seu ambiente de trabalho, insere-se nele e o interioriza, até que se torna parte de sua consciência prática. Tardif e Raymond, (2000, p. 217) pontuam que “a socialização é um processo de formação do indivíduo que se estende por toda a história de vida e comporta rupturas e continuidades”. Neste processo estão os saberes que são produzidos pela socialização, ou seja, imersos em diferentes mundos socializados - famílias, grupos, amigos, instituições, entre outros -, os professores constroem, aos poucos e em interação, sua identidade pessoa e social. O período de iniciação na docência, por exemplo, é um momento de relevância especial, pois conforme discutem Papi e Martins (2010), esse momento implica no confronto entre a realidade profissional, com suas contradições e em constante movimento, atravessado de fragilidades formativas. Nessa direção, Melo e Pimenta (2019, p. 67) postulam que “trata-se de um momento singular da carreira em que os saberes são colocados à prova, suas concepções e crenças pedagógicas”. Essa construção ocorre no decorrer da trajetória de formação dos professores, momento de crescimento e aperfeiçoamento para o profissional da Educação. 3 Desse modo, conhecer as experiências de socialização dos professores é relevante para a análise das concepções e das representações sociais compartilhadas no decorrer do exercício docente, pois esses profissionais são agentes que socializam e enfrentam diversas situações desafiadoras. Corroboramos com Dubar (2005, p. 18) quanto ao fato de que a socialização pode ser entendida como “um processo de construção, desconstrução e reconstrução de identidades ligadas às diversas esferas de atividade (principalmente profissional) que cada um encontra durante sua vida e das quais deve aprender a tornar-se ator”. Introdução A presente pesquisa está inserida nas discussões acerca da formação de docentes que atuam no ensino superior público, tema constante de debate proposto por autores como Cunha (1996, 1998), García (1999), Zabalza (2004), Almeida (2012), Pimenta e Anastasiou (2014). Com o intuito de contribuir com esses estudos, o presente artigo, é recorte de uma pesquisa do Mestrado em Educação, linha de pesquisa Formação, Linguagem, Memória e Processos de Subjetivação, desenvolvido no Programa de Pós-Graduação em Educação da Universidade Estadual do Sudoeste da Bahia/UESB, apresentando a dimensão da socialização profissional que está imbricada nos saberes docentes. 2 A temática da socialização profissional, ainda que não seja nova, requer um olhar atento quanto suas particularidades, visto ser um processo por meio do qual os profissionais passam a (re) construir valores, atitudes, conhecimentos e adquirem, segundo Melo e Pimenta (2019, p. 68) “habilidades que lhes permitam e justifiquem ser e estar numa determinada profissão.”. Seguindo essa linha de entendimento, a socialização profissional se configura enquanto uma atividade que permite a concretização dos ideais profissionais. Há, nesse processo, um movimento de traduzir em práticas profissionais os conhecimentos que são próprios da profissão. É importante que as dimensões dos saberes que os professores mobilizam/necessitam para e durante sua atuação docente sejam analisadas, visto que essas dimensões fazem parte Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 ç p : http://periodicos2.uesb.br/index.php/reed 89 em: http://periodicos2.uesb.br/index.php/reed -6889 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. ç p http://periodicos2.uesb.br/index.php/reed 89 Assim, podemos considerar que os professores vivenciam diversos espaços e situações de socialização, o que tem ligação direta com as dimensões temporais e identitárias dos saberes docentes e influenciam diretamente na tomada de decisões, na postura que adotam, bem como nas suas práticas. Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 em: http://periodicos2.uesb.br/index.php/reed -6889 Delineamento metodológico A presente investigação é de abordagem qualitativa, que conforme Minayo (2016, p. 21) “se aprofunda no mundo dos significados” e a realidade “precisa ser exposta e interpretada, em primeira instância, pelos próprios pesquisados, em segunda instância, por um processo compreensivo e interpretativo contextualizado.”. Nessa direção, não focamos em representatividade numérica do grupo pesquisado, mas no “aprofundamento da compreensão de um grupo social, de uma organização, de uma instituição, de uma trajetória etc.” (GOLDENBERG, 2004, p. 14). A atenção se voltou para os significados e para as relações sociais, partindo de uma perspectiva dialética que focaliza questões voltadas a sociedade, sua estrutura e seus sujeitos. Segundo Minayo (2016, p. 20): Esse conjunto de fenômenos humanos é entendido aqui como parte da realidade social, pois o ser humano se distingue não só por agir, mas, também, por pensar sobre o que faz e por interpretar suas ações dentro e a partir da realidade vivida e compartilhada com seus semelhantes. Nesse sentido, o percurso trilhado pela pesquisa qualitativa faz emergir aspectos subjetivos e são utilizados quando a busca é feita no sentido de captar percepções e entendimento sobre a natureza geral de determinada questão, o que dá margem à interpretação. O que podemos verificar, ainda de acordo com Minayo (2016, p. 20), é que “a pesquisa qualitativa responde a questões muito particulares”. Ela se ocupa, especialmente dentro das ciências sociais, “com o universo dos significados, dos motivos, das aspirações, das crenças, dos valores e das atitudes.”. Vale ressaltar que a pesquisa qualitativa não se volta especificamente para um produto, mas analisa os fenômenos, os processos, os percentuais macro e mínimos, bem como os contextos em que estes se constituíram. 4 É importante que na pesquisa qualitativa, os métodos não estejam estandardizados e sim, construídos e - por que não reconstruídos? Então, no decorrer do processo investigativo, fomos criando caminhos interpretativos, os quais tiveram as falas dos participantes como principais meios de interpenetração entre eles e o próprio mundo, numa dinâmica que envolve impressões, contextos, marcas e experiências vividas. O campo de pesquisa foram os cursos de Licenciatura em Pedagogia de uma universidade pública da Bahia. Essa escolha se justificou pelo fato de a instituição oferecer o Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. referido curso em todos os seus campi. Os participantes foram 06 professores atuantes nos cursos de Pedagogia e os critérios utilizados para a seleção desses professores levaram em consideração o fato de eles terem vasta experiência no campo da Educação e por atuarem no ensino superior público há mais de cinco anos. A expectativa foi de que, por possuírem uma grande bagagem pedagógica, esses participantes disponibilizariam um material de análise riquíssimo, advindo de suas respostas acerca da docência, formação, trajetória, entre outros. E essa expectativa foi consolidada. O instrumento de coleta de dados utilizado foi a entrevista semiestruturada, a qual conforme Minayo (2016, p. 59) “combina perguntas fechadas e abertas, em que o entrevistado tem a possibilidade de discorrer sobre o tema em questão sem se prender à indagação formulada”. Esse instrumento utilizado na presente pesquisa, que teve como matéria-prima as respostas dos interlocutores, contou com questões norteadoras estruturadas, em conformidade com o referencial teórico adotado. Embasamos nossa escolha da entrevista semiestruturada nos estudos de Bogdan e Biklen (1994, p. 134) que consideram como objetivo da pesquisa “recolher dados descritivos na linguagem do próprio sujeito, permitindo ao investigador desenvolver intuitivamente uma ideia sobre a maneira como os sujeitos interpretam aspectos do mundo”. Corroboramos também com Minayo (2016) ao destacar que a fala pode ser reveladora de condições de vida, de expressão de sistemas de valores e crenças. 5 Percebemos como uma das vantagens de se utilizar a entrevista semiestruturada a possibilidade que os participantes têm de proferir suas respostas de acordo com a perspectiva pessoal, não tendo que se prender completamente as questões que foram previamente elaboradas. Algumas das questões orientadoras foram: Quais foram suas primeiras impressões no início da docência no ensino superior? Como você avalia sua carreira docente até o presente momento? Como docente do ensino superior, quais contextos e experiências foram mais significativos e ofereceram subsídios para seu exercício? Utilizamos como ferramenta de registro das entrevistas o gravador de voz do aparelho celular, bem como as notas de campo. Além de gravar as entrevistas realizadas, fizemos anotações consideradas pertinentes para a pesquisa e que descreveram o comportamento dos docentes. Para Bogdan e Biklen (1994, p. investigação visto que, em muitos casos, o comportamento expressa maior sinceridade do que as próprias palavras/respostas. investigação visto que, em muitos casos, o comportamento expressa maior sinceridade do que as próprias palavras/respostas. A fase do tratamento e análise do material diz respeito aos procedimentos utilizados para “valorizar, compreender, interpretar os dados empíricos, articulá-los com a teoria que fundamentou o projeto ou com outras leituras teóricas e interpretativas cuja necessidade foi dada pelo trabalho de campo” (MINAYO, 2016, p. 26). Então, após a fase da coleta, os dados foram analisados partindo do específico para o geral, característica própria da pesquisa de cunho qualitativo. Para isso, utilizamos como técnica a Análise de Conteúdo (AC) que, consoante Bardin (2011, p. 48), é um conjunto de técnicas de análises das comunicações, visando obter, por di t i t áti bj ti d d i ã d t úd d Para isso, utilizamos como técnica a Análise de Conteúdo (AC) que, consoante Bardin (2011, p. 48), é um conjunto de técnicas de análises das comunicações, visando obter, por procedimentos sistemáticos e objetivos de descrição do conteúdo das mensagens, indicadores (qualitativos ou não) que permitem a inferência de conhecimentos relativos às condições de produção/recepção (variáveis inferidas) destas mensagens. um conjunto de técnicas de análises das comunicações, visando obter, por procedimentos sistemáticos e objetivos de descrição do conteúdo das mensagens, indicadores (qualitativos ou não) que permitem a inferência de conhecimentos relativos às condições de produção/recepção (variáveis inferidas) destas mensagens. Na mesma linha de pensamento, Amado (2017, p. 302) destaca que a técnica da Análise de Conteúdo é “flexível, e adaptável às estratégias [...], uma técnica que aposta claramente na possibilidade de fazer inferências interpretativas a partir dos conteúdos expressos”. Por meio da técnica, buscamos interpretar os aspectos relacionados à dimensão da socialização profissional, visto que a Análise de Conteúdo permite a inferência na busca de interpretação das falas dos participantes, bem como a frequência, similaridade das expressões correspondentes ou não, em que aparecem, levando em consideração o contexto em que está inserido (MINAYO, 2016). 6 Consideramos pertinente destacar que as entrevistas foram realizadas no ano de 2019, antes do isolamento social decorrente da pandemia ocasionada pela disseminação da Covid-19 e para preservar a identidade dos/as convidados/as, utilizamos nomes fictícios. Vale salientar também, que, com o intuito de respeitar os aspectos éticos das pesquisas que envolvem seres humanos, as exigências do Comitê de Ética em Pesquisa (CEP) foram atendidas, visto que a pesquisa foi submetida e aprovada com parecer final número 34965219.4.0000.0055. 49), a pesquisa de campo “exige que o mundo seja examinado com a ideia de que nada é trivial e que tudo tem potencial para constituir uma pista.”, então, acreditamos que as notas de campo podem ser de grande valia para a Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Saberes docentes e experiências significativas para o exercício docente Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Quais são os saberes docentes? O que eles representam na prática dos professores? Eles se complementam? Quais são suas implicações na formação do professor e no ensino? Essas e tantas outras indagações partem do desenvolvimento da presente pesquisa, visto que consideramos de extrema importância reconhecer a pluralidade dos saberes docentes e sua influência na formação dos professores. Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Na concepção de Freire (1996, p. 21) Na concepção de Freire (1996, p. 21) O ato de cozinhar, por exemplo, supõe alguns saberes concernentes ao uso do fogão, como acendê-lo, como equilibrar para mais, para menos, a chama, como lidar com certos riscos mesmo remotos de incêndio, como harmonizar os diferentes temperos numa síntese gostosa e atraente. A prática de cozinhar vai preparando o novato, ratificando alguns daqueles saberes, retificando outros, e vai possibilitando que ele vire cozinheiro. A prática de velejar coloca a necessidade de saberes fundantes como o do domínio do barco, das partes que o compõem e da função de cada uma delas, como o conhecimento dos ventos[...] Na prática de velejar se confirmam, se modificam ou se ampliam esses saberes. (grifos nossos) A metáfora utilizada na obra “Pedagogia da Autonomia” faz referência ao processo formativo, evidenciando que as atribuições da profissão, bem como a aproximação com o exercício fazem parte da construção dos saberes, visto que a docência pressupõe conhecimentos e aptidões específicas que refletirão na atividade dos professores. Para o autor supracitado, os saberes dos professores podem ser confirmados, modificados ou até mesmo ampliados no decorrer de sua carreira profissional. “São saberes demandados pela prática educativa em si mesma, qualquer que seja a opção política do educador ou educadora” (FREIRE, 1996, p.21). 7 Nessa linha de pensamento, Pimenta e Anastasiou (2014. p. 71) afirmam que “esses saberes se dirigem às situações de ensinar e com elas dialogam, revendo-se, redirecionando- se, ampliando-se. No entanto, também contribuem para revê-las, redirecioná-las, transformá- las”. Esses saberes vão sendo configurados pelas demandas que surgem na prática dos profissionais e podem ser mobilizados, produzidos e fundamentados a partir do próprio contexto de trabalho e dos diversos processos que envolvem a formação do professor. Eles são os pilares para o professor desenvolver seu trabalho em sala de aula considerando o que expressam as autoras supracitadas, de que esses saberes devem ser mobilizados de maneira articulada aos percursos de formação inicial e contínua. Gauthier (2013, p. 343) destaca que “os saberes nos quais os professores se apoiam dependem diretamente das condições sociais e históricas nas quais eles exercem sua profissão”. Então, para que eles sejam compreendidos precisam estar relacionados com as condições que estruturam o trabalho do professor, pois servem de base para a prática docente. Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Tenho prazer em ver os estudantes seguindo seus fluxos... acompanhar um aluno na graduação, depois ver na pós-graduação é fantástico... faz a gente ver que por mais ‘trabalho’ que o nosso aluno possa nos dar, mas ele pode e pode muito... ver esse desenvolvimento me nutre. (Professora Bernadete) A participante demonstrou em seu relato o quanto considera gratificante influenciar positivamente na formação acadêmica dos estudantes. As falas da professora coadunam com Melo e Pimenta (2019, p. 55), pois demonstram que a socialização não se trata de um movimento linear, mas sim, “de uma dialética do ser e estar no contexto profissional, em encadeamento com o que já está estabelecido, do ponto de vista da dinâmica institucional, permeada ora por atitudes que indicam transformações, ora por adaptações à cultura dada”. A professora Bernadete admitiu ainda: Gosto de poder ser importante no processo de formação de meus alunos, não por vaidade, mas por produção de sentido em meu existir no mundo. Às vezes eu me pergunto: e quando eu partir dessa experiência carnal, minha passagem terá valido à pena para as pessoas com quem convivi? Quando percebo que sim, sinto sentido na profissão de professora. (Professora Bernadete). A satisfação da docente ao ver o crescimento do estudante é evidente e demonstra que para ela essa é uma maneira de validar sua existência, o que está diretamente relacionado com o fato de ser professora. É como se existir e professorar estivessem na mesma esteira, ambos com um grande “peso”. Consoante essa ideia, Tardif e Raymond (2000, p. 225) afirmam que “os indivíduos dão sentido à sua vida profissional e se entregam a ela como atores cujas ações e projetos contribuem para definir e construir sua carreira.”. 9 Para o professor Carlos, “o que sustenta a docência é manter vivo o aluno em mim”. Ao se colocar no lugar constante de aprendiz, ele valida que a socialização se relaciona com as condições objetivas e subjetivas que envolvem o trabalho do professor. Sobre essa questão, Cunha (2010, p. 83) pondera que “o exercício da docência dá-se fundamentalmente a partir de elementos que o professor traz de sua trajetória como aluno e/ou de esquemas prévios e/ou de rotina que se coloca como balizadora de seus fazeres”. Ademais, a fala do professor Carlos nos remete a ideia de ensinagem apresentada por Pimenta e Anastasiou (2014, p. 205) na qual “a ação de ensinar é definida na relação com a ação de aprender, pois, para além da meta que revela a intencionalidade, o ensino desencadeia necessariamente a ação de aprender.”. Para Tardif (2014, p. 54), o professor é um sujeito que sabe alguma coisa e esse saber é um “saber plural, formado de diversos saberes provenientes das instituições de formação, da formação profissional, dos currículos e da prática cotidiana”. É justamente a partir da ideia de pluralidade que o autor reitera que a compreensão das diferentes tipologias de saberes propostas por diversos autores só é possível quando as associamos à natureza diversa de suas origens, às diferentes fontes de sua aquisição e às relações que os professores estabelecem com eles. Assim, essa relação não é formada exclusivamente pela transmissão dos conhecimentos legitimados, mas pela integração de diferentes saberes que intercambiam cotidianamente entre si. Nesse contexto está a socialização, um processo que contribui de maneira significativa para a aprendizagem da cultura da organização universitária (GARCIA, 1999), sobretudo quando nos referimos aos primeiros anos de ensino do professor universitário. É importante levar em consideração que diversas experiências e contextos são significativos e oferecem subsídio para o exercício profissional. Essas experiências fazem parte do processo de socialização e concordamos com Melo e Pimenta (2019, p. 54), ao defenderem que este é um “processo por meio do qual o professor passa a fazer parte de um coletivo docente”. O profissional passa a perceber as relações de interdependência e de colaboração entre os pares como elementos imprescindíveis do seu trabalho, visto que as ações coletivas têm implicações nas práticas e potencializam o desenvolvimento profissional docente. 8 Como docentes do ensino superior, os participantes da pesquisa relataram suas vivências, reforçando o que Tardif (2014, p. 110-111) discorre quanto ao saber da experiência: “é um saber temporal, evolutivo e dinâmico que se transforma e se constrói no âmbito de uma carreira, de uma história de vida profissional, e implica uma socialização e uma aprendizagem da profissão”. Seguindo essa linha de compreensão, Gauthier (2013, p. 344) esclarece que, “a aquisição desses saberes é acompanhada de uma socialização profissional associada a uma experiência da prática docente”. Nessa perspectiva, fica evidente que o universo de trabalho dos professores é permeado por saberes que se entrecruzam e desempenham papéis essenciais no processo de ensino-aprendizagem. No que se refere às experiências e/ou contextos mais significativos para o exercício profissional, professora Bernadete relatou: Tenho prazer em ver os estudantes seguindo seus fluxos... acompanhar um aluno na graduação, depois ver na pós-graduação é fantástico... faz a gente Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 http://periodicos2.uesb.br/index.php/reed 89 Disponível em: http://periodicos2.uesb.br/index.php/reed SSN: 2675-6889 bastante para o desenvolvimento do processo de ensino-aprendizagem.”. Nessa direção, ao admitir que há uma necessidade constante de aprender sempre que ensina, o professor Carlos expressou que tem conduzido sua formação para além das questões técnicas. Ainda sobre os processos de socialização, a professora Magda afirmou: As experiências mais significativas no meu processo de formação de professora universitária se deram/dão por meio da relação que estabeleço com os meus pares, com os estudantes, com os funcionários, com as escolas- campo de estágio. Neste movimento eu tenho me feito e me refeito. (Professora Magda) Inferimos a partir desse depoimento que o saber advindo da experiência é, como afirma Tardif (2014, p. 111) “um saber social e construído pelo ator em interação com diversas fontes sociais de conhecimentos [...] de saber-ensinar provenientes da cultura circundante, da organização escolar, dos atores educativos, das universidades, etc.” Atrelados a essa ideia de saber experiencial estão os processos de socialização, que conforme Melo e Pimenta (2019, p. 57) “implicam assumir pertencimento a um determinado grupo, ou seja, atitudes vão sendo assumidas, e, aos poucos orientam a conduta pessoal e profissional.” Professor José destacou: Professor José destacou: o que me permitiu hoje, por exemplo, ter acesso a espaços de produção do saber, a dialogar com grandes figuras do cenário intelectual brasileiro e mundial foi a possibilidade de estar dentro da Universidade lecionando. (Professor José) o que me permitiu hoje, por exemplo, ter acesso a espaços de produção do saber, a dialogar com grandes figuras do cenário intelectual brasileiro e mundial foi a possibilidade de estar dentro da Universidade lecionando. (Professor José) 10 10 Sobre essa questão pontuada pelo professor, Pimenta e Anastasiou (2014, p. 88) consideram que “a docência na universidade configura-se como um processo contínuo de construção da identidade docente e tem por base os saberes da experiência, construídos no exercício profissional mediante o ensino dos saberes específicos das áreas de conhecimento.”. Nesse sentido, percebemos a importância de o docente ter clareza da sua prática enquanto professor e nos impactos da sua atuação na vida do aluno da licenciatura, que também será professor. Concordamos com Barreto, Oliveira e Seixas (2017, p. 64) que “o docente que reflete sobre os seus atos oferece maior probabilidade de acertos nas suas decisões.”. Na perspectiva de Mello e Pimenta (2019, p. 59) o exercício da docência não é algo simples. “Trata-se de um fazer complexo que exige do docente uma formação para além do domínio do campo científico de sua área de formação e atuação – imprescindível – mas não o Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Assim, refletir sobre as posturas que adota, as concepções e posicionamentos que defende, as ações que pratica, as ideias que (re) constrói é fundamental para que o professor não reduza sua prática a meras reproduções. Ademais, a atuação docente no ensino superior exige maturidade, abertura Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. para o novo, postura profissional, conhecimentos ligados às questões que permeiam a educação superior, entendimento sobre os processos de ensino e de aprendizagem, metodologia, currículo, didática, Pedagogia, políticas, entre tantas outras exigências. Os contatos, essas aproximações, alguns distanciamentos também que aconteceram, isso eu devo a possibilidade de estar dentro da Universidade, que é justamente esse espaço onde o professor – vou usar um termo chulo - não é mero dador de aulas. Dar aula é muito pouco para se qualificar o professor, embora muitos professores se reduzam a isso mesmo, essa condição de ser ministrador de aulas, um dador de aulas. Eu vejo o espaço da Universidade como um espaço em que devemos articular ensino, pesquisa e extensão. (Professor José, grifos nossos). Sobre a articulação mencionada acima pelo professor José, a professora Maria também afirmou “sempre tento fazer a integração ensino, pesquisa e extensão”. De acordo com Pimenta e Anastasiou (2014), o ensino e a pesquisa caracterizam o exercício da profissão docente. No caso específico do professor universitário, soma-se a isso o exercício profissional em campo. A fala da professora Maria revelou certeza de estar agindo de maneira coerente. Na mesma linha de pensamento, professor José acrescentou: Na mesma linha de pensamento, professor José acrescentou: Eu penso que a Universidade é esse espaço desse tripé, dessa tríplice articulação entre ensino, pesquisa e extensão. Ela precisa oferecer um retorno para a sociedade do que ela produz em termos de pesquisa, em termos de conhecimento. (Professor José) 11 Ao realizar um exercício de análise da própria atuação, a Professora Bernadete mencionou: avalio minha carreira como avalio a minha própria vida. Muitos acertos, muitos erros, muitas aprendizagens. Muitas coisas para aprender, viver, experimentar... penso que fiz uma carreira que acompanha a pessoa que sou. E sou feliz como professora, sem nenhuma tentativa de perfumaria. (Professora Bernadete). Essa afirmação nos remete a pensar que há uma forte relação entre a avaliação que muitos professores fazem de sua vida profissional e a vida profissional. As falas revelam uma grande aproximação com a afirmação categórica de Nóvoa (1995, p. 17) de que “é impossível separar o eu profissional do eu pessoal”. Consciente de que seu processo está apenas começando, Magda esclareceu: “Avalio a minha carreira de professora universitária como um processo em desenvolvimento, sendo que Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 eu ainda tenho muito por aprender.”. Esse posicionamento dialoga com as ideias freirianas de inconclusão do processo formativo. Nas palavras de Freire (1996, p. 57) A consciência do mundo e a consciência de si como ser inacabado necessariamente inscrevem o ser consciente de sua inconclusão num permanente movimento de busca. Na verdade, seria uma contradição se, inacabado e consciente do inacabamento o ser humano não se inserisse em tal movimento [...] É na inconclusão do ser, que se sabe como tal, que se funda a educação como processo permanente. A professora Magda destacou ainda: “necessito muito da colaboração dos colegas e dos estudantes para me constituir nessa itinerância, de formar-me professora universitária”. O depoimento continua evidenciando a consciência da professora quanto a importância da socialização. Nas palavras de Anastasiou e Pimenta (2014, p. 79), quando chegam à docência na Universidade, os professores “sabem sobre ser professor por intermédio da experiência de outros, colegas, pessoas da família. Tem experiência acumulada sobre as agruras da profissão, sua não valorização social e financeira, o desgaste emocional que acarreta [...]”. Professor José afirmou: “se eu posso avaliar a minha carreira (28 anos de magistério público), avalio como positiva, mesmo diante de todos os problemas que nós enfrentamos em sala de aula”. Os relatos do professor apontam uma tomada de consciência quanto à promoção de mudanças diante das diversas dificuldades que se apresentam durante o processo de ensino- aprendizagem, a partir das suas intervenções enquanto professor. 12 Professora Magda relembrou algumas dificuldades: “As dificuldades foram referentes à insegurança diante do novo, as novas rotinas, os novos processos, as demandas específicas da docência na educação superior, a utilização de infinitas siglas”. Essas dificuldades são comuns, e conforme Batista e Nunes (2016) a atividade docente é especialmente difícil para os professores que começam e ainda não têm a vivência que lhes permitam enfrentar os problemas próprios da profissão. Sobre essa questão, Tardif e Raymond (2000, p. 217) ratificam que “a inserção numa carreira e o seu desenrolar exigem que os professores assimilem também saberes práticos específicos aos lugares de trabalho, com suas rotinas, valores, regras, etc.”. Assim, “quanto às facilidades, destaco a acolhida que recebi alguns colegas, que foram essenciais para a minha ambientação na universidade”, relembrou Magda. Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. conceitual, pode servir ainda para encorajar os professores novatos no enfrentamento das dificuldades e emoções presentes nesse contexto.”. conceitual, pode servir ainda para encorajar os professores novatos no enfrentamento das dificuldades e emoções presentes nesse contexto.”. Professor Dante destacou: Do ponto de vista intelectual, diria que sou feliz em saber que atuei de forma positiva para vida de algumas pessoas durante suas trajetórias acadêmicas, principalmente dos alunos que orientei. (Professor Dante) Essa autoavaliação positiva de sua trajetória intelectual, atrelada ao fato de que entende a contribuição acadêmica na vida dos alunos vai ao encontro da discussão proposta por Abreu, Alencar e Couto (2019, p. 435), ao afirmarem que “a formação do aluno é uma construção coletiva sujeita a uma série de influências, sendo o professor o elemento fundamental nesse complexo de relações.”. Entendemos que, conforme Tardif (2014) é no espaço de trabalho que o professor desenvolve o domínio sobre o fazer docente, o que ensinar, como preparar e realizar suas aulas, bem como quais objetivos desejam atingir no que diz respeito ao aprendizado de seus alunos. Essas noções edificam a carreira docente, possuem maior propriedade na constituição de saberes voltados às suas práticas, proporcionando a estes docentes o domínio do seu próprio trabalho e o enfrentamento das condições precárias que, em muitos casos, impossibilitam a realização de um bom exercício profissional. 13 13 O conjunto de falas da professora reforça o que Batista e Nunes discutem (2016, p.174) “a convivência com professores experientes, além de proporcionar situações de aprendizagem técnica e Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. i í l h // i di 2 b b /i d h / d Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 docência, compreendida como prática social complexa, que exige o domínio de vários saberes”. Esse processo de socialização envolve, primordialmente, a construção de saberes ligados tanto a questões profissionais quanto as questões pessoais, as quais o professor levará consigo para o resto de sua vida e repercutirão no seu exercício profissional. Um longo caminho ainda deve ser percorrido para que haja a promoção e ampliação de espaços de diálogo sobre os saberes docentes e sua dimensão da socialização profissional, o que sugere a realização de novas pesquisas. Além disso, é importante levar em conta que o ciclo da pesquisa não se fecha, visto que toda pesquisa produz conhecimento e gera novas indagações. Conclusões O presente texto propôs uma discussão sobre a dimensão da socialização profissional dos saberes docentes, enfatizando a mobilização desses saberes com questões que envolvem a experiência e os contextos significativos para o exercício profissional. O estudo levou em consideração a pluralidade dos saberes imbricados nas práticas dos professores, saberes esses atravessados por fatores relacionados a questões temporais, identitárias e que envolvem também os processos de socialização profissional. Dessa forma, os resultados da pesquisa ratificaram que a socialização é um processo de formação que comporta tanto rupturas quanto continuidades. Ademais, está relacionada também a um processo de marcação e de incorporação dos indivíduos às práticas e rotinas institucionalizadas das equipes de trabalho. Nessa direção, corroboramos com Melo e Pimenta (2019, p. 59) ao afirmarem que “torna-se imperiosa a elaboração de conhecimentos teóricos e práticos relacionados à Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 Referências AMADO, João. Manual de investigação qualitativa em Educação. 3 ed. Coimbra: Imprensa da Universidade de Coimbra, 2017. AMADO, João. Manual de investigação qualitativa em Educação. 3 ed. Coimbra: Imprensa da Universidade de Coimbra, 2017. BARDIN, Laurence. Análise de conteúdo. São Paulo: Edições 70, 2011. BARRETO, Denise Aparecida Brito; OLIVEIRA, Albertina Lima; SEIXAS, Ana Maria. dimensão social e política da autonomia. Revista Educação em Questão, v. 55, p. 58-83, dez 2017. 14 14 BATISTA, Daniela Ribeiro Medeiros; NUNES, Claudio Pinto. Profissão docente: dificuldades encontradas por professores iniciantes. In: Berta Leni Costa Cardoso; Claudio Pinto Nunes. (Org.). Trabalho Docente, Políticas e Gestão Educacional. 1 ed. Curitiba: Editora CRV, 2016, p. 163-178. BOGDAN, Robert.; BIKLEN, Sari. Investigação Qualitativa em Educação. Porto: Porto Editora , v. Colecção Ciências da Educação, 1994. CUNHA, Luiz A. A universidade reformanda: o golpe de 1964 e a modernização do ensino superior. Rio de Janeiro: Francisco Alves, 1988. CUNHA, Luiz A. Ensino superior: a gratuidade necessária. Revista Brasileira de Política e Administração da Educação, Porto Alegre, v. 2, n. 12, jul./dez. 1996. CUNHA, Maria Izabel; ZANCHET, Beatriz Maria Boechio. A problemática dos professores iniciantes: tendência e prática investigativa no espaço universitário. Revista Educação. Porto Alegre, v. 33, p. 189-197, set/dez 2010. DE ABREU, Elzenita Falcão; DE ALENCAR, Helouisa Moulin.; COUTO, Leandra Lúcia Morais. Formação Acadêmica e influência dos professores nos projetos profissionais de universitários. Práxis Educacional. Vitória da Conquista, v. 15, n. 31, p. 430-451, 2019. Revista de Estudos em Educação e Diversidade. v. 2, n. 6, p. 1-15, out./dez. 2021. Disponível em: http://periodicos2.uesb.br/index.php/reed ISSN: 2675-6889 FREIRE, Paulo. Pedagogia da autonomia: saberes necessários à prática educativa. 25 ed. São Paulo: Paz e Terra, 1996. FREIRE, Paulo. Pedagogia da autonomia: saberes necessários à prática educativa. 25 ed. São Paulo: Paz e Terra, 1996. DUBAR, Claude. A socialização: construção das identidades sociais e profissionais. São Paulo: Martins Fontes, 2005. GAUTHIER, Clermot. Por uma teoria da Pedagogia. Pesquisas contemporâneas sobre o saber docente. 3. ed. Ijuí: Unijuí, 2013. GAUTHIER, Clermot. Por uma teoria da Pedagogia. Pesquisas contemporâneas sobre o saber docente. 3. ed. Ijuí: Unijuí, 2013. GOLDENBERG, Mirian. A arte de pesquisar: como fazer pesquisa qualitativa em Ciências Sociais. 8. ed. São Paulo: Record, 2001. GARCIA, Carlos Marcelo. Formação de professores: para uma mudança educativa. Porto, PT: Porto, 1999. GARCIA, Carlos Marcelo. Formação de professores: para uma mudança educativa. Porto, PT: Porto, 1999. MELO, Geovana Ferreira; PIMENTA, Selma Garrido. Socialização profissional de docentes na universidade: contribuições teóricas para o debate. Revista Linhas, v. 20, p. 51-77, 2019. MINAYO, Maria Cecília de Souza. Pesquisa social: teoria, método e criatividade. 1. ed. Petrópolis, RJ: Vozes, 2016. NÓVOA, António. Os professores e sua formação. Lisboa: Dom Quixote, 1995. PIMENTA, Selma Garrido; ANASTASIOU, Lea das Graças Camargo. Docência no Ensino Superior. 5. ed. São Paulo: Cortez, 2014. PAPI, Silmara de Oliveira Gomes; MARTINS, Pura Lúcia O. M. As pesquisas sobre professores iniciantes: algumas aproximações. Educação em Revista. Belo Horizonte, v. 26, n. 3, p. 39-56, dez 2010. 15 15 PIMENTA, Selma Garrido; ANASTASIOU, Léa das Graças Camargos. Docência no Ensino Superior. 5. ed. São Paulo: Cortez, 2014. TARDIF, Maurice; RAYMOND, Danielle. Saberes, tempo e aprendizagem do trabalho no magistério. Revista Educação & Sociedade, v. 21, p. 209-244, Dezembro XXI. 2000. TARDIF, Maurice. Saberes docentes e formação profissional. 17 ed. Rio de Janeiro: Vozes, 2014. ZABALZA, Miguel A. O ensino universitário: seu cenário e seus protagonistas. Porto Alegre: Artmed, 2004. Recebido em: 08 de junho de 2021. Aprovado em: 20 de dezembro de 2021. Aprovado em: 20 de dezembro de 2021.
https://openalex.org/W4309308927	https://www.frontiersin.org/articles/10.3389/fendo.2022.1048601/pdf	English	null	Intracellular VHHs to monitor and modulate GPCR signaling	Frontiers in endocrinology	2,022	cc-by	12,519	Abbreviations: GPCR, G protein-coupled receptor; VHH, VH of heavy-chain only antibodies; Nb, nanobody; sdAb single-domain antibody; CDR, complementarity-determining region. G protein-coupled receptor, conformations, intracellular VHHs, Cell signaling, G proteins, b-arrestins, biosensor TYPE Review PUBLISHED 16 November 2022 DOI 10.3389/fendo.2022.1048601 TYPE Review PUBLISHED 16 November 2022 DOI 10.3389/fendo.2022.1048601 Intracellular VHHs to monitor and modulate GPCR signaling OPEN ACCESS EDITED BY Ralf Jockers, Universite´ de Paris, France REVIEWED BY Ga´ bor Turu, Semmelweis University, Hungary Karim Nagi, Qatar University, Qatar CORRESPONDENCE Pascale Cre´ pieux pascale.crepieux@inrae.fr †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology RECEIVED 19 September 2022 ACCEPTED 24 October 2022 PUBLISHED 16 November 2022 CITATION Raynaud P, Gauthier C, Jugnarain V Jean-Alphonse F, Reiter E, Bruneau and Cre´ pieux P (2022) Intracellular VHHs to monitor and modulate GPCR signaling. Front. Endocrinol. 13:1048601. doi: 10.3389/fendo.2022.1048601 Pauline Raynaud 1†, Camille Gauthier 1†, Vinesh Jugnarain 1, Fre´de´ric Jean-Alphonse 1,2, Eric Reiter 1,2, Gilles Bruneau 1 and Pascale Cre´pieux 1,2 1Physiologie de la Reproduction et des Comportements (PRC), Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientiﬁque (CNRS), Institut Franc¸ais du Cheval et de l’Equitation (IFCE), Universite´ de Tours, Nouzilly, France, 2Inria, Inria Saclay-Ile-de-France, Palaiseau, France Single-domain antibody fragments, also known as VHHs or nanobodies, have opened promising avenues in therapeutics and in exploration of intracellular processes. Because of their unique structural properties, they can reach cryptic regions in their cognate antigen. Intracellular VHHs/antibodies primarily directed against cytosolic proteins or transcription factors have been described. In contrast, few of them target membrane proteins and even less recognize G protein-coupled receptors. These receptors are major therapeutic targets, which reﬂects their involvement in a plethora of physiological responses. Hence, they elicit a tremendous interest in the scientiﬁc community and in the industry. Comprehension of their pharmacology has been obscured by their conformational complexity, that has precluded deciphering their structural properties until the early 2010’s. To that respect, intracellular VHHs have been instrumental in stabilizing G protein-coupled receptors in active conformations in order to solve their structure, possibly bound to their primary transducers, G proteins or b-arrestins. In contrast, the modulatory properties of VHHs recognizing the intracellular regions of G protein-coupled receptors on the induced signaling network have been poorly studied. In this review, we will present the advances that the intracellular VHHs have permitted in the ﬁeld of GPCR signaling and trafﬁcking. We will also discuss the methodological hurdles that linger the discovery of modulatory intracellular VHHs directed against GPCRs, as well as the opportunities they open in drug discovery. KEYWORDS G protein-coupled receptor, conformations, intracellular VHHs, Cell signaling, G proteins, b-arrestins, biosensor Introduction classes, ranging from photons or ions to large glycoprotein hormones. Accordingly, they are major targets of the therapeutic arsenal, including treatments of hypertension, migraine, cancers, metabolic diseases, or disorders of the central nervous system, among other pathologies. But drug discovery of small chemicals with high potency, efﬁcacy and selectivity is reaching a limit, and in 2017, the 481 referenced drugs against GPCRs only targeted 107 out of 850 receptors of this protein family (10). Conventional antibodies now open new avenues in the ﬁeld, but still, GPCRs remain extremely difﬁcult targets since i/they are highly hydrophobic in nature because of their seven transmembrane helices, ii/most of them expose very limited regions outside the cell membrane, iii/their wide conservation across the animal kingdom goes together with a low immunogenicity, which precludes a strong immune response in heterologous animals. Until now, only two therapeutic monoclonal antibodies that recognize extracellular regions of GPCRs are on the market: mogamulizumab, an anti-neoplasic Ig directed against C-C chemokine receptor type 4 (CCR4) in T cell leukemia (11) and erenumAb, an antagonist of the Calcitonin gene-related peptide type 1 receptor (CALCRL) used to treat migraine (12). The remarkable properties of VHHs now raise exciting prospects for future therapies, because they could stimulate GPCR activity in vivo by binding their orthosteric ligand-binding site, or they could modulate the activity of the endogenous ligand by binding to allosteric sites of the receptor. But this line of research is still in its infancy and so far, only the apelin receptor (APLNR) (13) and the metabotropic glutamate receptor 2 (GRM2) (14) have been targeted with agonistic/ positive allosteric modulator VHHs, while others, such as CXCR4 (15), are recognized by antagonists. Interestingly, a VHH directed against the parathyroid hormone receptor (PTHR1) has recently been used as a tether of PTH(1–34), a bioactive N-terminal fragment of PTH, in order to improve its selectivity for the receptor (16). Antibody fragments are currently revolutionizing the speciﬁc targeting of molecules inside the cell. Among those, paramount importance has been given to variable fragments from single-chain antibodies encountered in the blood of some Camelidae and cartilaginous ﬁshes. These fragments represent the VHH (VH of heavy-chain only antibodies), and their recombinant analogues have been baptized as Nbs for nanobodies, or sdAbs for single domain antibodies. Introduction When compared to conventional immunoglobulins (Igs), they have smaller molecular weight (∼13 kDa) and size (diameter of 2.5 nm, length of 4 nm), and they consist in the minimum paratope able to recognize an epitope. They are composed of 4 framework regions (FR) and 3 complementarity-determining regions (CDR) that support complementarity to the epitope. A striking feature of these VHHs is the greater length of their CDR3 compared to that of a VH, that optimizes the contact surface with the epitope and compensates the lack of light chain. The length and ﬂexibility of CDR3 are properties that are advantageous to reach poorly accessible, cryptic epitopes (1). VHHs contain a canonical disulﬁde bond linking FR1 and FR3, and possibly additional bonds, for example between CDR2 and CDR3 in lama. These disulﬁde bonds are essential for thermal and thermodynamic stability (2), but apparently, reducing conditions do not compromise antigen binding afﬁnity, nor mechanical stability (3). In addition, because of “hallmark” hydrophilic residues at solvent-exposed positions, they are less prone to protein aggregation than single-chain variable fragments (scFv) (4). These properties are of uttermost importance when intracellularly expressed VHHs (intra-VHHs) are being studied, because they are placed in a mildly reducing environment in the cytosol. Although we are aware that the terminology of “nanobody” given by Ablynx in 2001 is widely accepted, in the case of intracellular Nb we prefer the term “intra-VHH” over “intrabodies”, that does not explicitly indicate the format of the antibody (VHH, scFv, Ig). This is the term that will be used in this review. In contrast, the use of intra-VHHs has been essentially limited to academic research. Remarkably, they have been instrumental in overcoming the major challenges associated to the conformational complexity of GPCR in their ligand- activated states. These receptors basally exist in transient conformational states due to rearrangements at the atomic level and at larger receptor portions (17). The corollary of GPCR intrinsic instability is that obtaining the necessary periodic organization required for resolving the 3D structure of ligand-bound GPCRs at high resolution has been a major stumbling block in the GPCR research ﬁeld. Intra-VHHs are a new type of biological tools that target proteins present at different sub-cellular compartments [reviewed in (5)], such as the GTP-bound form of RhoA at the inner face of the plasma membrane (6), or lamin lining the nuclear envelope (7). Intracellular VHHs to monitor and modulate GPCR signaling Raynaud P, Gauthier C, Jugnarain V, Jean-Alphonse F, Reiter E, Bruneau G and Cre´ pieux P (2022) Intracellular VHHs to monitor and modulate GPCR signaling. F E d i l 13 1048601 Front. Endocrinol. 13:1048601. doi: 10.3389/fendo.2022.1048601 COPYRIGHT © 2022 Raynaud, Gauthier, Jugnarain, Jean-Alphonse, Reiter, Bruneau and Cre´ pieux. 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 Endocrinology Frontiers in Endocrinology frontiersin.org 01 Raynaud et al. 10.3389/fendo.2022.1048601 Raynaud et al. Introduction They can be used to enhance, block or monitor (8) protein activation/activity, to track protein trafﬁcking inside the cell when fused to ﬂuorescent probes (7), to hinder protein/protein interactions, or to direct the antigen protein to degradation pathways (9). Some of them appear exquisitely sensitive to protein conformational dynamics, and this chaperone-like property has been particularly useful to decipher the conformational complexity of G protein-coupled receptors (GPCR) in their ligand-activated states. These transient conformational states are stabilized by ligand binding and by the subsequent coupling of their intracellular transducing partners, namely G proteins and b- arrestins (Figure 1). Hence, there is an allosteric communication whereby ligand binding induces conformational changes at the ligand-binding pocket that propagate to the inner side of the receptor. Allosteric communication goes also the other way GPCRs are involved in most physiological responses and as such, they bind ligands encompassing a vast array of biochemical Frontiers in Endocrinology frontiersin.org 02 Raynaud et al. 10.3389/fendo.2022.1048601 FIGURE 1 Intra-VHH interference on GPCR signaling. An intra-VHH may stabilize inactive conformations of the GPCR (upper panel) and dissociate upon stimulation. The size of the aura indicates the degree of intrinsic instability. Intra-VHHs may also stabilize active conformations (bottom panel), and compete with G proteins and/or b-arrestins (not shown on the ﬁgure) for binding to the receptor, depending on their afﬁnity (left). Alternatively, intra-VHH and transducer binding may be compatible. In that case, GRK phosphorylation sites may be masked (middle) or the VHH may allosterically enhance Ga binding (right). FIGURE 1 Intra-VHH interference on GPCR signaling. An intra-VHH may stabilize inactive conformations of the GPCR (upper panel) and dissociate upon stimulation. The size of the aura indicates the degree of intrinsic instability. Intra-VHHs may also stabilize active conformations (bottom panel), FIGURE 1 Intra-VHH interference on GPCR signaling. An intra-VHH may stabilize inactive conformations of the GPCR (upper panel) and dissociate upon stimulation. The size of the aura indicates the degree of intrinsic instability. Intra-VHHs may also stabilize active conformations (bottom panel), and compete with G proteins and/or b-arrestins (not shown on the ﬁgure) for binding to the receptor, depending on their afﬁnity (left). Alternatively, intra-VHH and transducer binding may be compatible. In that case, GRK phosphorylation sites may be masked (middle) or the VHH may allosterically enhance Ga binding (right). in order to be able to lock the receptors in the active, intermediate, or inactive conformations. Expected insights onto GPCR activity and regulation displacement of the TM6, Nb80 disrupts the critical ‘ionic lock’ interaction between R1313.50 of TM3 and E2686.30 of TM6, which would otherwise maintain the receptor in the inactive state (24). Similar G protein mimetic intra-VHHs targeting corresponding regions of muscarinic acetylcholine receptor M1 (CHRM1) (28) and mu-type opioid receptor (OPRM1) (29) have also been structurally described. Partial agonism was demonstrated with intra-VHH Nb71 bound to ADRB2, of which the TM6 had lesser outward displacement (30). Alternatively, acting as a negative allosteric modulator (NAM), intra-VHH Nb60 also had its CDR3 inserted in the allosteric cytoplasmic pocket of ADRB2 (31). However, the inactive state of the receptor was maintained since residues of the CDR3 of the intra-VHH bridged ‘the ionic lock’. Upon agonist binding, GPCRs undergo an ensemble of conformational state transitions enabling the recruitment of G proteins and b-arrestins, that propagate the membrane signal to a complex intracellular signaling network. G protein-coupled receptor kinases (GRKs) also are direct binding partners that desensitize the receptor by phosphorylation and promote b- arrestin-dependent G protein uncoupling and internalization. In addition, GPCRs make direct interactions with less conventional partners, such as HOMER protein homologs (32), PDZ domain- containing protein GIPC (33), Na(+)/H(+) exchange regulatory cofactor NHERF1 (34), vesicle-fusing protein NSF (35) or Multiple PDZ domain protein (MPDZ) (36), that also deserve interest since they generally intervene in receptor targeting to speciﬁc subcellular compartments and cell signaling (37). Like G proteins, GRKs and b-arrestins, their interaction with GPCRs could also be modulated by intra-VHHs. Since 2011, more than a hundred structures of active GPCR conformationally stabilized with intra-VHHs directly or via stabilization of G proteins, have been solved. By stabilizing discrete receptor conformational states, in the close future, these single domain antibodies are expected to provide signiﬁcant insights onto the dynamics of the signaling network and integrated cell response induced by G protein and b-arrestin binding to a ligand- activated GPCR. However, very few VHHs directed against the intracellular parts of GPCRs have been extensively studied functionally. Here, we highlight the outcomes of intra-VHH binding to GPCR intra-cellular regions, or to their transducers, on the receptor signaling properties and trafﬁcking. We will also discuss the methodological pitfalls that preclude extensive studies on GPCR modulation by intra-VHHs, and conclude with the therapeutical avenues that they open in drug discovery. Introduction For class A GPCRs, displacement of the transmembrane domains (TM), in particular TM6, results into a range of ‘open-active’ or ‘close- inactive’ states on the intracellular side of the GPCR, which in turn determines the degree of activity of the receptor, via G protein or b-arrestin coupling (25, 26). For instance, the structure of ADRB2 stabilized in the active conformation by Nb80 showed that this intra-VHH possesses G protein mimetic properties (24). Upon agonist binding, Nb80 was capable of maintaining the receptor-activated form by having its CDR3 inserted in the hydrophobic allosteric cytoplasmic pocket, formed by TM segments 3, 5, 6 and 7, otherwise occupied by the C-terminal a-helix of Gas (27). Leading to major outward round, since, reciprocally, transducer binding enhances the afﬁnity of the receptor for the ligand (18, 19) (Figure 2). A decisive breakthrough has been achieved on the G protein and ligand binding pocket allosteric dialog (20–22), with the use of Nb80. Indeed, binding of this antibody to the b2-adrenergic receptor (ADRB2) induces a closed conformation of the orthosteric site that traps the agonist while decreasing its dissociation rate (20, 22). Reciprocal, allosteric coupling of the two regions is essential for full receptor activation. It explains why the crystal structure of ADRB2 bound to a potent agonist unexpectedly matched with the inactive crystal structure of the receptor (23), unless it was stabilized intracellularly (24). Structural studies have provided detailed insights into the critical epitopes on GPCRs that could be targeted by intra-VHHs Frontiers in Endocrinology frontiersin.org 03 Raynaud et al. 10.3389/fendo.2022.1048601 Intra-VHHs to modulate GPCR activity The Nb80 intra-VHH and Gas binding regions on ADRB2 overlap, which explains that the conformation of ADRB2 stabilized by Nb80 or by the Gabg trimer are structurally very close (24, 27). As such, this intra-VHH displaces Gas and stabilizes active conformations of ADRB2 (27). Eighteen other anti- ADRB2 intra-VHHs have been selected from llama immunization with ADRB2 embedded in lipids and covalently bound to the BI-167107 high afﬁnity agonist. These VHHs have been functionally characterized and most of them decreased or inhibited isoproterenol-induced cAMP production and b- arrestin recruitment (39). However, their inhibitory effect likely results from different causes, since 12 of them exhibited preference for agonist-bound ADRB2, which suggests a probable steric hindrance of transducer binding, while the 6 remaining ones, including Nb60, selectively bound to inactive conformations immobilized in the presence of antagonists that do not support transducer recruitment (39). The Nb71 intra- VHH preferentially binds to Isoproterenol-activated receptor, and decreased cAMP production. However, it impaired phosphorylation of ADRB2 by GRKs, hence precluding b- arrestin recruitment and proper receptor intracellular trafﬁcking, which suggests that Nb71 recognizes the carboxy- terminal region of ADRB2 and not the Gs binding region as Nb80 for example, although both intra-VHHs functionally inhibit cAMP production. Four additional anti- ADRB2 intra- VHHs with even better afﬁnity than Nb80 have been isolated from a yeast synthetic library by combining magnetic-bead and ﬂuorescence-activated cell sorting (FACS) enrichments, and they all inhibited the activity of a cAMP-responsive reporter gene, in response to adrenaline, up to 45% of Emax (see Nb.c200 in Figure 3) (40). The human cytomegalovirus US28 receptor is an interesting example of a receptor exhibiting high constitutive activity. The latter is due to structural instability of the inactive state, and is only modestly enhanced by ligand binding (46). This observation suggests that constitutively active US28 and ligand-bound receptor have distinct conformations. In agreement, the conformation-speciﬁc VUN103 intra-VHH competes with Gq and b-arrestin 2 that constitutively interact with US28, and severely impairs inositol phosphate accumulation, nuclear factor of activated T cells (NFAT), signal transducer and activator of transcription 3 (STAT3) and NF-ϰB activation. In contrast, Nb7 does not affect constitutively active US28, but rather stabilizes US28 bound to its fractalkine CX3CL1 ligand, and inhibits the cell signaling it mediates (44) (Figure 3). Although both Nb7 and VUN103 bind ICL2 and ICL3 (46), they seem to recognize distinct active conformations of US28. Expected insights onto GPCR activity and regulation Since anti-GPCR intra-VHHs stabilize selective conformations of the receptor, they have been named “confobodies”, a registered trademark of Confo Therapeutics (38). They can recognize intracellular regions of the receptor devoted to signal transduction (Figure 1). Hence, their expected modulatory effect is a competition with direct transducers such as G proteins and b-arrestins by steric hindrance. Alternatively, they may also mask regulatory GRK phosphorylation sites on the receptor. In addition, some intra-VHHs may bind to an active conformation of the receptor without competing with the transducers, by positive allostery. So far, no intra-VHH A B FIGURE 2 Allosteric communication between the ligand-binding pocket and the intracellular sites of an activated GPCR. (A) By mimicking occupancy of the receptor by a transducer, an intra-VHH increases its afﬁnity for the ligand. (B) A VHH may also allosterically enhance the efﬁcacy of the ligand to stimulate some signaling pathways, hence inducing a biased response when compared to the reference ligand alone. FIGURE 2 Allosteric communication between the ligand-binding pocket and the intracellular sites of an activated GPCR. (A) By mimicking occupancy of the receptor by a transducer, an intra-VHH increases its afﬁnity for the ligand. (B) A VHH may also allosterically enhance the efﬁcacy of the ligand to stimulate some signaling pathways, hence inducing a biased response when compared to the reference ligand alone. Frontiers in Endocrinology frontiersin.org 04 10.3389/fendo.2022.1048601 Raynaud et al. enhancing the afﬁnity of a transducer for a GPCR has been identiﬁed, nor, to our knowledge, intra-VHH forcing a new receptor conformation by induced ﬁt. (OPRK1) with the Nb6 and Nb39 intra-VHHs (Figure 3) (42, 45). The proﬁle of dose-response curves obtained in the presence of the salvinorin A (SalA) agonist indicates that Nb6 binds preferentially to inactive OPRK1, whereas Nb39 binds to the receptor only when fully active, and, as expected, enhances SalA binding afﬁnity (42). Accordingly, increasing concentrations of the JDTIc antagonist prevent Nb39 binding to OPRK1 but has no effect on Nb6 binding that recognizes inactive conformations. Interestingly, domain swapping experiments grafting the OPRK1 intracellular loop (ICL3) 3 to seven heterologous receptors physiologically coupled to Gs, Gi or Gq, confer not only Gq recruitment ability but also antagonistic Nb6 binding (45), suggesting that this system can be used to monitor the activation state of Gq-coupled GPCRs. Thus, the Nb6 intra- VHH could be used similarly as the mini-Gq biosensor, and may possibly help de-orphanising orphan Gq-coupled GPCRs (45). Intra-VHHs to modulate GPCR activity The VUN103 and Nb7 intra-VHH discriminate respectively between constitutively active and agonist-stimulated conformations of the receptor, because Nb7 has no afﬁnity for the apo-conformation. Competition between VUN103 and Nb7 and the transducers hampers US28 signaling and expression of its target genes. Agonists are in bright green, antagonist are in light grey. FIGURE 3 Functional action of intra-VHHs against OPRK1, OPRD1 and OPRK1 opioid receptors, ADRB2 and US28. Nb39 reveals the location of opioid receptors when stimulated by a peptide ligand (bright green) or a cell-permeant non-peptide ligand (black dot). Nb60 locks ADRB2 in an inactive conformation that precludes interaction with transducers. Nb80 stabilizes conformationally active ADRB2. Nb.c200 stabilizes ADRB2 even when stimulated by its natural, low-afﬁnity agonist, adrenaline. Nb71 stabilizes active conformations of ADRB2 and prevents b-arrestin recruitment by limiting access to GRKs. US28 is a constitutively active GPCR. The VUN103 and Nb7 intra-VHH discriminate respectively between constitutively active and agonist-stimulated conformations of the receptor, because Nb7 has no afﬁnity for the apo-conformation. Competition between VUN103 and Nb7 and the transducers hampers US28 signaling and expression of its target genes. Agonists are in bright green, antagonist are in light grey. Intra-VHHs to modulate GPCR activity Importantly, by interfering with US28 constitutive activity, VUN103 partially inhibited the growth and US28- dependent signaling of glioblastoma spheroids as well as glioblastoma cells. Most G protein mimics described above have afﬁnity for an agonist-bound GPCR, but none of them is able to allosterically enhance receptor-Ga interaction. By using viral evolution of genetically actuating sequences (VEGAS), intra-VHHs against D (2) dopamine receptor (DRD2), the pH-sensing GPR68 and the 5-hydroxytryptamine receptor 2A (HTR2A) receptor have been obtained (43). One of these, VGS-Nb2, was qualiﬁed as positive allosteric modulator (PAM). It directly associates to HTR2A and stimulates a serum-responsive element (SRE) responsive reporter gene in the absence of ligand and regardless of Gaq expression, hence inducing an active conformation of the receptor. This response was still enhanced after stimulation by 5HT via Gq, coupling, but not b-arrestin 2, whose recruitment to the receptor was decreased. Hence, intra-VHHs isolated by VEGAS are able of inducing receptor activity in the absence of ligand. Biological characterization of intra-VHH is easier for GPCR with diversiﬁed ligands and well-described pharmacology. By using agonists and antagonists of a given receptor, it is possible to conclude whether a VHH preferentially stabilizes inactive forms of the receptor or if it binds constitutively the active receptor and is displaced by further recruitment of G proteins, or whether it only binds activated conformations and then prevents G protein binding, provided that it has a sufﬁcient afﬁnity for the receptor, as seems to be the case of Nb80 (estimated Kd ≃140 nM) (41). Several authors have developed a bioluminescence resonance energy transfer (BRET) assay to monitor the interaction of the intra-VHH and a GPCR (42–44). This is the case of the interaction of the kappa-type opioid receptor Frontiers in Endocrinology frontiersin.org 05 Raynaud et al. 10.3389/fendo.2022.1048601 FIGURE 3 FIGURE 3 Functional action of intra-VHHs against OPRK1, OPRD1 and OPRK1 opioid receptors, ADRB2 and US28. Nb39 reveals the location of opioid receptors when stimulated by a peptide ligand (bright green) or a cell-permeant non-peptide ligand (black dot). Nb60 locks ADRB2 in an inactive conformation that precludes interaction with transducers. Nb80 stabilizes conformationally active ADRB2. Nb.c200 stabilizes ADRB2 even when stimulated by its natural, low-afﬁnity agonist, adrenaline. Nb71 stabilizes active conformations of ADRB2 and prevents b-arrestin recruitment by limiting access to GRKs. US28 is a constitutively active GPCR. Intra-VHHs as conformational biosensors to explore subcellularly localized GPCR signaling With the use of Nb6B9, a high-afﬁnity derivative of Nb80, agonist-activated D1A dopamine receptor (D1DR) or b1-adrenergic receptor (ADRB1) has also been observed at the Golgi apparatus in addition to the plasma membrane (70). variants thereof (52–54). But it is not directly informative of receptor conformation. By providing important advance onto active receptor trafﬁcking, intra-VHH conformational biosensors provide insights directly at the level of the receptor on the mechanisms underlying signaling bias. GPCR signaling and trafﬁcking are closely connected, since it has been unambiguously demonstrated that GPCR signaling does not exclusively take place at the plasma membrane, but also occurs in several intracellular compartments, ultimately leading to distinct biological outcomes (55–59). This conceptual advance has been enabled by repurposing intra-VHHs sensitive to GPCR conformational dynamics initially developed for structural biology. These antibodies can be converted to genetically- encoded conformational biosensors by fusion to a ﬂuorescent protein derived from green-ﬂuorescent protein (GFP). With these tools, it becomes possible to detect where activated receptors are located inside the cell. The ﬁrst anti-GPCR intra- VHH biosensor was derived from Nb80, that contributed to the resolution of activated ADRB2 3D structure (24). By tracking isoproterenol-activated ADRB2 inside the cell, this biosensor has uncovered that receptor stimulation leads to cAMP production, not only from the plasma membrane but also from endosomes, once the receptor is internalized (60). This ﬁnding was conﬁrmed with Nb37-GFP, that recognizes the alpha-5 helical domain of Gas in the guanine nucleotide-free form that is an activation intermediate and is important for GPCR binding (61). At intra-cellular concentrations that do not inhibit Gas activation, the Nb37-GFP biosensor is resident in the cytosol, and is recruited to the cell membrane upon ADRB2 activation. Even more importantly, this biosensor detects ligand-activated ADRB2 wherever it is located in the cell, hence providing precious informations on the sites of persistent ADRB2 cAMP signaling, such as the endosomes (Figure 4) (60). Similar observations were extended to the gastric inhibitory polypeptide receptor (GIPR), that also induces cAMP production once in the early endosomes (62). Single-molecule imaging with the Nb37-GFP biosensor has further uncovered that the alpha2A adrenergic receptor (ADRA2A) coupling to G protein is trapped to deﬁned hot spots of interactions inside the plasma membrane, where signaling mainly occurs. These nanodomains are partially constituted by actin ﬁbers, microtubules and clathrin-coated pits (63). Intra-VHHs as conformational biosensors to explore subcellularly localized GPCR signaling proteins and b-arrestins) with various efﬁcacy or kinetics, according to the ligand (47–50). The high conformational dynamics and heterogeneity of GRKs and b-arrestins and their multiple modes of interactions with GPCR contribute to this diversity (51). The ability of a ligand to selectively stimulate a signaling pathway over the other, when compared to a reference ligand, has been mainly approached as an indirect measure of G protein vs b-arrestin-dependent downstream signaling, and estimated by calculation with the operational model and GPCR intrinsic instability is the major driver of biased signaling. The latter is determined by the selection of discrete conformational states, stabilized by a given ligand, that will ultimately lead to the recruitment of different transducers (e.g. G Frontiers in Endocrinology frontiersin.org 06 Raynaud et al. 10.3389/fendo.2022.1048601 signal ampliﬁcation that happens when measuring downstream signaling. Therefore, these intra-VHH biosensors may potentially provide a more reliable access to efﬁcacy and potency of each ligand-receptor pair. Biosensors to monitor OPRM1, OPRD1, OPRK1 opioid receptors activity have been developed, either to monitor receptor activation state by different ligands, by quantifying biosensor interaction (42, 66), or to track activated receptor inside the cell (45, 66, 67). One of the latter ones has been developed to discriminate the side-effects of non-peptide clinically-relevant drugs versus the beneﬁcial therapeutical effects of natural peptide ligands of OPRM1 (67, 68). This biosensor, derived from Nb33, is almost similar to Nb39, the intra-VHH that binds selectively to activated OPRM1 and OPRK1 described above (29, 42, 69). By total internal reﬂection ﬂuorescence (TIRF) time- lapse experiments in neurons, the Nb33 biosensor reveals that met-enkephalin- or b-endorphin-activated OPRM1 or OPRD1 signals transiently from the plasma membrane, and then from receptor internalized in the endosomes. In contrast, non-peptide opioid drugs, such as morphine or etorphine, induce an additional, very early stimulation wave in the Golgi, that is independent of receptor trafﬁcking (Figure 3). Similar results have been gathered when probing OPRK1 with the Nb39 conformational biosensor in cells stimulated with the SalA permeant drug or with Dynorphin, the OPRK1 natural ligand (45). These observations, emphasizing the notion of spatio- temporal bias, have wide implications for the development of neuro-modulatory drugs that generally penetrate the cell, hence are prone to illegitimate signaling from the Golgi. The latter might be involved in the well-known side-effects of opioids such as toxicity and addiction. Intra-VHHs as conformational biosensors to explore subcellularly localized GPCR signaling Nanoclustering depends on the receptor conformational state, as demonstrated by single-molecule tracking of active and inactive conformers of ADRB2 bound to GFP-tagged Nb80, Nb37 or Nb60 biosensors in PC12 cells (64). Intra-VHHs directed against the C-C chemokine receptor type 7 (CCR7) have been recently derived by random mutagenesis of CDR1 and CDR3 of Nb80 (71). Interaction of Nb1, Nb5 and Nb38 with CCR7 was tracked by bimolecular ﬂuorescence complementation (BiFC) using split-YFP, regardless of the receptor activation state. They co-localized with CCR7 at the plasma membrane in resting conditions, and also in membrane rufﬂes and endosomes upon CCL19 stimulation, conﬁrming that they do not recognize selective conformations of the receptor. Accordingly, they also modestly interfered with Gi binding to the receptor and Ca2+ response. Frontiers in Endocrinology Intra-VHHs to mimic transducer activity The NbSmo8 biosensor was the ﬁrst intra-VHH biosensor directed against a class F GPCR. Similarly to anti-class A biosensors, it has been shown to bind the Smoothened homolog (SMO) only when activated by agonist binding (65). Ligand binding induces conformational modiﬁcations of GPCRs that lead to the recruitment of intracellular transducers, among which G proteins and b-arrestins have Diverse ligands stabilizing discrete active states of the receptor induce its interaction with the conformational intra-VHH biosensors in a concentration-dependent manner, without the Frontiers in Endocrinology frontiersin.org 07 Raynaud et al. 10.3389/fendo.2022.1048601 FIGURE 4 Mechanisms of action of intra-VHHs against GPCR transducers. Nb37 recognizes the nucleotide-free form of active Gas, and has been used as a conformational biosensor to track receptor activation and signaling throughout the cell compartments. Nb35 stabilizes the interface between Ga and Gbg, and prevents GTP release. Nb5 binds to Gbg. It has a pronounced effect on Gbg signaling (adenylate cyclase, pERK and pAkt) and on the K+ current of the GIRK channel in the striatum. Nb32 is an anti-b-arrestin 1 VHH. Nb35 and Nb32 have been used to reconstruct the structural arrangement of the megacomplex composed of an agonist-stimulated ADRB2-V2R chimera simultaneously bound to Ga and to b- arrestin 1. FIGURE 4 Mechanisms of action of intra-VHHs against GPCR transducers. Nb37 recognizes the nucleotide-free form of active Gas, and has been used as a conformational biosensor to track receptor activation and signaling throughout the cell compartments. Nb35 stabilizes the interface between Ga and Gbg, and prevents GTP release. Nb5 binds to Gbg. It has a pronounced effect on Gbg signaling (adenylate cyclase, pERK and pAkt) and on the K+ current of the GIRK channel in the striatum. Nb32 is an anti-b-arrestin 1 VHH. Nb35 and Nb32 have been used to reconstruct the structural arrangement of the megacomplex composed of an agonist-stimulated ADRB2-V2R chimera simultaneously bound to Ga and to b- arrestin 1. been the most extensively studied. Intra-VHHs stabilizing G proteins or b-arrestins have served as tools to address the following questions, that are theoretically applicable to every GPCRs: i/the cryogenic electron microscopy (cryo-EM) structure of activated GPCR complexes with G proteins or b- arrestins, ii/the sub-cellular localization of signaling receptor, iii/ the modulation of transducer activity and signaling outcomes, iv/understanding the relationships between b-arrestin conformational transitions and its activity, v/modulating the interactions of b-arrestins with their binding partners. Intra-VHHs to mimic transducer activity such as the thyrotropin receptor (TSHR) (72, 73), the lutropin- choriogonadotropic hormone receptor (LHCGR) (74) or the adenosine receptor A2a (ADORA2A) (75) among others, in active, transducer-bound conformation. In contrast to the paradigm establishing that b-arrestins binding precludes Ga binding on the receptor in order to desensitize it, it has been demonstrated by single-particle electron microscopy that Gas and b-arrestin 1 can bind simultaneously the GPCRs that strongly interact with b-arrestins, such as the vasopressin V2 receptor (AVPR2) (76). In agreement, Nb35 has been used to stabilize the Gabg trimer within an activated ADRB2/AVPR2 chimeric receptor in complexation with both transducers (77) (Figure 4). The AVPR2 moiety represents the carboxyterminal region of the AVPR2 receptor that confers long-lasting interaction with b-arrestins, in contrast to ADRB2 that only transiently interacts with them (78). This study has profound been the most extensively studied. Intra-VHHs stabilizing G proteins or b-arrestins have served as tools to address the following questions, that are theoretically applicable to every GPCRs: i/the cryogenic electron microscopy (cryo-EM) structure of activated GPCR complexes with G proteins or b- arrestins, ii/the sub-cellular localization of signaling receptor, iii/ the modulation of transducer activity and signaling outcomes, iv/understanding the relationships between b-arrestin conformational transitions and its activity, v/modulating the interactions of b-arrestins with their binding partners. The structure of Gs-bound ADRB2 complexes has been resolved with the Nb35 as a structural chaperone (27). Because it recognizes the interface between Gas Ras domain and Gbg and prevents GTP dissociation when the trimer is bound to agonist-occupied receptor, Nb35 has helped solving the high resolution 3D structures of other active, Gs-coupled GPCRs, Frontiers in Endocrinology frontiersin.org 08 10.3389/fendo.2022.1048601 Raynaud et al. biological impact because it revisits the role of b-arrestins as desensitizing agents presumably precluding Gs access to the receptor, by showing that b-arrestin and Gs binding is in fact not mutually exclusive. Gs occupies the core domain of the receptor, while b-arrestin 2 remains bound to the phosphorylated carboxy-terminal region of the receptor, in a so-called “tail” conformation. b-arrestins can also adopt a “core” conformation whereby they engage three structural elements, including the ﬁnger loop, into the core (transmembrane helices and intracellular loops) of the receptor for desensitization, their classically assigned role (77). Intra-VHHs to mimic transducer activity Hence, by demonstrating that a GPCR coupled to b-arrestins can continue to signal through Gs, even if internalized, this “megaplex” hypothesis has provided a mechanistic clue on endosomal signaling, as presented above. adopts a partially active conformation that can be allosterically modiﬁed to accommodate b2-adaptin interaction and subsequent internalization. The use of Ib30 as a biosensor to monitor b-arrestin 1 conformational changes has been extended to other GPCRs (82, 84, 85). Finally, bona ﬁde camelid anti-b-arrestin 1 intra-VHHs came out, and one of them, Nb32, has been very useful to investigate the functional outcomes of b-arrestin 1 binding to the GPCR core and/or to the tail (Figure 4) (86). This antibody binds b-arrestin 1 only when associated to a receptor. When comparing a b-arrestin 1 deleted of its ﬁnger loop, hence devoid of its ability to bind the core domain of the receptor, and the Nb32-stabilized b-arrestin 1, it appeared that b-arrestin 1 binding to the tail predominantly drives the receptor to internalization and promotes signaling, but is not able to promote G protein desensitization, as expected from the megaplex model (76, 77). Because of its negative regulatory action on Ga through re- association, the Gbg complex has raised interest as intra-VHH target. Therefore Nb5, that recognizes Gb subtypes 1-4, has been developed (79). By suppressing Gbg signaling, Nb5 inhibits Gbg- regulated G protein-gated inward rectiﬁer potassium (GIRK) channels signaling in medium spiny neurons of the striatum excited with DRD2- or CHRM4-mediated inhibitory post- synaptic current (IPSC). In response to apelin, Nb5 indirectly limits the inhibition of the Gi-coupled apelin receptor on forskolin-stimulated cAMP, by suppressing the inhibitory action of Gbg on adenylate cyclase. Nb5 also inhibits downstream kinases such as Akt and Erk. But despite its inhibitory action on Gbg functioning, this intra-VHH has no effect on GTP-bound Ga (Figure 4) (79). So far, the anti-b-arrestin antibody fragments that have been described allosterically modulate b-arrestin/GPCR interaction by stabilizing selective receptor conformations. In the future, it will be of great interest to stabilize selective, fully or partially engaged conformations of b-arrestins instead. In this way, it should be possible to control independently receptor-mediated signaling and trafﬁcking. Beyond b-arrestin/GPCR interaction, only scFv5 has been shown to impair the interaction of b-arrestin 2 with one of its non-receptor interaction partner (80). This ﬁnding opens the promising possibility of interfering with b-arrestin signaling partners. Intra-VHHs to mimic transducer activity This is of paramount interest because b-arrestins undergo interactions with hundreds of signaling proteins, including at least 17% of direct interactions, leading to diversiﬁed, yet essential biological responses (87–89). Hence, intra-VHHs directed against precise interfaces of b-arrestins might help to decipher the speciﬁcity of their signaling outputs. Intracellular antibodies against b-arrestins exhibiting interesting biological properties have also been characterized, although the ﬁrst ones were not VHHs but scFv or Fabs. For example, scFv5 disrupted the interaction of b-arrestins with clathrin and inhibited the endocytosis of 8 different GPCRs, suggesting a generic value of this scFv as a tool to assess GPCR internalization (80). In addition, a Fab, Fab30, that recognized an active “tail” conformation of b-arrestin 1 when bound to a phosphorylated carboxyterminal region of V2R (81), was isolated. From Fab30, an intra-cellular scFv, named Ib30 by the authors, has been derived as a biosensor to monitor b-arrestin 1 recruitment to chimeric as well as GPCRs with a native carboxyterminal region, and then to track b-arrestin trafﬁcking inside the cell (82). Importantly, Ib30 confers a gain-of-function in b-arrestin 1 conformation, since it restores its translocation to endosomes and its ability to stimulate ERK MAP kinases, otherwise impaired upon agonist-stimulation of the AVPR2 T360A phosphorylation mutant (83). Structural analysis and molecular dynamics indicate that only Lys11 of b-arrestin 1 phosphate sensor remains engaged by ionic interactions with the phosphorylated receptor and not Lys 294 in the polar core, nor Arg25, that are also involved in fully activated conformation of b-arrestin 1. This analysis explains that, when bound to the AVPR2 T360A mutant, b-arrestin 1 Methodological limitations and opportunities Methodological hurdles that linger the development of VHHs against GPCR, such as the selection of the antigen (90), the advantages of synthetic libraries versus libraries from immunized animal (40, 91, 92), the comparison of display methods (5, 38, 40) have been previously reported. Here, we focus on the speciﬁcities that make anti-GPCR intra-VHH expression inside the cell and characterization particularly challenging (Figure 5). One is that the anti-GPCR intra-VHHs isolated so far are generally conformational antibodies that recognize non-linear epitopes within the inner core domain of the receptor, as shown for Nb80. However, the intracellular loops, especially ICL3, and the carboxyterminal region of GPCR are intrinsically disordered, which may be a disadvantage to select speciﬁc, high-afﬁnity VHHs. Frontiers in Endocrinology frontiersin.org 09 Raynaud et al. Raynaud et al. 10.3389/fendo.2022.1048601 inactive conformations of the receptor, it would be bound to the GPCR prior to agonist stimulation. Therefore, if its afﬁnity is high enough, it should prevent transducer binding and lead to functional inhibition of downstream signaling. But if it is not, then it should be displaced by the ligand-activated transducers, without any detectable functionaloutcome,whichmayleadtobewilderingconclusions.One alternative is to express the VHH intracellularly from an inducible promoter, as previously done with anti-US28 VUN13 intra-VHH (44), which also minimizes off-target reactions. Quite interestingly, destabilizing mutations of an intra-VHH that are silent only in the presence of the speciﬁc antigen have been reported (94). These mutations constitutively drive the intra-VHH to the proteasome- degrading pathway, unless the antibody is bound to its cognate antigen. Hence, an intra-VHH may be permanently degraded until its cognate GPCR target adopts the suitable active conformation upon ligand binding. This work is of broad potential applications because the destabilizing mutations are present in the structurally conserved framework regions of the antibody. In addition, several optically-controlled intra-VHHs have been engineered [reviewed in (95)]. Additional endeavors have to be pursued, based on the introduction of the antibody with a cell-penetrating peptide (96), or the transfection of in vitro-transcribed RNA, to detect the intra- VHH as soon as 3 hours after introduction (97). The key parameter to be monitored when isolating an antibody against a selected target is direct antibody/antigen binding. But in vitro binding by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) or interferometry can be a challenging task when the target is a GPCR that cannot be easily produced and puriﬁed in native, active conformation. Methodological limitations and opportunities Hence, this strategy raises hopes to the selection and characterization of ligands selective of a given receptor conformation of interest, in a cell-free system. reproducibility between independent experiments, especially because the stoichiometric ratio between the receptor and the VHH, by altering ligand binding afﬁnity, indirectly alters the efﬁcacy to stimulate a given functional readout. with an intra-VHH, then the afﬁnity of not only its natural ligand will increase but also any other biased ligand with interesting signaling properties. This is the purpose of reverse pharmacology, where a receptor is locked in an active conformation with an intra- VHH, to screen for (allosteric/biased) ligands endowed with new pharmacological properties when compared to the reference agonist. A proof-of-concept of reverse pharmacology has been successfully applied to ADRB2 fused to the transducer-mimicking Nb80 and to the OPRM1-Nb33 fusion, in a fragment-based screening of 1000 small molecules (38, 100, 101). This comparative fragment screen has led to the pharmacological classiﬁcation of multiple novel ligands as agonists, antagonists, inverse agonists, etc, on the basis of their efﬁcacy to stimulate readouts of interest (100). Fragment-derived compounds binding the orthosteric site of ADRB2-Nb80 fusion with afﬁnity in the nM range were selected, and they discriminate accurately the active and basal states of the receptor. Hence, this strategy raises hopes to the selection and characterization of ligands selective of a given receptor conformation of interest, in a cell-free system. In several instance, the question remains as whether conformational intra-VHHs that have been reported are really speciﬁc of one receptor, or if they recognize active/inactive conformations that are common to several receptors of the same family (ex, the opioid receptors, adrenergic receptors, etc) or even receptors from distinct classes (ex, class A GPCR vs class F). As indicated above, Nb6B9 recognizes D1DR in addition to ADRB1 and ADRB2 (70). For class A GPCR, cross- reactivity could reﬂect the relative conformational conservation of their intracellular half, that accommodates to a limited number of G proteins and b-arrestins (98, 99). Methodological limitations and opportunities Assaying intra-VHH-GPCR binding in cell can be an alternative by using BRET (42–44) or the developing Nanobit complementation technology (83, 93). Cytometry is also an efﬁcient means (39), but low level of endogenous or exogenously expressed recombinant receptor and limited intracellular afﬁnity of the VHH may lead to signal below the detection threshold. In addition, in vitro afﬁnity may not be linearly correlated to efﬁcacy in the reducing milieu of the cytoplasm, as shown for the anti-ADRB2 Nb.c203, which exhibits moderate afﬁnity maturation in yeast when compared to the other intra-VHHs selected, but was the most efﬁcient to decrease the Emax of adrenaline to produce cAMP. Similarly, the folding of a VHH produced in bacteria and expressed from inside the cell may be different, which adds another level of variability when assessing afﬁnity. These observations underscore the necessity to test several intra-VHHs to obtain a maximum effect on receptor signaling. Since the VHH is generally expressed inside the cell from a transfected plasmid, it can be difﬁcult to accurately control its quantity from the beginning of the experiment. If it recognizes The fact that it is very difﬁcult to date to control precisely the expression level of an intra-VHH severely compromises FIGURE 5 Examples of methodological challenges associated to functional studies on intra-VHHs that modulate GPCR signaling, and the existing solutions. Frontiers in Endocrinology frontiersin.org 10 Raynaud et al. 10.3389/fendo.2022.1048601 with an intra-VHH, then the afﬁnity of not only its natural ligand will increase but also any other biased ligand with interesting signaling properties. This is the purpose of reverse pharmacology, where a receptor is locked in an active conformation with an intra- VHH, to screen for (allosteric/biased) ligands endowed with new pharmacological properties when compared to the reference agonist. A proof-of-concept of reverse pharmacology has been successfully applied to ADRB2 fused to the transducer-mimicking Nb80 and to the OPRM1-Nb33 fusion, in a fragment-based screening of 1000 small molecules (38, 100, 101). This comparative fragment screen has led to the pharmacological classiﬁcation of multiple novel ligands as agonists, antagonists, inverse agonists, etc, on the basis of their efﬁcacy to stimulate readouts of interest (100). Fragment-derived compounds binding the orthosteric site of ADRB2-Nb80 fusion with afﬁnity in the nM range were selected, and they discriminate accurately the active and basal states of the receptor. Therapeutic opportunities The explosion of structures of GPCRs in active conformations since 2011 has paved the way for the rational, structure-based design of new drugs of potential therapeutic interest. In the ADRB2-Nb80 complex, a b hairpin conformation of the VHH CDR3 engages interactions with the receptor overlapping the Gas binding site (24). This prompted research groups to launch rational design programs using CDR3 b hairpin peptidomimetics to stabilize active conformations of ADRB2 for ligand screening (102). Two of these peptidomimetics inhibited cAMP production, as expected from competition with endogenous Gas. Nevertheless, the effect was modest when compared to Nb80 inhibitory effect. In drug discovery, one moonshot is to enhance binding afﬁnity of biased agonists of therapeutical interest because, by stimulating various signaling pathways with distinct efﬁcacy when compared to the natural agonist, they are expected to lead to less side-effects (Figure 6). If a GPCR is stabilized in a deﬁned active conformation FIGURE 6 Potential therapeutical applications of anti-GPCR intra-VHHs. A GPCR immobilized by an intra-VHH in an active conformation of biological interest may serve as a target for drug discovery of new biased ligands, be they allosteric or orthosteric (left). In vivo, provided that intra-VHH may be vectorized into the appropriate target cell, they could allosterically modulate the pharmacological properties of the endogenous ligand, which, for example, would respect the endocrine physiological rhythms and control. FIGURE 6 Potential therapeutical applications of anti-GPCR intra-VHHs. A GPCR immobilized by an intra-VHH in an active conformation of biological interest may serve as a target for drug discovery of new biased ligands, be they allosteric or orthosteric (left). In vivo, provided that intra-VHH may be vectorized into the appropriate target cell, they could allosterically modulate the pharmacological properties of the endogenous ligand, which, for example, would respect the endocrine physiological rhythms and control. Frontiers in Endocrinology frontiersin.org 11 Raynaud et al. 10.3389/fendo.2022.1048601 TABLE 1 Summary of intra-VHHs modulating GPCR signaling and/or on trafﬁcking cited in this paper. Intra- VHH Target Signaling Reference Nb80 Active ADRB2 Gas mimic Disrupts the ionic lock between TM3 and TM6. Decreases cAMP response (Gas competition) and b-arrestin recruitment (24, 39) Nb80-GFP Active ADRB2 Probes receptor activity from the plasma membrane and from the endosomes (60) Nb6B9 Active ADRB2, ADRB1, D1DR Nb80 high afﬁnity derivative. Therapeutic opportunities Detects active D1DR and ADRB1 at the plasma membrane and in the Golgi (70) Nb60 Inactive ADRB2 Prevents cAMP response and b-arrestin recruitment (39) Nb71 Partially active ADRB2 Partial outward shift of TM6. Decreases cAMP response. Inhibits receptor phosphorylation by GRKs and b- arrestin recruitment (30, 39) Nbc.200 ADRB2 stimulated with adrenalin Decreases cAMP response (40) Nb6 Inactive OPRK1 Binds ICL3. Displaced by Gq recruitment (42) Nb39 Active OPRK1 No effect on Gi binding (42, 45) Nb39-GFP Active OPRK1 Detects signaling from plasma membrane and from endosomes with dynorphin. and also from Golgi if non-peptide opioid (67) VUN103 Constitutively active US28 Binds ICL2 and ICL3. Displaces Gq and b-arrestin 2. Impairs IP3 accumulation, NFAT, NF-KB and STAT3 activity (44) Nb7 Ligand-activated US28 Binds ICL2 and ICL3. Neutral to constitutive activity (44) VGS-Nb2 Active HTR2A PAM even in the absence of Gq. Stimulates SRE activation, decreases b-arrestin 2 recruitment (43) NbSmo8-GFP Active SMO Detects active SMO at the plasma membrane (65) Nb33-GFP Active OPRM1. OPRD1 Detects signaling from plasma membrane and from endosomes with met-Enk. and also from Golgi if non- peptide opioid (67) Nbl Inactive and active CCR7 Slightly interferes with Gi binding. Detects CCR7 at the plasma membrane in basal conditions and also in membrane rufﬂes and vesicules upon CCL19 stimulation (71) Nb38 Inactive and active CCR7 Inhibits Gi binding. Detects CCR7 at the plasma membrane in basal conditions and also in membrane rufﬂes and vesicles upon CCL19 stimulation (71) Nb37-GFP Alpha-5 helical domain of nucleotide-free Gas Detects active Gas at the plasma membrane and at the endosomes (ADRB2 and GIPR). Identiﬁes hot spots of Gas-dependent signaling in the plasma membrane (ADRA2). (60, 62, 63) Nb35 GPCR bound Gas and Gbg interface Prevents Gas and Gbg dissociation upon activation. Stabilizes the megaplex (27, 76, 77) Nb5 Gb subtypes 1-4 Suppresses Gbg signaling, Akt and ERK signaling (79) Nb32 Activated b-arrestin 1 Stabilises b-arrestin binding to the GPCR core and promotes desensitization (77, 86) Only the targets for which a signaling outcome has been measured are indicated. Below the dark line, the intra-VHHs recognizing G proteins or b-arrestins are also indicated. The exquisite properties of anti-GPCR VHHs, such as speciﬁcity, afﬁnity, short serum half-life, open exciting opportunities for future therapeutics. Obviously, these properties are shared with intra-VHHs. Therapeutic opportunities However, in the latter case, even more critically than for in vitro applications, a major issue to be overcome is their functional delivery into target cells in vivo, although some progress are being made, as reviewed in (107). This is why, to date, no example of intracellularly delivered VHH has been reported. These attempts still need more efforts for success, probably because peptidomimetics for ADRB2 fail to fully stabilize active conformations of the receptor (103, 104). Importantly, intra-VHHs have the unique potential to isolate GPCR agonists, not only as small chemical compounds, but also in the VHH format, recognizing the extracellular regions of a receptor. As indicated above, VHHs with agonistic have only been described for APLNR and GRM2. Hence, intra-VHHs may increase the discovery of new agonistic VHHs to GPCR of clinical relevance. Another application of intra-VHHs in drug discovery for GPCR can be foreseen through the example of atypical chemokine receptor 3 (ACKR3), that is naturally biased towards GRK and b-arrestin signaling, because of the absence of a kink of TM4 that leads to a clash of ICL2 and Ga (105, 106). Hence, it is conceivable that an intra-VHH stabilizing this compact conformation of the Ga-binding cleft would increase the probability to isolate new biased ligands of this receptor. 6. Keller L, Bery N, Tardy C, Ligat L, Favre G, Rabbitts TH, et al. Selection and characterization of a nanobody biosensor of GTP-bound RHO activities. Antibodies (2019) 8(1):8. doi: 10.3390/antib8010008 Funding PR is funded by a joint fellowship from Région Centre Val de Loire and INRAE PHASE Department; CG and VJ are funded by Conﬂict of interest The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be considered as a potential conﬂict of interest. Author contributions All authors signiﬁcantly contributed to conceptualization and writing of the manuscript, and to the design of the ﬁgures. All authors contributed to the article and approved the submitted version. Conclusions Intra-VHHs have widely proven their utility as chaperones for structural studies of GPCR by X-ray crystallography or cryo- EM. In contrast, the characterization of anti-GPCR intra-VHHs with functional properties inside the cell is still poorly developed. Frontiers in Endocrinology frontiersin.org 12 Raynaud et al. 10.3389/fendo.2022.1048601 the SELMAT grant of Région Centre Val de Loire ARD2020 Biomédicaments Program. CG is co-funded by the INTACT grant from Région Centre Val de Loire; GB and ER are funded by INRAE, FJ-A and PC are funded by the CNRS. This publication was funded with support from the French National Research Agency under the program “Investissements d’avenir” Grant Agreement LabEx MabImprove: ANR-10-LABX-53; “ARD2020 Biomédicaments” and “APR-IR INTACT” grants from Région Centre Val de Loire; Bill & Melinda Gates Foundation. So far, the functional impact of only very few intra-VHHs on GPCR signaling activity has been characterized, when considering that only ADRB2, OPRK1 and US28 and, to a lesser extent, HTR2A, have been targeted to date (Table 1). One clear bottleneck is the difﬁculty to properly characterize their pharmacological properties in a complex cell system and to assess speciﬁcity despite likely intracellular off-targets. Screening for drugs that bind to conformationally stabilized binding pockets within GPCR might lead to more selective and efﬁcient therapeutics. Intra-VHHs indirectly mimic the effect of biased agonists, from the intracellular side. 5. Cheloha RW, Harmand TJ, Wijne C, Schwartz TU, Ploegh HL. Exploring cellular biochemistry with nanobodies. J Biol Chem (2020) 295(45):15307−27. doi: 10.1074/jbc.REV120.012960 Acknowledgments In the future, exploration of the functionality of intra-VHHs on a given GPCR will need to be systematically coupled to resolution of its structure. Only in this condition will it be possible to approach the issue of conformation/activity relationship, to correlate a (biased) ligand to a receptor conformation or ensemble of conformations, to a signaling pathway, or even network, and ultimately, integrated biological response. Still, one caveat is that the conformations that are co-crystallized do not necessarily predict accurately the conformations that are preferentially stabilized in a complex cell system. The authors would like to acknowledge Région Centre Val de Loire and the MAbImprove Labex for their constant support of our research on antibody fragments. 7. Rothbauer U, Zolghadr K, Tillib S, Nowak D, Schermelleh L, Gahl A, et al. Targeting and tracing antigens in live cells with ﬂuorescent nanobodies. 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https://openalex.org/W4387218197	https://ojs.mahadewa.ac.id/index.php/nirwasita/article/download/2954/2184	Indonesian	null	DISKRIMINASI GENDER TERHADAP PEREMPUAN DALAM WAJAH POLITIK DI INDONESIA	Nirwasita	2,023	cc-by	3,432	Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 165 Diskriminasi Gender Terhadap Perempuan Dalam Wajah Politik D Indonesia Gender Discrimination Against Women in the Face of Politics in Indonesia Sulfiana1, Muhammad Syukur2, Ridwan Said Ahmad3 Universitas Negeri Makassar Jl. A. P. Pettarani, Tidung, Kec. Rappocini, Kota Makassar, Sulawesi Selatan 90222 Email : anasulfiana715@gmail.com, m.syukur@unm.ac.id, m.ridwan.said.ahmad@unm.ac.id Gender Discrimination Against Women in the Face of Politics in Indonesia Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 166 bahwa setiap warga negara berhak dalam menyampaikan pendapatnya, baik laki- laki dan perempuan. Sebagaimana dalam Undang-Undang Dasar 1945, Pasal 281 (2) menyebutkan “Setiap orang berhak bebas dari perlakuan yang bersifat diskriminatif atas dasar apa pun dan berhak mendapatkan perlindungan terhadap perlakuan yang bersifat diskriminatif.” Untuk mewujudkan hal ini, pemerintah membreakdown-nya kedalam undang- undang, sebagaimana dalam mandat Undang Undang No. 7 Tahum 1984, bahwa pemerintah berkewajiban memenuhi serta melindungi hak-hak yang melekat pada perempuan baik sebagai individu maupun kelompok. Indonesia pada dasarnya sudah memihak kepada kaum perempuan (Kiftiyah, 2019a). Gender Discrimination Against Women in the Face of Politics in Indonesia Sulfiana1, Muhammad Syukur2, Ridwan Said Ahmad3 Universitas Negeri Makassar Jl. A. P. Pettarani, Tidung, Kec. Rappocini, Kota Makassar, Sulawesi Selatan 90222 Universitas Negeri Makassar Jl. A. P. Pettarani, Tidung, Kec. Rappocini, Kota Makassar, Sulawesi Selatan 90222 Email : anasulfiana715@gmail.com, m.syukur@unm.ac.id, m.ridwan.said.ahmad@unm.ac.id Abstrak. Dalam masyarakat patriarkhis, laki-laki dianggap memiliki peran gender yang lebih privilege ketimbang peran gender perempuan. Pembedaan kepantasan tersebut, dirasa tidak adil bagi perempuan karena peran gender adalah hasil konstruksi sosial. Contoh nyatanya, lelaki lebih cocok berkiprah diranah publik sedangkan perempuan lebih cocok berkiprah diranah domestik, sehingga keterlibatan wanita dalam proses politik di Indonesia masih sangat minim. Adanya subordinasi gender menjadi penghalang bagi keterwakilan perempuan untuk berpartisipasi dalam ranah politik. Tujuan penelitian ini adalah membahas tentang diskriminasi gender terhadap perempuan dalam ranah politik. Penelitian ini merupakan penelitian kualitatif dengan menggunakan metode kajian pustaka atau studi kepustakaan yaitu berisi teori-teori yang relevan. Hasil penelitian ini menunjukkan bahwa budaya patriarki yang sudah berakar kuat sejak jaman dulu dan diwariskan turun-menurun membuat laki-laki merasa lebih superior daripada perempuan. Budaya patriarki menempatkan kedudukan laki-laki lebih tinggi daripada perempuan sehingga sering kali terjadi praktik-praktik ketidakadilan dan diskriminasi terhadap perempuan. Kata Kunci: Perempuan, Diskriminasi gender, Politik. Abstract. In a patriarchal society, men are considered to have a more privileged gender role than women's gender roles. This distinction of appropriateness is felt to be unfair for women because gender roles are the result of social construction. For example, men are better suited to work in the public sphere, while women are better suited to work in the domestic sphere, so that women's involvement in the political process in Indonesia is still very minimal. The existence of gender subordination is a barrier for women's representation to participate in the political sphere. The purpose of this study is to discuss gender discrimination against women in the political sphere. This research is a qualitative research using the method of literature review or literature study which contains relevant theories. The results of this study indicate that patriarchal culture, which has been firmly rooted since ancient times and has been passed down from generation to generation, makes men feel superior to women. Patriarchal culture places men in a higher position than women so that unfair and discriminatory practices often occur against women. Keyword: Woman, Gender Discrimination, Political. Keyword: Woman, Gender Discrimination, Political. PENDAHULUAN Seperti yang diketahui tulisan-tulisan tentang perempuan sudah sering kita dengar dan sudah banyak kita temui, tapi itu semua tidak menutup kemungkinan literasi tentang perempuan memanglah sangat penting dan harus selalu ditingkatkan. Isu-isu seputar perempuan tidak akan pernah habis untuk dibicarakan oleh para pemikir, baik para agamawan maupun pemikir sekuler. Pada abad pertengahan dan era modern telah menghasilkan teori yang berbeda di masyarakat tentang eksistensi kaum perempuan. Kehadiran kaum perempuan ditengah-tengah masyarakat memiliki fungsi serta peranan yang sangat penting. Akan tetapi yang terjadi adalah peran penting perempuan tidak menjadi perhatian lebih sehingga masyarakat tidak menyadari pentingnya peran tersebut. Akibatnya perempuan bisa menjadi kaum yang termarginalkan. Seperti yang diketahui tulisan-tulisan tentang perempuan sudah sering kita dengar dan sudah banyak kita temui, tapi itu semua tidak menutup kemungkinan literasi tentang perempuan memanglah sangat penting dan harus selalu ditingkatkan. Isu-isu seputar perempuan tidak akan pernah habis untuk dibicarakan oleh para pemikir, baik para agamawan maupun pemikir sekuler. Pada abad pertengahan dan era modern telah menghasilkan teori yang berbeda di masyarakat tentang eksistensi kaum perempuan. Kehadiran kaum perempuan ditengah-tengah masyarakat memiliki fungsi serta peranan yang sangat penting. Akan tetapi yang terjadi adalah peran penting perempuan tidak menjadi perhatian lebih sehingga masyarakat tidak menyadari pentingnya peran tersebut. Akibatnya perempuan bisa menjadi kaum yang termarginalkan. Tetapi dalam prakteknya pemenuhan terhadap kuota 30% perempuan dalam politik sangat sulit untuk dipenuhi. Keterbatasan akses/ Di negara Indonesia budaya patriarki masih tetap berjalan meskipun sedikit demi sedikit budaya tersebut terkikis karena perkembangan zaman yang semakin modern (modernisasi). Banyak kaum perempuan yang berani mengekspresikan pendapatnya di ruang publik. Dalam undang-undang secara jelas dituliskan merupakan salah satu kesulitan bagi perempuan untuk menunjukkan bahwasannya kepentingan perempuan tidak sepenuhnya dapat terakomodir dalam sistem politik. Dalam anggota dewan, tidak hanya dalam hal kuota dan kualitas, akan tetapi dalam hal tanggung jawab, merupakan salah satu kesulitan bagi perempuan untuk menunjukkan bahwasannya kepentingan perempuan tidak sepenuhnya dapat terakomodir dalam sistem politik. Dalam anggota dewan, tidak hanya dalam hal kuota dan kualitas, akan tetapi dalam hal tanggung jawab, Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 167 perempuan tidak diberi tanggung jawab yang signifikan. Kondisi tersebut adalah kendala untuk mengembangkan organisasi perempuan dalam memformulasikan kebijakan yang berpihak kepada kepentingan kaum perempuan. Oleh karena itu perempuan perlu diberikan ruang untuk dapat mengembangkan dirinya. laki dan kaum wanita.” laki dan kaum wanita.” METODE PENELITIAN Penelitian ini menggunakan metode kajian pustaka atau studi kepustakaan yaitu berisi teori teori yang relevan dengan masalah-masalah penelitian. Dalam mengkaji tulisan ini menggunakan metode penelitian kepustakaan (library research) dengan mengkaji kritis dan mendalam terhadap bahan-bahan pustaka yang relevan dengan materi tulisan ini, seperti buku dan jurnal yang layak dijadikan referensi. Seperti yang dikemukakan oleh Miqzaqon T dan Purwoko bahwa penelitian kepustakaan merupakan suatu studi yang digunakan dalam mengumpulkan informasi dan data dengan bantuan berbagai macam material yang ada di perpustakaan seperti dokumen, buku, majalah, kisah-kisah sejarah dan sebagainya (Assyakurrohim et al., 2022). Pembangunan pemberdayaan wanita merupakan komitmen nasional sebagai bagian integral dari pembangunan sumber daya manusia. Pemberdayaan wanita ini dimaksudkan untuk meningkatkan status, posisi dan kondisi wanita agar dapat mencapai kemajuan yang setara dengan laki- laki. Karena, rendahnya partisipasi wanita di dalam bidang politik. Di samping itu, masih adanya berbagai bentuk diskriminasi dan kesenjangan terhadap kaum wanita yang bersumber dari ketimpangan sosio- kultural masyarakat. Data kuantitatif dan kualitatif menunjukkan bahwa indeks pembangunan gender dan indeks pembedayaan gender yang rendah. Padahal, peluang bagi kaum wanita untuk berkarya sebenarnya telah diatur di dalam UUD 1945 Pasal 27 dan dalam hadits Rasulullah SAW yang menjelaskan bahwa “menuntut ilmu itu wajib bagi kaum laki- Teori Tentang muted group theory (teori kebungkaman). Muted group theory atau teori kelompok yang dibungkam ialah cara- cara praktis dari sebuah kelompok dominan dalam upaya untuk menekan, membungkam, dan merendahkan kata- kata atau pun ide dari kelompok Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 168 subordinat. Teori ini digagas oleh Edwin Ardener dan Shirley Ardener yang menekankan kepada bagaimana manusia dengan perbedaan status tertentu mampu menimbulkan tingkat perhatian yang berbeda dan tidak sama rata dengan lainnya. pembungkaman yaitu: pembungkaman yaitu: 1) Ridicule (ejekan), meremehkan pendapat yang diberikan oleh perempuan mengenai gagasannya. 2) Ritual (ritual), keterlibatan konteks tradisi budaya dalam beberapa kegiatan yang menempatkan perempuan dengan kedudukan yang lebih rendah daripada laki-laki. Dalam hal ini, perbedaan status tersebut dapat berbentuk hierarki contohnya seperti gender, ras, etnis, agama, dan sebagainya yang menyebabkan mereka dengan kelompok subordinat atau kelompok marginal harus menerima konstruksi sosial yang terbentuk diciptakan oleh kelompok dominan. Kemudian teori ini dikembangkan oleh Kramae (dalam Em Griffin, 2011: 483), yang menelaah lebih dalam mengenai teori kelompok yang dibungkam dengan berfokus pada perbedaan bahasa yang digunakan oleh perempuan dan laki-laki. Dalam muted group theory terdapat anggapan bahwa bahasa ada diciptakan untuk laki-laki. Menurut Kramae bahasa dalam pengertian ini diartikan secara harfiah sebagai a man- made construction, sehingga hal tersebut menyebabkan perempuan yang menjadi bagian dari kelompok marginal, sulit untuk mengungkapkan pikirannya. Kramae membagi empat tahapan dari proses 3) Control (kontrol), di mana adanya pendapat bahwa laki-laki lebih dapat mengontrol sosial. 4) Harassment (pelecehan), yakni ketika perempuan tidak dapat dengan bebas untuk melakukan kegiatannya di ranah publik karena bahaya kekerasan pelecehan yang mengintai (Ginting & Rahmiaji, n.d.). 4) Harassment (pelecehan), yakni ketika perempuan tidak dapat dengan bebas untuk melakukan kegiatannya di ranah publik karena bahaya kekerasan pelecehan yang mengintai (Ginting & Rahmiaji, n.d.). Penyebab Terjadinya Diskriminasi Gender Terhadap Perempuan. Dalam pembangunan pemberdayaan perempuan yang terjadi selama ini permasalahan mendasar yang masih dialami adalah rendahnya partisipasi perempuan dalam pembangunan, di samping masih adanya berbagai bentuk praktik diskriminasi terhadap perempuan. Rendahnya kualitas hidup perempuan terjadi di berbagai lini, antara lain sosial budaya, lingkungan, pendidikan, kesehatan, ekonomi, dan politik. Menurut Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 169 ahli bahasa, Hockett (dalam Corbett, 1991: 1) mengartikan gender adalah sebuah kata benda yang tercermin dalam perilaku kata- kata terkait. Artinya, gender bukan merupakan kodrat manusia, namun lebih kepada hasil dari konstruksi sosial maupun kultural. Identitas gender itu sendiri dapat dipahami sebagai cara pandang konsep kultural dalam membedakan peran, perilaku, mentalitas, dan karakteristik emosional yang tercipta antara laki-laki dan perempuan. Menurut Fakih (2013:13), diskriminasi gender dapat dimanifestasikan dalam lima bentuk antara lain sebagai berikut. tanggungjawab berat yang harus dipikulnya secara berkesinambungan (Politik 11.Pdf, n.d.). Eksistensi Perempuan Dalam Partisipasi Dalam Ranah Politik. Sejak era reformasi, partisipasi politik kaum perempuan dalam proses pengambilan kebijakan merupakan bagian yang penting bagi pemerintah dan lembaga legislatif. Dalam sistem demokrasi yang inklusif masyarakat mempunyai peran yang sangat penting untuk dapat terwujudnya partisipasi politik dari kaum perempuan agar lebih luas dan lebih merata. Persoalan akuntabilitas dan representasi merupakan persyaratan yang mutlak bagi terwujudnya demokrasi. Meskipun pada saat ini hak-hak politik kaum perempuan banyak yang telah mendapatkan pengakuan, akan tetapi hal tersebut tidak dapat menjamin bahwasannya hak perempuan dalam keikut sertaanya pada partisipasi politik telah bipraktekkan sebagaimana mestinya. Ini menunjukkan bahwa adanya peran keterwakilan perempuan didalam politik praktis, dan kebijakan-kebijakan yang ada memiliki sensivitas gender. 1. Marginalisasi atau proses peminggiran karena adanya perbedaan jenis kelamin antara laki-laki dan perempuan. 2. Subordinasi yakni perempuan selalu ditempatkan dalam posisi yang lebih rendah dibandingkan dengan laki-laki. 3. Pelabelan yang pada umumnya berbentuk stereotip tertentu di masyarakat dan telah membuat seseorang dirugikan. 4. Kekerasan atau violence merupakan bentuk tindakan kekerasan baik fisik maupun psikis yang dilakukan oleh salah satu jenis kelamin atau institusi. Meskipun perempuan telah terlibat dalam politik praktis, akan tetapi keterlibatan perempuan secara mandiri 5. Beban ganda yang menempatkan perempuan memiliki tugas dan Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 170 tidak dapat begitu saja terwujud meskipun hak-hak perempuan dalam dunia politik sudah diakui. Perempuan sebagai warga negara seharusnya memiliki hak yang sama untuk dapat berpartisipasi dalam proses demokrasi secara utuh. Kehadiran perempuan dalam dunia politik praktis yang telah dibuktikan dengan adanya keterwakilan perempuan di parlemen menjadi syarat mutlak untuk proses pengambilan sebuah kebijakan publik yang ramah dan sensitif pada kepentingan kaum perempuan. Tanpa adanya keterwakilan perempuan di parlemen yang memiliki jumlah memadai akan mengakibatkan timbulnya kecenderungan untuk menempatkan kepentingan laki-laki sebagai pusat dari pengambilan kebijakan. Oleh karena itu, aktifnya perempuan dalam ranah politik sangat membantu masa depan perempuan untuk melindungi hak-hak kaum perempuan dan mengembangkan potensi kaum perempuan. perempuan-perempuan yang menjabat sebagai kepala daerah. Keterlibatan perempuan tidak hanya sampai disitu, akan tetapi mereka juga mulai aktif bergabung di partai-partai politik yang ada. Akan tetapi, keterlibatan perempuan masih harus tetap diperjuangkan. Karena, semakin banyak perempuan yang terjun secara langsung dalam politik, maka semakin besar kesempatan untuk memperjuangkan hak-hak dan perlindungan terhadap perempuan yang selama ini belum secara maksimal diberikan oleh Negara. Adanya pendapat tersebut membuktikan bahwa politik tidak membedakan antara laki-laki dan perempuan. Eksistensi Perempuan Dalam Partisipasi Dalam Ranah Politik. Dalam dunia politik laki-laki dan perempuan mempunyai kedudukan yang setara. Kesetaraan gender dalam politik tersebut membuat perempuan menjadi agent of change yang mempunyai potensi besar untuk membuat perubahan menjadi lebih baik. Oleh sebab itu peran perempuan harus diawali dengan pemberdayaan diri kemudian diberikan kedudukan dan tanggung jawab. Kedudukan yang diberikan kepada perempuan dibutuhkan untuk dapat meningkatkan posisi perempuan harus didukung pula dengan kesempatan, Keterlibatan perempuan dalam dunia politik memiliki sebuah peningkatan. Banyak para perempuan yang terjun langsung dalam politik praktis. Ada beberapa perempuan yang menjabat sebagai menteri pada kebinet Gotong Royong, selain itu banyak Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 171 pendidikan, materi, kesempatan dan keterwakilan politik (Kiftiyah, 2019b). kelamin wanita yang tidak banyak dilibatkan dalam proses-proses politik, khususnya “pengambilan keputusan” di mana hasil dari keputusan tersebut dalam banyak kasus akan mengena kepada mereka. Idealnya semua komponen bangsa harus terlibat. Sangat tidak adil, bahkan, melanggar hak asasi manusia, jika wanita masih juga dimarginalisasikan atau didikriminasikan untuk berpartisipasi dalam lembaga-lembaga politik formal (Muslimat, 2020). Dalam realitas politik di banyak negara termasuk Indonesia, terdapat persoalan dalam masalah peran dan posisi gender antara laki-laki dan wanita. Selama ini jika kita bicara dunia politik, yang terjadi adalah diskriminasi atau peminggiran politik terhadap wanita di dalam kehidupan demokrasi atau dunia politik. Konsep-konsep seperti kompetisi, partisipasi politik serta kebebasan sipil dan politik dalam realitas politik ternyata hanya terbatas pada dunia laki-laki (dunia maskulin). Kalaupun wanita terlibat di sana, mereka pun harus masuk dan berperilaku politik dalam dunia laki-laki. Diskriminasi seperti ini lebih didasarkan pada apa yang disebut sebagai keyakinan gender. Hal ini menjadi dasar ketidakadilan di berbagai tingkatan mulai dari rumah tangga, sekolah, tempat kerja, masyarakat, hingga lingkungan pemerintahan atau negara. Hambatan Terhadap Partisipasi Politik Pada Perempuan Dalam negara yang menganut sistem nilai patriarkal, seperti Indonesia, kesempatan perempuan untuk menjadi politisi relatif terbatasi karena persepsi masyarakat mengenai pembagian peran antara laki-laki dan perempuan, yang cenderung bias kearah membatasi peran perempuan wanita pada urusan rumah tangga. Namun demikian, pada masa perjuangan kemerdekaan, kebutuhan akan kehadiran banyak pejuang, baik laki-laki maupun perempuan, membuka kesempatan luas bagi para wanita untuk berkiprah di luar lingkup domestik dengan tanggungjawab urusan rumah tangga. Masyarakat menerima dan menghargai para pejuang perempuan yang ikut Diskriminasi seperti ini lebih didasarkan pada apa yang disebut sebagai keyakinan gender. Hal ini menjadi dasar ketidakadilan di berbagai tingkatan mulai dari rumah tangga, sekolah, tempat kerja, masyarakat, hingga lingkungan pemerintahan atau negara. Berbicara mengenai partisipasi politik wanita di Indonesia, khususnya keterlibatan mereka dalam lembaga- lembaga politik formal, yang terjadi adalah “representasi” wanita yang rendah di dalamnya. Masalahnya sangat jelas, yakni ada kelompok masyarakat yang berjenis Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 172 berperan di medan perang, dalam pendidikan, dalam pengobatan, dan dalam pengelolaan logistik. Kesempatan ini memberi kemudahan pada perempuan untuk memperjuangkan isu-isu yang berhubungan dengan kepentingan mereka atau yang terjadi di sekitar mereka, selain isu politik. berbagai bidang (Politik 5.Pdf, n.d.). Naik dan menurunnya keterwakilan perempuan didalam parlemen dapat dipicu karena kebijakan yang sama, karena pada dasarnya gairah kaum perempuan dalam dunia politik jika tidak ditompang dengan upaya- upaya serta kebijakan yang lebih memacu jumlah keterwakilan akan berakibat menurun, hal ini dapat dilihat dari penggunakan kebijakan yang sama dari pemilu 2009 dan 2014. Kekuatiran keterwakilan perempuan menurun itu bukan berarti hilang pada 2019 meski 40 Persen perempuan masuk kedalam daftar pencalonan legislatif dipemilihan umum 2019, karena terkadang partai politik mengikut sertakan perempuan kedalam busaran pemilihan hanya sebagai pemenuhan syarat saja, masih banyak partai politik yang belum peka terhadap isu gender (Priandi & Roisah, 2019). Kurangnya representasi perempuan dalam bidang politik antara lain disebabkan oleh kondisi budaya yang patriakal yang tidak diimbangi kemudahan akses dalam bantuk tindakan afirmatif bagi perempuan, seperti pemberian kuota. GBHN, dan berbagai instrumen politik dan hukum tidak secara eksplisit menunjukkan diskriminasi terhadap perempuan namun tidak pula memberikan pembelaan dan kemudahan bagi perempuan dalam berbagai bidang, termasuk politik. Hambatan Terhadap Partisipasi Politik Pada Perempuan Undang-Undang Dasar 1945, Bab X, Ayat 27 menyatakan bahwa “Semua warganegara adalah sama di hadapan hukum dan pemerintah,” sedangkan Ayat 28 menjamin “Kebebasan berkumpul dan berserikat, dan kebebasan menyatakan pendapat baik secara lisan maupun tertulis.” Sekalipun demikian, dalam kondisi yang patriakhal perempuan menghadapi beberapa kendala untuk mensejajarkan diri dengan laki-laki dalam Pemahaman dan wawasan politik yang dimiliki kader perempuan masih kurang terhdap dunia politik yang mereka masuki tersebut. Hal ini menyebabkan di dalam diri perempuan tersebut terbentuk persepsi/pandangan yang mengangap bahwa perempuan tidak pantas untuk menjadi seorang pemimpin dan perempuan tidak boleh melangkahi laki-laki seperti Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 173 menjadi pemimpin. Persepsi seperti ini masih menjangkiti diri perempuan karena kurang memahami pentingnya peranan mereka dalam pengambilan keputusan politik partai. mentalitas dan melahirkan pola pikir bahwa laki-laki selalu diutamakan, dan perempuan hanya sebagai pelengkap saja. Walau sudah merdeka dan menganut asas demokrasi 1945, tapi berdasarkan realitas yang ada, salah satunya dapat kita lihat pada kancah dalam bidang politik. Masih banyak masyarakat yang menyamakan kepemimpinan dengan ketidiktatoran ala jaman penjajahan, sehingga sangat sulit untuk dihilangkan diskriminasi gender terhadap perempuan, khususnya kesempatan terhadap kaum perempuan untuk eksis di ranah publik. KESIMPULAN Priandi, R., & Roisah, K. (2019). UPAYA MENINGKATKAN PARTISIPASI POLITIK PEREMPUAN DALAM PEMILIHAN UMUM DI INDONESIA. Jurnal Pembangunan Hukum Indonesia, 1(1) tps://doi.org/10.14710/jphi.v1i1.10 6-116 KESIMPULAN Berbicara tentang perempuan tidak akan pernah habisnya. Banyak literasi dan pemikir-pemikir yang mengangkat isu-isu tentang keperempuanan, itu membuktikan pentingnya hal ini untuk selalu diperbincangkan. Berbicara perihal gender, seringkali kita mendegar dengan istilah kesetaraan gender. Yang harus diseterakan menurut saya adalah peran setiap gender. Karena peran bukan kodrat tapi melainkan konstruksi bukan ciptaan Tuhan tapi ciptaan manusia. Kita tidak bisa mengelak lahir sebagai perempuan, tapi kita bisa saja memberontak jika tidak diperlakukan layaknya manusia. Dalam undang-undang No. 2 tahun 2008 yang memuat kebijakan yaitu mengharuskan partai politik dalam pendirian maupun kepengurusan ditingkat pusat harus menyertakan keterlibatan 30 % perempuan. Kebijakan untuk memberi ruang lebih kepada perempuan sekitar 30% pada legislatif bertujuan untuk menghilangkan diskriminasi bagi perempuan atau menghindari dominasi dari laki-laki, dan juga diharapkan keterwakilan perempuan bisa menghasilkan peraturan atau undang- undang yang bisa mengakomodir perempuan maupun anak yang dalam tataran masyarakat Indonesia masih terjadi diskriminasi. Sehingga kedepannya Kesetaraan gender sulit direlisasikan khususnya dalam ranah politik, karena adanya budaya patriarki dan miskonsepsi tentang kepemimpinan. Budaya patriarki yang sudah berakar kuat sejak jaman dahulu dan diwariskan secara turun temurun, membuat laki- laki merasa superior dibandingkan perempuan. Dampak dari budaya ini, menjadikan Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 Jurnal Nirwasita Vol.4 No.2 September 2023 e-ISSN 2774-6542 Hal: 165-174 174 174 Representasi Standardisasi Kecantikan Wanita dalam Film. Kiftiyah, A. (2019a). Perempuan dalam Partisipasi Politik di Indonesia. 14(1). Kiftiyah, A. (2019b). Perempuan dalam Partisipasi Politik di Indonesia. 14(1). Muslimat, A. (2020). Rendahnya Partisipasi Wanita di Bidang Politik. Jurnal Studi Gender dan Anak, 7(02), 131. https://doi.org/10.32678/jsga.v7i 02.181 Politik 5.pdf. (n.d.). Politik 11.pdf. (n.d.). Priandi, R., & Roisah, K. (2019). UPAYA MENINGKATKAN PARTISIPASI POLITIK PEREMPUAN DALAM PEMILIHAN UMUM DI INDONESIA. Jurnal Pembangunan Hukum Indonesia, 1(1) tps://doi.org/10.14710/jphi.v1i1.10 6-116 harus selalu dibekali diri literasi tentang isu-isu keperempuanan. Dan juga menyadari penuh posisi dan peran kita di segala aspek bidang dan kehidupan, agar terciptanya suatu kesetaraan gender. Literasi harus selalu diadakan dalam lapisan masyarakat, Maka kita sebagai orang yang terpelajar harus bisa menekan angka deskriminasi yang terjadi terhadap budaya patriarki, dan memutus akar budaya patriarki itu sendiri. Agar dapat terjalin suatu kehidupan masyarakat yang aman, damai, dan tentram. Kiftiyah, A. (2019a). Perempuan dalam Partisipasi Politik di Indonesia. 14(1). Kiftiyah, A. (2019b). Perempuan dalam Partisipasi Politik di Indonesia. 14(1). Muslimat, A. (2020). Rendahnya Partisipasi Wanita di Bidang Politik. Jurnal Studi Gender dan Anak, 7(02), 131. https://doi.org/10.32678/jsga.v7i 02.181 Politik 5.pdf. (n.d.). Politik 11.pdf. (n.d.). DAFTAR RUJUKAN Priandi, R., & Roisah, K. (2019). Assyakurrohim, D., Ikhram, D., Sirodj, R. A., & Afgani, M. W. (2022). Metode Studi Kasus dalam Penelitian Kualitatif. Jurnal Pendidikan Sains dan Komputer, 3(01), 1–9. https://doi.org/10.47709/jpsk.v3i 01.195 Ginting, S., & Rahmiaji, L. R. (n.d.). Assyakurrohim, D., Ikhram, D., Sirodj, R. A., & Afgani, M. W. (2022). Metode Studi Kasus dalam Penelitian Kualitatif. Jurnal Pendidikan Sains dan Komputer, 3(01), 1–9. https://doi.org/10.47709/jpsk.v3i 01.195 Ginting, S., & Rahmiaji, L. R. (n.d.). PARTISIPASI POLITIK PEREMPUAN DALAM PEMILIHAN UMUM DI INDONESIA. Jurnal
https://openalex.org/W3204627769	https://air.unimi.it/bitstream/2434/870456/2/Boffi%20et%20al%20-%202021%20-%20Nature%20experiences%20of%20older%20people.pdf	English	null	Nature Experiences of Older People for Active Ageing: An Interdisciplinary Approach to the Co-Design of Community Gardens	Frontiers in psychology	2,021	cc-by	17,658	Nature Experiences of Older People for Active Ageing: An Interdisciplinary Approach to the Co-Design of Community Gardens Marco Bofﬁ1, Linda Pola 1, Natalia Fumagalli 2, Elisabetta Fermani 2, Giulio Senes 2 and Paolo Inghilleri 1 1 Department of Cultural Heritage and Environment, University of Milan, Milan, Italy, 2 Department of Agricultural and Environmental Sciences, University of Milan, Milan, Italy In Western countries, the concepts of healthy ageing and active ageing, that is, concepts that favour health, participation, and security to increase the quality of life of older people, have become key issues. Hence, the effective design of public green spaces in urban areas is crucial, as visiting these areas encourages social relations and interactions in natural, healthy contexts. Consistent with these perspectives, existing landscape design criteria emphasise the importance of considering not only the functional and aesthetic elements, but also the ecosystemic and biophilic relationships between people and the environment, producing positive effects for both. To maximise the impact of such design criteria, proper engagement strategies are desirable, both to assign an active role to older people themselves and to inform the ﬁne-tuning of the design process according to the speciﬁc needs of the local population. This study presents an interdisciplinary co-design method that encourages and informs a biophilic approach by describing the experiences of people in natural environments, actual and designed, through the application of attention restoration theory (ART). The case study was developed through six focus group meetings with older people for the co-design of a restorative area in a community garden in the Ortica district in Milan (Italy). Results show how the main needs expressed by participants can be classiﬁed into the ART factors of “compatibility” (a multifunctional garden), “fascination” (sense of contact with nature), and “being away” (metaphorical escape from nursing homes). The garden designed includes biophilic principles that respond to such needs, and speciﬁc links with designed elements were identiﬁed. For example, “being away” (e.g., isolation from daily routine and visual occlusion of the surroundings) and “compatibility” (e.g., pergola and aesthetic value) are the factors that include the elements that more satisfactorily answer previous needs. “Fascination” also includes many positive aspects, allowing space for improvements (e.g., more water elements, interaction with animals). Implications of the method are discussed, including the importance of subjective experience in informing design, the use of different psychological constructs to describe it, and the methodological alternatives for psychological assessment. Keywords: citizen engagement, attention restoration, biophilic design, focus group, participatory design ORIGINAL RESEARCH published: 27 September 2021 doi: 10.3389/fpsyg.2021.702525 Edited by: Jack Leon Nasar, The Ohio State University, United States Reviewed by: Birgitta Gatersleben, University of Surrey, United Kingdom Annalisa Setti, University College Cork, Ireland Correspondence: Marco Bofﬁ marco.bofﬁ@unimi.it Reviewed by: Birgitta Gatersleben, University of Surrey, United Kingdom Annalisa Setti, University College Cork, Ireland Reviewed by: Birgitta Gatersleben, University of Surrey, United Kingdom Annalisa Setti, University College Cork, Ireland *Correspondence: Marco Bofﬁ marco.bofﬁ@unimi.it Specialty section: This article was submitted to Environmental Psychology, a section of the journal Frontiers in Psychology Received: 29 April 2021 Accepted: 27 August 2021 Published: 27 September 2021 Citation: BofﬁM, Pola L, Fumagalli N, Fermani E, Senes G and Inghilleri P (2021) Nature Experiences of Older People for Active Ageing: An Interdisciplinary Approach to the Co-Design of Community Gardens. Front. Psychol. 12:702525. doi: 10.3389/fpsyg.2021.702525 September 2021 \| Volume 12 \| Article 702525 Frontiers in Psychology \| www.frontiersin.org 1 Nature Experiences of Older People Bofﬁet al. INTRODUCTION relationship with local cultural aspects. In such perspectives, the interdisciplinary exchange between the social sciences and design sciences is much needed today, which is in line with the concept of cities as artefacts that can be properly interpreted only by referring to both their spatial features and the social dynamics taking place there (Romice et al., 2016; Inghilleri, 2021). Although such perspectives are producing some results in the ﬁeld of research and related experimental initiatives, the social sciences still play a limited role in design processes, which occur in everyday contexts. This not only diminishes the overall quality of design projects, but also becomes an obstacle to the full actualization of the positive potential of some technical solutions. For an eﬀective interdisciplinary co-design approach, we can consider urban design as a recursive process that can be divided into ﬁve main design phases (analyze, plan, design, assess, and communicate) that cross six key actions (observe and interpret, measure and compare, model and simulate, strategize, design, and communicate) (Piga, 2017). According to such a scheme, the contribution of social sciences can occur during the entire design process, oﬀering theories and models to observe and interpret the interactions between people and their environment and then providing tools for measuring and comparing that interaction consistently with the chosen theories. If such aspects are those traditionally faced by scholars in the ﬁeld of environmental psychology, a further contribution to the participatory and communicative aspects of the process can come from community psychology. From this point of view, the social sciences can support designers and planners from the very beginning to the end of the process. However, such an inﬂuence is bidirectional, as the design sciences can inform about the needs emerging in speciﬁc contexts, contributing to the creation of integrated perspectives and tools that can more eﬀectively build knowledge about the people–environment interaction. To maximise the impact of design criteria, proper engagement strategies are desirable, both to assign an active role to older people and to inform the ﬁne-tuning of the design process according to the speciﬁc needs of local populations. An increasingly ageing population is a challenge for most of the European countries. Such a demographic change requires appropriate measures to help older people remain healthy and active, with these measures being consistent with the concepts of healthy ageing and active ageing that have been adopted in many countries to deﬁne appropriate guidelines. INTRODUCTION Healthy ageing refers to a “holistic perspective that includes subjective experiences and meanings, functional deﬁnitions emphasising autonomy, participation, and well-being” (Sixsmith et al., 2014, p. 1). Active ageing can be deﬁned as “the process of optimising opportunities for health, participation, and security to enhance the quality of life as people age” (WHO, 2002 p. 12). As is recommended in the global age-friendly cities guide published by the WHO (2007), one way to ensure that our cities and communities meet the needs of the ageing population is to involve them in co-designing processes, which can be particularly fruitful in the case of nature-based solutions (Mahmoud and Morello, 2021). The relevance of natural elements to human health is sustained by a wide and growing body of research in the ﬁeld of healthcare for older adults, showing that interactions with the urban green environment have a wide range of positive health outcomes and beneﬁts. Spending time in natural surroundings encourages physical activity and engagement, increases energy, ﬁghts depression, boosts memory, improves physical health, reduces stresses, provides restorative experiences, and encourages social relations (Kaplan, 1995; Berman et al., 2008; Pearson and Craig, 2014; Capaldi et al., 2015; Lawton et al., 2017). This scenario emphasises the importance of properly designing green public spaces in urban areas, taking into account both the physical features of the environment and the subjective factors that are of importance to sensitive populations. In fact, in cities of today, the satisfactory experience of nature requires deliberate design and development decisions (Kellert, 2016). In this ﬁeld, at the intersection between built and natural environments, two emerging design methods, biophilic design and biophilic urbanism, represent an inter-scalar approach to increasing connectivity to the natural environment. Biophilic design attempts to achieve the beneﬁcial eﬀects of contact with nature in the modern built environment by working, for example, at the architectural scale. The goal of biophilic urbanism is to reduce the contemporary urban disconnect from nature, making the experience of the natural world an integral part of ordinary city life; it aims to ameliorate the urban environment at the district or municipal scale. Rather than focusing on a single building, landscape, or human use, biophilic urbanism encompasses a vast array of ecological systems and human activities. They both deﬁne criteria and strategies to provide experiences of nature in urban areas that evoke positive physiological and psychological responses (Eid et al., 2021). Frontiers in Psychology \| www.frontiersin.org DESIGN CRITERIA FOR A RESTORATIVE GARDEN Biophilia, biophilic design, and biophilic urbanism oﬀer an integrated and inter-scalar approach to fostering a renovated consciousness of our relationship with nature. They emphasise the importance of the world beyond ourselves in fostering our well-being, highlighting the notion of a relationship with the environment, in general, and with nature, in particular. From a design perspective, this must not be conceived as a mere decorative intervention, but rather as a systemic approach to landscape conception (Kellert, 2016). Biologist Wilson (1984) asserted that humans have an innate predisposition, which he called biophilia, to connect with other forms of life; speciﬁcally, an innate and genetically determined aﬃnity of human beings with the natural world. From an evolutionary standpoint, biophilia has promoted the successful adaptation to environmental conditions across hundreds of millennia in which humans have lived in close association with nature. “Biophilic rules persist from generation to generation, atrophied and ﬁtfully manifested in the artiﬁcial new environments into which technology has catapulted humanity” (Wilson, 1993, p. 32). According to Letourneau (2013), present- day manifestations of biophilia in humans include the quickness and decisiveness with which humans learn particular things about certain kinds of plants and animals (Wilson, 1984); the attraction of modern humans to open spaces with lush vegetation, scattered trees, and conspicuous water features; the aversion of modern humans toward closed spaces (Heerwagen and Orians, 1993; Ulrich, 1993); the preference of modern humans for viewing natural landscapes over urban scenes (Ulrich, 1993); the stress-reducing and restorative eﬀects of visiting, or even just viewing non-threatening, natural settings or water features, on modern humans (Berto et al., 2018) the therapeutic eﬀects of contact with nature on modern humans (Ulrich, 1993); the enhancement of creativity, mental discipline, and higher-level cognitive functioning in modern humans associated with an exposure to nature (Ulrich, 1993; Faaber-Taylor et al., 1998; Bassi et al., 2018). The increasing attention to the therapeutic and regenerative eﬀects that contact with nature has on humans has now opened numerous lines of research on the use of gardens in the health and care sectors and on a speciﬁc design approach named “healing garden design.” The formal design of gardens, seen as oﬀshoots of internal environments, where the built prevails over the naturalistic component, does not exploit the regenerative and therapeutic potential that these spaces can oﬀer. INTRODUCTION It is worth noting that, although a great emphasis is traditionally placed on the “organic or naturalistic dimension” (Kellert, 2008, p. 5) of the biophilic approach, by referring to forms that recall the human predisposition toward Overall, the goal of the current study is to comprehend how the key features of the experiences of older people, in their relationship with nature, aﬀect their evaluation of biophilic environments. The understanding of this relationship can be achieved by integrating traditional top-down biophilic design criteria with bottom-up co-design data and by expanding the investigation area from the single site, typical of biophilic design, to the broader context, as suggested by biophilic urbanism. As no consolidated assessment tool has been developed in the ﬁeld of biophilia, we borrowed the interpretative categories of the experiences in nature from attention restoration theory (ART) (Kaplan and Kaplan, 1989), a well-established model that has provided many quantitative instruments in the ﬁeld of environmental psychology. From a theoretical perspective, the goal is to emphasise the points of contact between biophilic design and ART, with a strong link with actual designed physical elements. We aim to reconnect the early phases of the design practise (i.e., analyse, plan, and design; see Piga, 2017) with the existing principles of ART. From the methodological perspective, the goal is to develop a method that can be practically included It is worth noting that, although a great emphasis is traditionally placed on the “organic or naturalistic dimension” (Kellert, 2008, p. 5) of the biophilic approach, by referring to forms that recall the human predisposition toward nature, this concept also encompasses a “place-based or vernacular dimension” (Kellert, 2008, p. 6), which involves a September 2021 \| Volume 12 \| Article 702525 Frontiers in Psychology \| www.frontiersin.org 2 Nature Experiences of Older People Bofﬁet al. in the design process. We aim to provide a qualitative tool to inform co-design activities with a theory-driven interpretation of the experiences of both citizens in nature and their reactions to design project proposals. The case study was developed in the Ortica district in Milan to redesign a portion of its community garden named “San Faustino,” creating a healing garden. extension of biophilia that tends to include natural systems and processes in buildings and constructed landscapes in order to provide human beings with their much-needed exposure to nature. INTRODUCTION This has led scholars and practitioners to reﬂect on the environmental qualities (e.g., light, colours, shapes, materials, and vegetation) that can have a positive impact on human physiology and psychology in order to design spaces capable of improving the experiences of diﬀerent peoples. In the following section Design criteria for a restorative garden, we summarise the biophilic design criteria that can be considered when designing a community garden, with a speciﬁc focus on the needs of older people. We illustrate the ART (Kaplan and Kaplan, 1989), which is an eﬀective conceptual and empirical bridge between design and psychology. In section Focus groups for citizens engagement, we reﬂect on the implications of adopting focus groups as a method for co-design, subsequently proposing an analysis approach that includes ART both for the observation of needs and for design project assessments. In section Method, we present the method, in general, and its main steps. In section Results, we illustrate the results obtained from each step. In section Discussion and conclusion, we discuss the implications of the case study and reﬂect on the future development of the method. Much of the literature available about biophilic design concerns architecture, but this design approach also ﬁnds a place in landscape architecture (Davidson, 2013); for biophilic design to be truly eﬀective, it should extend beyond buildings into “life between buildings” (Moore and Marcus, 2008) and integrate ecology in a sustainable landscape design. Hence, biophilic urbanism oﬀers a key contribution, as it develops biophilia at the urban scale. Bringing in biophilia at the urban scale implies the need to focus on the experience in the environment and to facilitate the relationship with nature through urban planning and design tools. Therefore, urban planning strategies and the design of urban spaces can foster direct (e.g., green infrastructures, parks, and green roofs) or indirect contact with nature (e.g., natural materials and green buildings) through the application of natural models and processes to the built spaces (Totaforti, 2020). Frontiers in Psychology \| www.frontiersin.org September 2021 \| Volume 12 \| Article 702525 DESIGN CRITERIA FOR A RESTORATIVE GARDEN Frost (1992) speaks of “do-not-touch environments,” referring to those types of green spaces with a predominantly aesthetic function, as they are unable to involve and stimulate those for whom these spaces are intended and can inhibit interaction. The design of a healing/restorative garden is the combination of two conceptual components: the healing/restorative process and the space in which this process is supported (Marcus and Barnes, 1999). This suggests how the quality of both the environment and the experiences they oﬀer can aﬀect the healing process. One important interaction between individuals and their environment is the restoration of our attention, our energy, and ourselves by experiencing or viewing nature (Clay, 2001). An urban lifestyle, from a psychological perspective, imposes a high demand on our cognitive systems: even if relaxing settings and activities may provide restorative opportunities, nature seems to be particularly useful for this purpose (Kaplan and Berman, The evolutionary theory proposed by Wilson recognised the human life support function that nature plays in providing ecosystem services (Wang et al., 2021). It has oriented architectural and urban planning toward biophilic design, an September 2021 \| Volume 12 \| Article 702525 Frontiers in Psychology \| www.frontiersin.org 3 Nature Experiences of Older People Bofﬁet al. 2010). “Restorative environments” refer to those natural places that favour a shift toward more positively toned emotional states and positive changes in physiological activity levels, cognitive functioning, and behaviour (Kaplan and Kaplan, 1989). They not only permit, but also promote restoration, thus enabling a faster, more complete recovery of depleted resources than environments that are relatively free of demands but which lack positive features (Hartig, 2004). It is an eﬀect of the relationship between a user and setting without any therapeutic program or deﬁned therapeutic activities (Haller, 2004). Research into the restorative beneﬁts of contact with nature has generally looked at three main areas of contact: active, less active, and passive. Physical rehabilitation and engagement in horticultural therapy are examples of “active” interactions with the garden, while sitting in the garden, observing plants and animals, and listening to nature sounds are examples of “less-active” modes of interaction. However, the garden can also be experienced “passively” by viewing it through a window from the inside (Stigsdotter et al., 2011). support if needed, but also independence, challenge, and learning (Carstens, 1993). DESIGN CRITERIA FOR A RESTORATIVE GARDEN Some examples of the translation of such features into design include accessibility (physical and experiential) to the main and secondary areas that lead to diﬀerent kinds of experiences, the legibility of the layout (what to do and where), the opportunity to make choices, graded diﬃculties (physical and psychological limits of users), security, respect for anthropometric measures (raised beds that can be easily reached by people on wheelchairs), and microclimatic regulation for use at diﬀerent times of the day and for most of the year. Regenerative environment: one which helps to renew or revitalise psychological and physical resources, give new energy, enhance resilience, recover the capacity to fend oﬀ distraction and coercion, and renew the cognitive powers of a person (Kaplan, 1983). This concept leads to some speciﬁc characteristics of the environment like complexity, richness (biodiversity), sensory stimulation, attractive and more wild- looking destinations, and the opportunity for wandering. Most of the research projects related to restorative environments derive from the biophilia hypothesis and ART. Both have an evolutionary approach, maintaining that we are adapted through human evolution to function well in natural environments, although some natural environments will better serve restoration than other kinds of environments (Stigsdotter et al., 2011). Human tendencies to love and take care of nature are aﬀected by attention, i.e. the ability to focus on natural stimuli eﬀortlessly, actually to be fascinated by nature (Kaplan, 1995), and empathy, i.e. to feel connected emotionally to the various life forms, and to participate in their condition. Attention restoration theory (Kaplan and Kaplan, 1989) suggests that the ability to concentrate can be restored by exposure to natural environments and describes four diﬀerent factors, the combination of which encourages “involuntary” or “indirect attention” and enables our “voluntary” or “directed” attention capacities to recover and restore ourselves (Kaplan, 1995). The Kaplans have established that, in order to be restorative, a place must have the following features (Kaplan and Kaplan, 1989): fascination, which is achieved through a setting that easily engages attention, thereby allowing fatigued attention to rest; being away, which is provided by a setting that is either physically or conceptually diﬀerent from everyday settings of an individual; extent, which is provided by a setting that is complex enough to engage the mind and promote exploration; compatibility, which is achieved when the design of a setting supports the intended use by the occupant. DESIGN CRITERIA FOR A RESTORATIVE GARDEN g pp y g Ecosystem value: one with the enhancement of the ecological functions and beneﬁts that the natural ecosystems provide to support native life forms. “Healthy ecosystems” seem to be more relevant than “nature” for restorative experiences, and it is due to synergic the compatibility between environmental attitudes and healthy ecosystems that triggers restorative processes (Giusti and Samuelsson, 2020). The design should derive from the study of local natural models, focus on nature-based solutions, and aim for multifunctionality and sustainability. The sustainability of a designed garden carries a positive message, perceived at a subliminal level by the user; it communicates the care for the well-being of all living beings (Marcus and Barnes, 1999). An excessively artiﬁcial garden that requires continuous maintenance and communicates artiﬁciality and control, on the contrary, is perceived in a negative way. Frontiers in Psychology \| www.frontiersin.org FOCUS GROUPS FOR CITIZEN ENGAGEMENT In order to link the general design criteria to the actual experiences of the local population in nature, a co-design approach was developed. The term co-design often includes a wide range of design practises (Sanders and Stappers, 2008). For the purpose of this work, we consider it as a collective task, including the analysis of a challenge in the present context and the attempt to address it through a creative act (e.g., a physical transformation, a new service, or a product) to improve a future context (Zamenopoulos and Alexiou, 2018). In such a perspective, the crucial aspect expressed by this term is in its connective, cooperative, and collaborative nature, which includes both the professionals involved in the design process and involved non-professionals through consultations. In the planning ﬁeld, to help the restorative process and to support the needs of users, the design has to follow speciﬁc criteria that result from diﬀerent disciplines: landscape architecture, medical sciences, ecopsychology, and landscape ecology. The design criteria can be summarised as follows (Fumagalli et al., 2020): In this general framework, we chose focus groups for implementing the method. A focus group discussion is a technique where a relatively homogeneous group of individuals is purposely selected to provide information about speciﬁc topics of interest (Hughes and DuMont, 1993). Focus group Prosthetic environment: one which compensates for cognitive deﬁcits and positively inﬂuences the functional status and behaviour of older people whose ability to interact with their surroundings declines beyond their 80th year. It has to guarantee the optimal functioning of individuals by oﬀering September 2021 \| Volume 12 \| Article 702525 Frontiers in Psychology \| www.frontiersin.org 4 Nature Experiences of Older People Bofﬁet al. TABLE 1 \| Actors and roles in the co-design process. Actors involved in the process of co-design of public spaces Roles Psychologists Research design, focus group design, and execution (data collection and analysis), informing the landscape design phase Designers Research design, informing the focus group design, transforming focus group results into physical environmental choices, design plan, and execution Focus group’s participants Sharing their personal experience, beliefs, and attitudes TABLE 1 \| Actors and roles in the co-design process. discussions are widely used in co-design interventions, but it is not uncommon to ﬁnd the terminology “focus group discussion” used synonymously with “group interviews” or “workshops” (Hanington and Martin, 2012; Kpamma et al., 2017; Salvia and Morello, 2020). FOCUS GROUPS FOR CITIZEN ENGAGEMENT • to interpret such experiences by referring to a speciﬁc theoretical model, ART, which is a widely consolidated and employed theory in applied and research contexts; • to interpret such experiences by referring to a speciﬁc theoretical model, ART, which is a widely consolidated and employed theory in applied and research contexts; • to interpret such experiences by referring to a speciﬁc theoretical model, ART, which is a widely consolidated and employed theory in applied and research contexts; • to inform the design project through the interpretation of such experiences with the factors of ART; • to include information consistent with the principles of biophilic urbanism in the design process. METHOD This article illustrates the co-design process developed for a project that aimed to redesign a portion of a community garden (CG) named “San Faustino,” located in the Ortica district in Milan, as a sustainable restorative natural space for older people. This study area, part of the broader CG, was designed to beneﬁt older people through exposure to and contact with nature. The study area is mainly dedicated to the guests of two nursing homes participating in the research project, even though the CG is open to the public who can also access this area. This article illustrates the co-design process developed for a project that aimed to redesign a portion of a community garden (CG) named “San Faustino,” located in the Ortica district in Milan, as a sustainable restorative natural space for older people. This study area, part of the broader CG, was designed to beneﬁt older people through exposure to and contact with nature. The study area is mainly dedicated to the guests of two nursing homes participating in the research project, even though the CG is open to the public who can also access this area. The research team adopted a multidisciplinary approach to the co-design process, integrating psychology and landscape design. Local stakeholders were involved in the process, namely, institutional representatives, private nursing homes, and neighbourhood associations. The research activity included the following main steps: In this study, ART represents a common framework that allows us to build a bridge between psychology and design, thus envisioning the landscape with an experiential approach that relies on the interactions with nature described by participants and integrating the prosthetic aspect of the environment for psychophysical comfort and stimulation. In the light of what has been described, the goals of this contribution are: • the development of the research design: the research team developed the materials and methods consistently with the objectives of the study. FOCUS GROUPS FOR CITIZEN ENGAGEMENT The relationship between the researcher and the participants distinguishes focus groups from other participatory methods. During a focus group discussion, in fact, the researchers adopt the role of “facilitators” or “moderators” rather than the role of “investigators” (Smithson, 2000). Their roles are more peripheral than central (Bloor et al., 2001; Sim, 2002; Smithson, 2007): they are there to moderate and stimulate the group discussion between participants and not between the researchers and the participants. p p From this point of view, in the process of the co-design of public spaces, citizens are involved as experts on their own relationships with such spaces (see Table 1). Indeed, they are invited to discuss their own experiences in living or carrying out speciﬁc activities in these spaces. The sum of the attitudes, beliefs, and opinions of the participants can provide new critical insights and lead to a greater understanding of the topics of interest (Massey, 2011). The role of the researcher, then, is to facilitate the exchange of personal views among participants, utilising the topics raised to further feed the discussion. Such discussions revolve around personal experiences in or attitudes and beliefs about those spaces, without explicitly asking for design solutions. The kinds of data obtained in this way can be interpreted at two levels: articulated data and attributional data (Massey, 2011). “Articulated data” refer to the explicit answers of participants to issues proposed during the discussion, corresponding to what Braun and Clarke (2006) deﬁne as an inductive thematic analysis. Conversely, “attributional data” are theory driven, which means that they are not explored directly but, instead, indirectly, and then categorised. At the second level, consistent with a deductive thematic analysis, psychologist researchers have the responsibility to translate the narrations of participants into consistent and theoretically grounded concepts; designers, on the other hand, are in charge of transforming the results into physical environmental features (Boﬃand Rainisio, 2017). The wide availability of data oﬀered by the focus group makes it a preferable methodological choice compared with other co-design techniques, as it favours the integration of theoretical models during the analysis phase. This “biophilic co-design approach” allows us to link general biophilic principles to the speciﬁc needs of a local community. First-Round Participants and Procedure First-Round Participants and Procedure The ﬁrst round of focus groups involved carrying out three focus group meetings that were held with potential users of the CG. The participants were recruited through local stakeholders: local administrative institutions, district associations, and nursing homes provided three lists of people aged over 65 who were likely to access the CG due to geographical proximity. Snowball sampling was used for district residents and members of associations. The residents of the nursing homes were recruited with the support of local healthcare personnel, selecting those with cognitive functions to allow a meaningful interaction. The ﬁrst focus group involved older people living in the Ortica district or nearby (six participants, ﬁve women and one man; age range, 65–71; acceptance rate, 43%); a second focus group involved older people actively involved in local associations, even if they were not residing in the district (10 participants, 6 women and 4 men; age range, 65–82; acceptance rate, 100%); a third focus group involved older people hosted in the nursing homes next to the CG (seven participants, ﬁve women and two men; age range, 70–84). District residents in the ﬁrst focus group did not know one another, except for a married couple. The local activists in the second focus group were members, among others, of an association active in the CG. The guests of the nursing homes were acquaintances. Two trained psychologists led all the focus groups to facilitate interactions and took notes during the discussions; the third focus group included two additional facilitators already familiar to the participants. All the participants were Italian native speakers and long-time residents of Milan or, in the third focus group, of close municipalities before moving to the nursing homes. The characteristics of the three groups are consistent with respective target populations, even though district residents were not balanced for gender. No discussed issues or group interactions suggested a related eﬀect. The participants signed written consents, allowing the anonymous analyses of the recorded audio. All the subjects gave their informed consent before participating; the study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of University of Milan (Project Green Space for Active Living. Older Adults perspectives) on April 19, 2019. The audio recording started after signing the consent, with each focus group meeting lasting ∼2 h. Site Analysis and Preliminary Design The ﬁrst step in the design process, before proceeding with the focus group phase, was the site analysis. This step consisted of research activity that looked at the existing conditions of the project site, along with any potential future conditions. The aim was to identify the values of the place and the critical issues to be mitigated or resolved and to better understand the relationship of the place itself with the neighbourhood context. Therefore, the physical characteristics (e.g., site boundaries, contours, dimensions, site features, and microclimate) and natural features (e.g., typology of vegetation, composition) of the site and the typology of current users were analysed. All the information acquired allowed the designer to understand the existing opportunities and problems in the site. By relating what emerged from the site analysis to the design criteria of a restorative garden, which were previously illustrated, it was possible to develop preliminary design proposals and produce some material for an initial discussion with the participants of the focus groups. First-Round Participants and Procedure The ﬁrst two focus groups took place in the meeting room of a well-known local cooperative; the third focus group was held in a local nursing home. During the ﬁrst part of the focus group on topics 1 and 2, the cartographic and photographic materials facilitated the sharing of the personal image of the district and the CG among the participants, as the emerging issues were placed on the maps. In the last part of the discussion on topic 3, the Frontiers in Psychology \| www.frontiersin.org METHOD This included the recruitment of participants, location identiﬁcation, a site analysis, and a preliminary design; • the ﬁrst round of focus groups, which aimed to collect information about needs and desired experiences of users; • to collect the experiences of diﬀerent groups of older people in nature to complement the criteria oﬀered by biophilic design with contextual data; • the stimulation of the CG design project by sharing ﬁndings from the ﬁrst-round focus groups with the team; • the ﬁnal design project, including related communication materials; September 2021 \| Volume 12 \| Article 702525 Frontiers in Psychology \| www.frontiersin.org 5 Bofﬁet al. Nature Experiences of Older People • the second round of focus groups, aimed to gather feedback on the ﬁndings from the participants and collect their reactions to the design project. • the second round of focus groups, aimed to gather feedback on the ﬁndings from the participants and collect their reactions to the design project. (Piga, 2011; Piga and Morello, 2015) (A0 size, placed around the participants), a CG map (A1 size), and postcards reproducing the main landmarks of the CG (placed on the table among the participants); (topic 3), a set of postcards representing evocative images of nature or people in nature consistent with the design criteria, for example, an older person smelling a ﬂower, a group of older people chatting on a bench, or even the image of some birds on a tree (placed on the table among the participants). This section presents the method of the study, including both the design of the CG (the preliminary design and its ﬁne-tuning) and the involvement of the citizens through co-design (materials, participants, and procedures that allowed us to collect data and their analysis criteria). The following section describes the results. September 2021 \| Volume 12 \| Article 702525 First-Round Analysis The data for the analysis included written notes and the comments placed on the maps and postcards by the facilitator or the participants themselves. The audio recording supported the analysis, providing the original context for the data. A thematic analysis (Braun and Clarke, 2006) was performed on the qualitative data collected during the diﬀerent phases of the focus group, adopting a hybrid approach that combines inductive and deductive analyses (Fereday and Muir-Cochrane, 2006). Data on the ﬁrst two topics, namely, the image of the district and of the CG, were analysed with an inductive thematic approach; no pre-existing coding frame was applied to identify the main themes introduced by the participants. Such a thematic analysis was integrated with a representation of the themes on the map of the district to emphasise the spatial connexion of each theme with a speciﬁc location in the district. The third topic about the desired experiences of the participants in nature was analysed with a deductive thematic approach; the a priori codes for classifying the data were represented by the four dimensions of ART (being away, compatibility, extent, and fascination). Each sentence used by the participants to describe such experiences was assigned to a single ART dimension. In some cases, these theory-driven themes were further divided into data-driven sub-themes to track relevant aspects of the results, thus informing the design project (e.g., for the compatibility and fascination dimensions). The same approach was carried out by the psychologists for all the focus groups and subsequently interdisciplinary validated. Second-Round Participants and Procedure The second round of focus groups involved the realisation of three more focus group meetings, one for each target group of potential users engaged during the ﬁrst round. Contacts collected for the ﬁrst round, including the former participants, were invited again. The ﬁrst focus group with older people living in the Ortica district or nearby included three of the former six participants, and no new member joined the group (three participants, two women and one man; age range, 65– 70; acceptance rate, 23%). The second focus group with older people involved in local associations included all the former participants (10 participants, 6 women and 4 men; age range, 65– 82; acceptance rate, 100%). The third focus group with the guests of the nursing homes included all the former participants and ﬁve new members (12 participants, 9 women and 3 men; age range, 70–84). Fine Tuning of the CG Design Project g g The results of the ﬁrst-round focus groups were the basis for a multidisciplinary exchange, stimulating the revision of the initial design in consideration of the experiences collected. The four factors of ART were the basis for the discussion and the implementation of the ﬁnal version of the design. First Round of Focus Group Discussions First-Round Materials In order to carry out the focus groups, a semi-structured schedule was built to investigate three speciﬁc topics: the image and characteristics of the district (not investigated with the older people hosted in the nursing homes), for example, using sentences to stimulate the discussion, such as “Describe your typical day in the district”; the image and characteristics related to the CG, e.g., “I go there when I want to feel...”; expectations about the study area and the desired experience of living in nature, e.g., “Imagine that, in 1 year from now, the CG is frequently attended by older people: why is it successful?” The image of a place is deﬁned as a mental product that is culturally elaborated, resulting from observation, perception, orientation, and experience; far from being permanent, it is likely to be the result of continuous confrontations, exchanges, and agreements between individuals (Lynch, 1960). Considering our goals, the images of the district and the garden were as important as the experience of the participants in nature. The semi-structured schedule was integrated by a set of cartographic and photographic materials according to the three topics explored. They included: (topic 1), a map of the Ortica district (A0 size) and postcards reproducing the main landmarks of the district (placed on the table among the participants); (topic 2), four photographs of the study area as it was before the transformation in order to evoke the visual context of the garden according to the principles of experiential simulation in design September 2021 \| Volume 12 \| Article 702525 6 Nature Experiences of Older People Bofﬁet al. participants were asked to describe their relationship with nature and, through the selection of the postcards with evocative images, to reﬂect on the kind of experiences they would like to enjoy while interacting with the study area. around the participants); a map of the study area renovated according to the design project (A1 size); a set of postcards reproducing the main elements of the design project; a CG map, including the study area renovated according to the design project (A1 size); a set of postcards reproducing the main landmarks of the CG (existing and renovated). Frontiers in Psychology \| www.frontiersin.org Second Round of Focus Groups Second-Round Materials For the second round of focus groups, a new semi-structured schedule was designed: the ﬁrst part aimed at presenting the results of the previous phase and the design proposal; the second part aimed at investigating the reactions to the designed study area, for example, using sentences stimulating the discussion such as “What elements of the design projects impressed you the most? What does it remind you of?”; the third part aimed at exploring the image of the renovated CG, e.g., “Imagine you are describing the CG to a friend....” As in the previous round, in addition to the semi-structured schedule, the procedure included additional cartographic and photographic materials for facilitation: four pictures of the study area as it was before the transformation (A0 size, placed around the participants); four pictures of the study area according to the design project to evoke the future condition (A0 size, placed First-Round Analysis The characteristics of the groups and their inﬂuence on the discussed issues were comparable with those observed during the ﬁrst round. The same facilitators were involved, including the landscape designer for the ﬁrst phase of the focus groups. During the ﬁrst phase of the meetings, the participants were presented with the results of the ﬁrst round of investigation to reinforce their active roles in the process. During this phase, the room in which the focus group took place was set up to evoke the current visual context of the project: four photographs of the study area before the transformation were placed around the table of the focus groups. At the end of the presentation, the four pictures around the participants were substituted by four pictures of the study area according to the revised design in order to evoke the future condition. At the same time, the map of the study area according to the design project was placed on the table. The landscape designer introduced the biophilic design principles and showed the features of the design solution. After answering a few questions from the participants, the designer left the room, and the second phase started. The participants were asked to indicate which elements impressed them the most and why, providing explanations for what they considered speciﬁc strengths or weaknesses according to their experiences in nature. In order to support this part of the process, postcards illustrating the main elements of the project were used. The third and last parts of the focus group meetings were dedicated to exploring the preﬁgured image of the CG, considering both current and future elements. To support this phase, a map of the CG was proposed, including the study area according to the design project. RESULTS This section presents the results of the ﬁrst-round focus groups, their impact on the design proposal, and the results of the second-round focus groups. As the main interest of this study is the integration of data on the experiences of older people in nature into the entire design process, the emphasis is on the most relevant results obtained in each phase and their use in the subsequent phases. For a more detailed analysis of the ﬁrst-round focus groups, see Fumagalli et al. (2020). From the ﬁrst-round focus groups, we present: citizens (...) We also have very nice exchanges, nice discussions ]. This evidence has an important impact on the co-design process of the study area. For those citizens living in the area but not actively involved in local associations, the district where the CG is located is, overall, hardly considered to be one of the available resources that satisfy personal needs, even if no speciﬁc critical aspects are mentioned. However, they cited a few local places for social aggregation, a few green areas, and almost no services (“My typical day? I walk around with no destination”). Although the district is recognised as undergoing a requaliﬁcation process, older people living in the Ortica district or nearby did not mention the transformation as a chance to meet their own personal needs. In such a perceived void that characterises the area, the new regenerative garden included in the CG becomes less salient. Indeed, for older people living in the Ortica district or nearby, the CG is in an almost empty space, without meaningful places that function as attractors or reference points to reinforce its appeal. The district map resulting from the perception of older people who are actively involved in local associations oﬀers, instead, a complex and multifaceted representation. They show a deeper knowledge of local urban spaces and their transformations. Such an aspect is visible on the map in quantitative terms (a higher number of salient elements placed in the area), in qualitative terms (a wider spectrum of categories of the taxonomy; a more nuanced list of strengths, weaknesses, and 2-fold issues), and in spatial terms (a more homogenous distribution of the elements throughout the area with no empty spaces). RESULTS For the older people actively involved in local associations, the space surrounding the CG is richly populated by meaningful places, which are not always positive but are a vivid source of debate that suggests the relevance of the area in their perception. • a description of the image of the Ortica district, where the CG is located, and the green areas identiﬁed as landmarks by the participants; • a description of the image of the community garden where our study area is located; • expectations about the study area and desired experiences of potential users in nature with the study area. From the second-round focus groups, we present: • the coherence between the desired experiences that emerged from the ﬁrst-round focus groups and the study area according to the design project; g j • the CG image in the light of the introduction of the study area according to the revised design project. Second-Round Analysis The data construction and analyses for the second round followed the same criteria as those previously described. An inductive thematic analysis was performed on the experiences in nature elicited by the design project, collected during the second September 2021 \| Volume 12 \| Article 702525 7 Nature Experiences of Older People Bofﬁet al. phase. A deductive thematic analysis was carried out on the image of the renovated CG explored during the third phase. local swimming pool, which is associated with the café service without mentioning the natural experience itself (“Sometimes, just to have a quick walk around, I go to the pool garden, where there is a café”). Both of these places were frequented sporadically. The older people actively involved in local associations also described a district with few green areas, but, unlike the older people living in the Ortica district or nearby, they had daily routines where the relationship with green elements was more present. In addition to the botanical garden, the older people actively involved in local associations also cited the CG, where they acted as volunteers [“We wanted to work in a green and beautiful place that made us feel good. I studied agriculture and it seemed nice to put my knowledge into practise at the service of citizens (...) We also have very nice exchanges, nice discussions”]. Frontiers in Psychology \| www.frontiersin.org First Round of Focus Groups The Image of the Ortica District and Its Green Areas The Image of the Ortica District and Its Green Areas The image of the Ortica district that emerged from the personal experiences of the participants allowed us to identify the distinctive features of the place, both physical and symbolic. The results are summarised in Figures 1, 2. Each place mentioned during the discussion was included in a bottom-up taxonomy composed of places for social aggregation, services, places relevant to mobility issues, places of ongoing urban transformation, and green areas. In addition, each place was categorised as a strength, a weakness or a 2-fold issue, including both strengths and weaknesses. The maps resulting from such a classiﬁcation highlighted two very diﬀerent perspectives. Older people actively involved in local associations, although not all living in the Ortica district or nearby, showed greater knowledge of the strengths and weaknesses of the area. However, there was a general lack of awareness of the available resources and of weaknesses of the district from the older people living in the Ortica district or nearby. This general level of knowledge was associated with the frequency of reported access to local green areas. In fact, even if green areas are considered as a general positive aspect of a district by both groups, older people living in the Ortica district or nearby did not report spending much time there. Two green areas were mentioned by the older people living in the Ortica district or nearby, namely the botanical garden, which is identiﬁed as a place mostly suitable for children, and the greenery surrounding the The Image of the Community Garden The Image of the Community Garden The ﬁrst round of focus groups also allowed us to characterise the CG where the study area is located. Consistent with the perceived void in the district, the CG was barely known by older people living in the Ortica district or nearby. However, the older people actively involved in local associations and the guests of the nursing homes had good knowledge of the CG and could highlight which elements of ﬂora, fauna, human artefacts, and other elements characterised their imaginations about it. Regarding the plants, most of the participants mainly mentioned trees (e.g., cherry and elm) rather than other greenery. September 2021 \| Volume 12 \| Article 702525 8 Bofﬁet al. Nature Experiences of Older People FIGURE 1 \| A map resulting from the older people living in the Ortica district or nearby. The community garden (CG) position is marked in blue. map resulting from the older people living in the Ortica district or nearby. The community garden (CG) position is marked in blue. FIGURE 2 \| A map resulting from the older people actively involved in local associations. The CG position is marked in blue. FIGURE 2 \| A map resulting from the older people actively involved in local associations. The CG position is marked in blue. FIGURE 2 \| A map resulting from the older people actively involved in local associations. The CG position is marked in blue. September 2021 \| Volume 12 \| Article 702525 Frontiers in Psychology \| www.frontiersin.org 9 Nature Experiences of Older People Bofﬁet al. The trees were seen as key elements characterising the site in contrast with the urban context (“it is the background that gives you the idea of not being in the city”). Various animals were cited, mainly in positive terms (e.g., pheasants and bees), even though mice were seen as potentially damaging for the area. The discussion about human artefacts often highlighted a 2-fold perception, as they were perceived as positive elements but could not adequately manage to fulﬁl the potential of the garden (e.g., the vegetable gardens should be kept in a smaller dedicated area, the artiﬁcial pond should be refurbished, and the hives were not working properly); the arbour was the only element perceived as completely positive. Finally, some general concerns were expressed about mobility within the CG. The Image of the Community Garden For further information about the results that emerged on this topic, see Fumagalli et al. (2020). elements, was widely identiﬁed by the participants as the kind of experience in nature most capable of giving them pleasure, joy, and peace. The third most important dimension of ART is being away. The opportunity to escape from everyday life through immersion in a green space is important for all the targets interviewed, although it seems to be a particularly relevant experience for the guests of the nursing homes. In fact, the older people living in nursing homes seem particularly sensitive to the desire for escape as a reaction to the perceived contraction of their space-time freedom and physical skills [“We would use it just to break out a little bit (...) it would be the only opportunity to go out for a little bit”; “I would like to enjoy nature alone by myself”]. Finally, the extent, linked to the need for cohesion between the elements that compose a natural landscape, seemed to be the least important dimension. It is worth noting that the study area is small, that could make the need for feeling safe while exploring less relevant. Expectations About the Study Area and the Desired Experience of Living in Nature The results regarding the preﬁgured experience that the participants associated with the study area have been discussed in depth in Fumagalli et al. (2020). A summary of the main ﬁndings is presented here and in the ﬁrst row of Tables 2–5, separately for each ART factor, in order to provide a more complete overview of the results of the co-design process. Design Proposal The needs that emerged from the ﬁrst round of the focus groups were compared with the preliminary project. On the basis of the collected information, the design solutions were realigned or adapted, accommodating, as far as possible, the necessities of users. The experiential ambitions in the garden emerged from the focus groups are summarised in two trends in terms of design: g p Compatibility emerges as the ART dimension most preﬁgured across the groups, although the analysis underlines the diﬀerences among them. Compatibility, which is connected to the possibility of using the area as a place for interpersonal exchanges, is particularly important in the imaginations of the older people living in the Ortica district or nearby [“This could be an exchange area. It’s refreshing to be in contact with other generations. It’s a joy to see the kids (...) This dimension is more important to me than just relaxing”]. Compatibility appears to be less relevant for the older people actively involved in local associations and for the guests of the nursing homes. In this regard, we must recall that, as presented in the previous paragraphs, the older people living in the Ortica district or nearby ﬁnd it diﬃcult to identify signiﬁcant places within their social context that can meet their socialisation needs [“An outdoor- listening group could be instituted (...) Many older people just need to talk”]. On the contrary, the map of the older people actively involved in local associations identiﬁes various places of socialisation in their daily routines in addition to the CG. Finally, concerning the guests of the nursing homes, a wide range of activities is planned daily for indoor spaces shared by the guests [“Here, there are already groups to be in the company of others, while I would like to sit there and see the leaves move (...) reconnect with nature”]. (1) a multifunctional garden compatible with the needs and attitudes of the identiﬁed targets, consistent with the compatibility factor of ART: a place (a) of aggregation and relational exchanges, b) for manual activities, and (c) for and where to ﬁnd cultural initiatives; (2) a garden that can create contact with nature, consistent with the being away, extent, and fascination factors of ART: a place (d) of fascination and mystery, (e) where other kinds of experiences from everyday life can be lived, and (f) for concentration and solitude. September 2021 \| Volume 12 \| Article 702525 Frontiers in Psychology \| www.frontiersin.org Design Proposal (2) a garden that can create contact with nature, consistent with the being away, extent, and fascination factors of ART: a place (d) of fascination and mystery, (e) where other kinds of experiences from everyday life can be lived, and (f) for concentration and solitude. The contextual information about the image of the district allowed us to weigh the perception of the CG by diﬀerent groups, supporting the creation of a synthesis to inform the design proposal. One of the most relevant aspects in this regard was the factor of compatibility. The participants living in the district assigned it great importance, imagining many activities to carry out in the CG. Yet, many of them could be consistently satisﬁed by other facilities, which already exist in the district but of whose existence they do not know. This prominence given to compatibility is combined with the lower relevance assigned to being away, which is, instead, the most important aspect for the guests of the nursing homes who have few alternatives for experiencing it. The combined interpretation of the data on the district and the CG led the research team to reduce the weight of compatibility, as many of the needs expressed by residents could be addressed by other types of interventions in the district. The ART dimension of fascination is the second most relevant experiential dimension associated with the study area, according to the desires and preﬁgurations of the participants. A multisensory experience related to contact with natural elements is highly desirable for the targets [“The greenery does not involve only the sight but even other senses (...) It attenuates noises(...) Especially for me, since I am very anxious and nervous, it is very important”]: being in touch with the colours and scents of ﬂowers and fruits, with the vitality of plants, animals, and aquatic y y The design process, ﬁrstly, developed a concept plan that deﬁned the functional areas (what to do and where) and their connexions to ensure the eﬃcient use of the space, and secondly, a master plan that speciﬁed works in their entirety. A summary of the main design choices is presented in the second row of September 2021 \| Volume 12 \| Article 702525 Frontiers in Psychology \| www.frontiersin.org 10 Nature Experiences of Older People Bofﬁet al. Design Proposal TABLE 2 \| Comparison of the needs and reactions of the participants to the design proposal [attention restoration theory (ART), compatibility]. ART dimension: compatibility Needs emerged in ﬁrst-round focus groups 1. To have an aesthetically pleasant place for intra-generational and inter-generational social exchanges (“If there is beauty, the possibility of interpersonal exchanges is interesting”) 2. To have a place for knowledge in itself (“I like to look at plants [...]Find out whichplants are there”) 3. To have a place to implement manual skills (“An area where you can do your vegetable garden [...]Hand activities for plant care...”) Design solutions 1. Although mainly dedicated to the older people, the whole garden is attractive for the different generations to facilitate inter-generational social exchanges. Every design element, linked to active or passive activities was made available to users with high levels of physical limitations, also to guarantee the older people’s autonomy and ease of orientation (visible paths, landmarks, clear visibility of the whole garden). 2. An illustrative panel, at the beginning of the main path, illustrates the layout of the area with the main attractions, the strategic design elements and speciﬁed their functions. Main trees are identiﬁed with a common name tag for an easy recognition 3. The manual activities mainly concerntaking care of the plants, watering them, weeding, but without a speciﬁc commitment. This freeuse of the space allows visitors to contribute to its maintenance and care(that are formally entrusted to a specialised company, technical partner of the project). The garden does not propose speciﬁc activities, such as horticultural ones, as it is designed as a restorative garden where there are no prescribed therapeutic or occupational activities Designed elements highlighted in second-round focus groups 1. The pergola (Supplementary Figure 1) is recognised as the design element most suitable to satisfy socialisation needs. The participants show some doubts about its comfort. In this regard, some participants propose to implement the pergola through some form of covering, to allow people to use it even during less favourable weather conditions, such as wind or rain. Another potential weakness in this area is an adequate shading (“the trees that you will plant are already big, right?”). Participants seem to appreciate also the design philosophy introduced to them, about designing a place open to everyone (“This is the spirit… design for all”). TABLE 2 \| Comparison of the needs and reactions of the participants to the design proposal [attention restoration theory (ART), compatibility]. 1. To have an aesthetically pleasant place for intra-generational and inter-generational social exchanges (“If there is beauty, the possibility of interpersonal exchanges is interesting”) 2. To have a place for knowledge in itself (“I like to look at plants [...]Find out whichplants are there”) 2. To have a place for knowledge in itself (“I like to look at plants [...]Find out whichplants are there”) 3. To have a place to implement manual skills (“An area where you can do your vegetable garden [...]Hand activities for plant care...”) 3. To have a place to implement manual skills (“An area where you can do your vegetable garden [...]Hand activities for plant care...”) 1. Although mainly dedicated to the older people, the whole garden is attractive for the different generations to facilitate inter-generational social exchanges. Every design element, linked to active or passive activities was made available to users with high levels of physical limitations, also to guarantee the older people’s autonomy and ease of orientation (visible paths, landmarks, clear visibility of the whole garden). 1. Although mainly dedicated to the older people, the whole garden is attractive for the different generations to facilitate inter-generational social exchanges. Every design element, linked to active or passive activities was made available to users with high levels of physical limitations, also to guarantee the older people’s autonomy and ease of orientation (visible paths, landmarks, clear visibility of the whole garden). 2. An illustrative panel, at the beginning of the main path, illustrates the layout of the area with the main attractions, the strategic design elements and speciﬁed their functions. Main trees are identiﬁed with a common name tag for an easy recognition 3. The manual activities mainly concerntaking care of the plants, watering them, weeding, but without a speciﬁc commitment. This freeuse of the space allows visitors to contribute to its maintenance and care(that are formally entrusted to a specialised company, technical partner of the project). The garden does not propose speciﬁc activities, such as horticultural ones, as it is designed as a restorative garden where there are no prescribed therapeutic or occupational activities 3. The manual activities mainly concerntaking care of the plants, watering them, weeding, but without a speciﬁc commitment. This freeuse of the space allows visitors to contribute to its maintenance and care(that are formally entrusted to a specialised company, technical partner of the project). Second Round of the Focus Groups Coherence Between the Desired Experiences That Emerged in the First-Round Focus Groups and the Designed Study Area Evaluation The results of the second-round focus groups concern the reactions to the design proposal for the study area. The contents are categorised according to the ART dimensions, and a summary of the main ﬁndings is presented in the third row of Tables 2–5 separately for each ART factor and consistently with the results of the ﬁrst round and the related design elements (these are included in the Supplementary Materials). It includes references to the postcards of the main design elements of the project, which was proposed as a tool for discussion. The postcards are reproduced in the Supplementary Materials. In general, the needs of the ﬁrst round of the focus groups are met according to the inductive thematic analysis based on the ART factors. Overall, the factor of compatibility appears satisﬁed in its diverse components (the pergola for social relations; the labels describing the plants for knowledge needs; the free access to taking care of the plants for the desire for manual activities). Some weaknesses (Supplementary Figure 2) 2. To respond to this need, participants propose to add information plaques about the variety of plants in the study area (“will there be a card for each plant? -...someone entering the garden wants to ﬁnd out new information”) 3. The designed area is preﬁgured to respond to the need for doing manual work on the greenery (“I imagine myself going there to do manual activities, such as using the mower”). This expectation is not speciﬁcally linked to any of the design elements presented are mentioned, speciﬁcally regarding the comfort experienced under the pergola for temperature (shade to protect from the sun) and bad weather (wind, rain). The elements classiﬁed under the fascination factor are all positively evaluated: the contact with ﬂowers, fruits, animals, and water is recognised as valuable. The most debated negative aspects regard health issues (possible pollution of the soil connected to the fruit trees), the presence of undesired animals (pigeons and mosquitos), and the lack of more water elements (the drinking fountain is considered positive but not suﬃcient). Being away is appreciated by the participants, and no downsides are mentioned. The factor of extent did not emerge during the ﬁrst round, yet it was included in the design proposal according to the design criteria; interestingly, the reactions to the design project elicited some comments about the increased coherence of the area. Tables 2–5, separately for each ART factor and consistently with the contents that emerged from the ﬁrst round of the focus groups. The project was represented through tables and realistic renderings that were as similar as possible to the actual desired result for clear and legible communication. Frontiers in Psychology \| www.frontiersin.org TABLE 2 \| Comparison of the needs and reactions of the participants to the design proposal [attention restoration theory (ART), compatibility]. The garden does not propose speciﬁc activities, such as horticultural ones, as it is designed as a restorative garden where there are no prescribed therapeutic or occupational activities Designed elements highlighted in second-round focus groups 1. The pergola (Supplementary Figure 1) is recognised as the design element most suitable to satisfy socialisation needs. The participants show some doubts about its comfort. In this regard, some participants propose to implement the pergola through some form of covering, to allow people to use it even during less favourable weather conditions, such as wind or rain. Another potential weakness in this area is an adequate shading (“the trees that you will plant are already big, right?”). Participants seem to appreciate also the design philosophy introduced to them, about designing a place open to everyone (“This is the spirit… design for all”). They also highlight as a strength the design solutions intended to favour an inter-generational use of the space (Supplementary Figure 2) Design Proposal They also highlight as a strength the design solutions intended to favour an inter-generational use of the space (Supplementary Figure 2) 2. To respond to this need, participants propose to add information plaques about the variety of plants in the study area (“will there be a card for each plant? -...someone entering the garden wants to ﬁnd out new information”) 3. The designed area is preﬁgured to respond to the need for doing manual work on the greenery (“I imagine myself going there to do manual activities, such as using the mower”). This expectation is not speciﬁcally linked to any of the design elements presented TABLE 2 \| Comparison of the needs and reactions of the participants to the design proposal [attention restoration theory (ART), compatibility]. ART dimension: compatibility TABLE 2 \| Comparison of the needs and reactions of the participants to the design proposal [attention restoration theory (ART), compatibility]. ART dimension: compatibility Designed elements highlighted in second-round focus groups The Image of the Community Garden in the Light of the Introduction of the Study Area According to the Design Project Being in contact with fruits (“it would be nice to also have spontaneous fruit trees”) 3. Being in contact with animals [“if older people, who have a dog or a cat, found small animals in the garden (...) for those who have not lived in the city but have lived in the countryside it is like returning to their roots”] 4. Being in contact with aquatic elements (“it would be nice to have a small body of water”) 4. Being in contact with aquatic elements (“it would be nice to have a small body of water”) 4. Being in contact with aquatic elements (“it would be nice to have a small body of water”) Design solutions 1. The design included large areas of wildﬂowers along the main path, a sensory garden with numerous brightly coloured blooms, two raised ﬂower beds with edible and cut ﬂowers and several hedges of ornamental shrubs that seasonally and gradually produce abundant blooms 1. The design included large areas of wildﬂowers along the main path, a sensory garden with numerous brightly coloured blooms, two raised ﬂower beds with edible and cut ﬂowers and several hedges of ornamental shrubs that seasonally and gradually produce abundant blooms 2. The project included an orchard, along the main path, a row of mulberry trees and a naturalistic hedge with native shrubs that produce wild fruits, for birds and small mammals, but also edible for people 3. Many design elements serve as habitats and food reserves for mammals, pollinators, and beneﬁcial insects. The project included a bird garden with bird feeders and a hedge of mixed native berry-producing shrubs, beneﬁcial to birds 4. The project included a small, clearly visible, and accessible coloured water fountain. For technical and maintenance reasons it was not possible to insert a pond or a water basin 4. The project included a small, clearly visible, and accessible coloured water fountain. For technical and maintenance reasons it was not possible to insert a pond or a water basin Designed elementshighlighted in second-round focus groups 1. The design project of the study area includes an area dedicated to ﬂowers (Supplementary Figure 3). This element is considered an important and positive element responding to the need of the potential users (“It is beautiful… my soul could be blessed with all that beauty, all those perfumes...”). The Image of the Community Garden in the Light of the Introduction of the Study Area According to the Design Project On the other hand, the participants underlined the CG location as a negative element; it seems to be far away from frequented paths of their daily lives. ﬂowering, the autumn with its colours, winter, and every season;” “I also would go there to get out from the community: even if socialisation is important, solitude for me means a sense of liberation;” “what attracts the most is that it’s like seeing a piece of the countryside in an urban area”). On the other hand, the participants underlined the CG location as a negative element; it seems to be far away from frequented paths of their daily lives. The Image of the Community Garden in the Light of the Introduction of the Study Area According to the Design Project In this regard, participants propose to increase the close contact with ﬂowers, offering the possibility of being surrounded by them (“I would like to lie down in the middle of this meadow in bloom”) 2. The orchard area (Supplementary Figure 4) is very appreciated (“It’s very important for me to have fruit trees… it represents even more clearly the fruitful interaction between nature and human being”; “stay in an orchard means to be in a living space)”. Some participants underline concerns about the salubrity of the ground (“it was an industrial area… would it be safe to eat fruits grown there?”) 3. The presence of elements such as the bird garden (Supplementary Figure 5) and the Benje’s hedge (Supplementary Figure 6), ﬁnalised to attract and protect the local animals, has been underlined as a strength. Yet, there are concerns about these same elements. Respectively, participants point out how the bird feeders may also attract pigeons (“I like this project element, as long as the pigeons do not come”) and they seem worried about the time needed for the Benje’s hedge to respond to its function (“How long do we have to wait… years?”). In general, the presence of mosquitoes also emerges as a potential weakness, so the introduction of bat houses is proposed as a mitigation action 4. Even if the presence in the project of a drinking fountain (Supplementary Figure 7) is recognised as positive (“The pink drinking water seems cool!!”), the lack of additional water elements is seen as a disadvantage (“For example, in the botanical garden there is a pool of water; water is important for children and people.”; “there was a pond here... are there frogs?”) interaction with nature (“Our experience in the new area will be much less interactive; we will be afraid to take a ﬂower. In our area, we have an interaction, because we feel it belongs to us,” said by a participant involved in local associations). ﬂowering, the autumn with its colours, winter, and every season;” “I also would go there to get out from the community: even if socialisation is important, solitude for me means a sense of liberation;” “what attracts the most is that it’s like seeing a piece of the countryside in an urban area”). Frontiers in Psychology \| www.frontiersin.org Designed elementshighlighted in second-round focus groups The Image of the Community Garden in the Light of the Introduction of the Study Area According to the Design Project This element is considered an important and positive element responding to the need of the potential users (“It is beautiful… my soul could be blessed with all that beauty, all those perfumes...”). In this regard, participants propose to increase the close contact with ﬂowers, offering the possibility of being surrounded by them (“I would like to lie down in the middle of this meadow in bloom”) 2. The orchard area (Supplementary Figure 4) is very appreciated (“It’s very important for me to have fruit trees… it represents even more clearly the fruitful interaction between nature and human being”; “stay in an orchard means to be in a living space)”. Some participants underline concerns about the salubrity of the ground (“it was an industrial area… would it be safe to eat fruits grown there?”) 3. The presence of elements such as the bird garden (Supplementary Figure 5) and the Benje’s hedge (Supplementary Figure 6), ﬁnalised to attract and protect the local animals, has been underlined as a strength. Yet, there are concerns about these same elements. Respectively, participants point out how the bird feeders may also attract pigeons (“I like this project element, as long as the pigeons do not come”) and they seem worried about the time needed for the Benje’s hedge to respond to its function (“How long do we have to wait… years?”). In general, the presence of mosquitoes also emerges as a potential weakness, so the introduction of bat houses is proposed as a mitigation action 4. Even if the presence in the project of a drinking fountain (Supplementary Figure 7) is recognised as positive (“The pink drinking water seems cool!!”), the lack of additional water elements is seen as a disadvantage (“For example, in the botanical garden there is a pool of water; water is important for children and people.”; “there was a pond here... are there frogs?”) ﬂowering the autumn with its colours winter and every season;” interaction with nature (“Our experience in the new area will be 1. Being in contact with ﬂowers (“It would be the great to have pathways through the ﬂowers”) 1. Being in contact with ﬂowers ( It would be the great to have pathways through the ﬂowers ) 2. Being in contact with fruits (“it would be nice to also have spontaneous fruit trees”) 2. The Image of the Community Garden in the Light of the Introduction of the Study Area According to the Design Project A further goal of the second-round focus groups was to understand if and how the introduction of the designed study area inside the CG changed its image. The positive elements that prevail in deﬁning the identity of the CG, in general, are the possibility of living contemplative experiences of being away from everyday life and of ﬁnding real contact with natural elements (“For me, the CG would be to breathe and admire the September 2021 \| Volume 12 \| Article 702525 11 Nature Experiences of Older People Bofﬁet al. TABLE 3 \| Comparison of the needs and reactions of the participants to the design proposal (ART, fascination). TABLE 3 \| Comparison of the needs and reactions of the participants to the design proposal (ART, fascination). ART dimension: fascination Needs emerged in ﬁrst-round focus groups 1. Being in contact with ﬂowers (“It would be the great to have pathways through the ﬂowers”) 2. Being in contact with fruits (“it would be nice to also have spontaneous fruit trees”) 3. Being in contact with animals [“if older people, who have a dog or a cat, found small animals in the garden (...) for those who have not lived in the city but have lived in the countryside it is like returning to their roots”] 4. Being in contact with aquatic elements (“it would be nice to have a small body of water”) Design solutions 1. The design included large areas of wildﬂowers along the main path, a sensory garden with numerous brightly coloured blooms, two raised ﬂower beds with edible and cut ﬂowers and several hedges of ornamental shrubs that seasonally and gradually produce abundant blooms 2. The project included an orchard, along the main path, a row of mulberry trees and a naturalistic hedge with native shrubs that produce wild fruits, for birds and small mammals, but also edible for people 3. Many design elements serve as habitats and food reserves for mammals, pollinators, and beneﬁcial insects. The project included a bird garden with bird feeders and a hedge of mixed native berry-producing shrubs, beneﬁcial to birds 4. The project included a small, clearly visible, and accessible coloured water fountain. For technical and maintenance reasons it was not possible to insert a pond or a water basin Designed elementshighlighted in second-round focus groups 1. The design project of the study area includes an area dedicated to ﬂowers (Supplementary Figure 3). No needs emerged The width of the area and the presence of adult trees within the CG are, at the design level, an important starting point to enhance the “extent”factor. Every area is physically and visually connected; two different paths allow to explore the whole garden easily and safely; the layout of the area is legible and the message of what can be done and where is clear. The vegetation and architectural elements that build the restorative setting contribute to the functioning of the place and to the feeling of being immersed in coherent and unique environment Designed elementshighlighted in second-round focus groups Designed elementshighlighted in second-round focus groups During the second-round this dimension of ART is deﬁned more precisely. The designed study area is perceived to have internal coherence and to be suitable for orientation between the different designed zone. In particular, the presence of a double path is appreciated by the preﬁgured users. Yet, some mobility concerns are expressed about the accessibility of the main path through the garden for older people using walkers or wheelchairs the needs of compatibility and fascination, whereas being away and extent are less relevant for citizens critically observing the design proposal, which is consistent with their expectations of experiences in nature. A key feature of the method is the possibility of linking such factors to speciﬁc design elements included in the design project by assigning physical elements to the attributional categories used for the analysis; hence, the components of the designed study area in the CG are categorised through the four dimensions of ART, relying on perceptions of the participants. The integration of articulated data allowed us to add observations on speciﬁc functional aspects that, even if less relevant in terms of restoration, can be considered for the comfort of prospective users. Another distinctive characteristic of the method is that it places the speciﬁc study area into a wider context, oﬀering a representation of the image of the neighbourhood and the CG. This oﬀers some further elements for the development of the design project, which can either aﬀect its physical features in the design phase or inform the guidelines for the eﬀective usage and maintenance of the site in the management phase (Niemelä, 1999; Cadenasso and Pickett, 2008; Philips, 2013). The method is conceived as a practical tool to apply with a multidisciplinary team when designing green areas. ART dimension: extent Needs emerged in ﬁrst-round focus groups Design solutions ART dimension: being away Needs emerged in ﬁrst-round focus groups Design solutions Being away from everyday life [“A place for reﬂecting, stopping and contemplating (...) Even being alone just for an hour would be nice to me”] The ﬁrst design action was to identify the major disturbing elements in the surrounding urban contextand reduce their impact. Vegetal structures have been introduced to mitigate or screen the neighbouring buildings, enhancing more pleasant views. The second step was to design an interesting and pleasant setting (lush and various vegetation), inviting to interaction (sensory garden, bird garden, natural playground etc,) and to exploration (more wild areas), accessible to all kind of users (a main path in permeable paving and an “explorative” secondary one on mown grass), where it is possible to do different activities (multifunctional) Designed elementshighlighted in second-round focus groups The designed area is appreciated for its ability to respond to this type of need (“it’s like an oasis, you feel far away from the city; I would even go there alone, for contemplation”). Coherently all the participants mention as a positive aspect the introduction of a mitigation area designed to hide the presence of the adjacent nursing home (“It is essential to cover that building... It is a punch in the eye...”) TABLE 5 \| Comparison of the needs and reactions of the participants to the design proposal (ART, extent). DISCUSSION AND CONCLUSION The current study illustrates a case study for the application of a co-design method, which employs biophilia principles, and in which the impact on the participants is assessed using the four dimensions of ART. The method shows that the biophilic design project presented to the citizens elicited an experience of the natural environment that is consistent with the needs collected in the ﬁrst phase. Thanks to the semi- structured schedule and the deductive thematic analysis, it is possible to describe how the design project answers mainly to In addition to these general points, the adherence between the image of the CG and the designed study area has been the object of further examination, mentioning what appears to be a general reduction of extent. In this regard, the study area is perceived to be so well-designed that it could make the other areas look a bit worse (“now, you have to ﬁx the rest!;” “it’s a more fancy area, the rest is very wild...”). Another diﬀerence that made the new identity of the CG less homogeneous than before is that the study area appeared to some to be less inviting to an active September 2021 \| Volume 12 \| Article 702525 Frontiers in Psychology \| www.frontiersin.org 12 Nature Experiences of Older People Bofﬁet al. TABLE 4 \| Comparison of the needs and reactions of the participants to the design proposal (ART, being away). September 2021 \| Volume 12 \| Article 702525 No needs emerged In psychological terms, it is a rather well-established construct composed of multiple aspects, which are traditionally separated at least in two dimensions regarding hedonic pleasure and eudaimonic self-realisation (Ryan and Deci, 2001). Like restoration, well- being can be conceived as an individual measure that can be assessed and compared with other indicators, both objective and subjective. Indeed, when we think of the role of the social sciences in informing decisions aﬀecting the transformation of the physical environment, having data on psychological assessment oﬀers a clear advantage; “city planners may face choices between economic growth and increased air pollution. Certain political ideologies call for an emphasis on economics, whereas other ideologies call for an emphasis on other factors such as equality or environmental conservation. How can a policy maker weigh alternatives in a systematic way and move beyond a total reliance on intuition and ideology? Broad measures of well-being can provide a valuable source of information because they can reveal how well-being of people is aﬀected by the various policies” (Diener et al., 2009, p. 54–55). However, it is important not to misinterpret such an approach as the idea of policies simply maximising well-being (Stutzer, 2020) or any other psychological construct, including restoration. Such a technocratic approach would be detrimental for an eﬀective participatory approach, consequently diminishing the importance of developmental factors favouring well-being (Prilleltensky et al., 2001; Boﬃ et al., 2016) and neglecting the way institutions and inclusive procedures increase the value of procedural utility, which is a source of subjective well-being (Stutzer and Frey, 2006). Consistently, with this approach, an eudaimonic perspective on well-being would allow assessments, informing the design of places that are not only restorative from a cognitive point of view but also oﬀering the possibility to reach meaningful objectives for the growth and realisation of an individual (Rainisio et al., 2015; Boﬃand Rainisio, 2017). Th hi d l l h i d d f h users, including the guests of nursing homes. The issue of active ageing is a crucial aspect informing the development of the design proposal. With this perspective, two main classes of users are targeted; older people, both from the general population and from the nursing homes and who are the primary users, and their related family members of diﬀerent generations (siblings, children, and grandchildren) and health assistants, who are secondary users. No needs emerged Notwithstanding the heterogeneous population, the central issue of active ageing contributes to the narrowing of the design goals. This aspect is also emphasised by the relatively small dimensions of the area. It is necessary to test the method in more varied contexts and in bigger areas, where the needs can diﬀer to a larger extent and the identiﬁcation of a main design goal is more challenging. In this case, it would be possible to understand how the method helps in such a process. Despite such limitations, we argue that the method oﬀers some useful elements for a multidisciplinary approach to co-design on three diﬀerent levels. The ﬁrst level regards the importance of human-centred design (Bazzano et al., 2017), which places the experience of an individual at the centre of the design practise and emphasises the importance of empirical data. This helps professionals keep their focus on the needs of people, relying not on general assumptions but on contextual data from actual users and integrating environmental aspects with psychosocial factors. As discussed, this is a key aspect that can reinforce the interaction between the social sciences and design sciences by creating a common ground. It is crucial to remember that, according to the methodology proposed, involving the citizens does not mean assigning them the role of analysing the current context or designing prospective solutions. They are, instead, invited to share personal behaviours, attitudes, and beliefs that reﬂect the personal meaning they assign to the stimuli oﬀered for discussion. The second level is related to the choice of the speciﬁc theoretical model to describe the experience of an individual that, in this case, is represented by ART. It proved to be a fruitful grid for interpreting the experiences of participants and provided insights and feedback for the designer, also because such a model is eﬀectively rooted in both disciplines (see Fumagalli et al., 2020 for more details of this analysis). All the contents included in the deductive thematic analysis ﬁt with the ART model, even though the theoretical category of extent did not include any content during the ﬁrst round. This notwithstanding, the main advantage of such approach is in oﬀering a categorisation of both subjective experiences and design elements linked to a well-established theoretical model, which facilitated comparisons across diﬀerent project sites and design teams. Relying on pre-deﬁned categories reduces the inﬂuence of idiosyncratic interpretations. No needs emerged Our results have some limitations that require reﬂection when considering the applicability of the method. In particular, as the eﬃcacy of the method is evaluated on a single case study, some aspects are worth mentioning. In the ﬁrst place, the landscape designer involved in the project is part of the research team and has a background in biophilic design. Such conditions are both desirable in our conception of a multidisciplinary approach to co-designs, but we cannot consider them as the standard circumstances observed in the professional ﬁeld. As a consequence, the capability of the average practitioner to design a garden following biophilia principles can vary, and this can signiﬁcantly alter the design outcome that is then tested during the second round of the focus groups. In addition, the interpretation of textual feedback categorised through the four dimensions of ART and their usage to ﬁne-tune the design project is part of a broader professional skill; such “ambiance empathy” allows the eﬀective merging of environmental features and human experiential factors and is also signiﬁcantly diﬀerent depending on the background of the practitioners (Piga, 2021). Hence, it is also desirable to apply the method with a broader and more heterogeneous sample of landscape designers to appreciate its actual impact. In the second place, the current design project concerns a community garden with a speciﬁc focus on older September 2021 \| Volume 12 \| Article 702525 Frontiers in Psychology \| www.frontiersin.org 13 Nature Experiences of Older People Bofﬁet al. attributional analysis. A useful alternative in such a perspective is, hence, the stress recovery theory (SRT) (Ulrich et al., 1991). However, another psychological construct less examined by biophilic design literature that can be usefully included in such methodology is well-being. Interestingly, whereas the construct of restoration is strongly integrated into design research and practise, the concept of well-being is often mentioned as a relevant goal for design and active ageing but seldom by referring to a speciﬁc construct (e.g., see Bazzano et al., 2017; Istat, 2020). As many disciplines deﬁne such notions in diﬀerent ways, referring to both objective indicators and subjective measures, the diﬀusion of speciﬁc psychological constructs is largely reduced in comparison to other ﬁelds of research. Often, “well-being” is most easily understood in terms of comfort when referring to urban planning and design. Frontiers in Psychology \| www.frontiersin.org ACKNOWLEDGMENTS The authors are grateful to the staﬀand managers of nursing homes Anni azzurri for their collaboration; they thank the nursing home guests, community garden members, garden visitors, and all other people involved in the focus group activities. The ﬁrst draft of this paper was presented at the symposium Greening cities shaping cities on October 12 and 13, 2020, at the Polytechnic of Milan; for more information visit www.greeningcities-shapingcities.polimi.it. ETHICS STATEMENT number of participants; a pre-deﬁned set of items that may not best ﬁt the speciﬁc context; less space for emerging data), which must be accurately taken into account when designing an engagement process that is psychologically sustainable for citizens (Boﬃet al., 2015). Our attempt was, instead, to develop a qualitative tool that allowed us to collect two types of data at the same time. “Articulated data” concern the explicit aspects investigated during the focus group discussion (e.g., landmarks and distinctive features of the Ortica district, which emerged during the focus group), which are self-evident in their content but must be organised in fruitful, bottom-up categories during the analysis depending on the exploratory goals. “Attributional data” are, instead, strictly linked to a speciﬁc theoretical model, and their structure is not manifested to the participants but is applied by the researcher (e.g., the four ART categories presented about the desired experiences in nature). The combination of these two categories oﬀers a wide array of content that can simultaneously inform the designers from diﬀerent perspectives. The studies involving human participants were reviewed and approved by Ethics Committee of University of Milan. The patients/participants provided their written informed consent to participate in this study. SUPPLEMENTARY MATERIAL The datasets presented in this article are not readily available because data include an audio recording of the focus groups that would allow the identiﬁcation of participants. Requests to access the datasets should be directed to Marco Boﬃ, marco.boﬃ@unimi.it. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg. 2021.702525/full#supplementary-material No needs emerged Hence, having a proper theoretical model as a reference allows teams to make speciﬁc hypotheses and test them with qualitative and quantitative data using already established methods, which is a remarkable advantage compared with using an inductive thematic analysis. Other valuable options consistent with the biophilic approach are available in psychological literature. The most investigated construct linking psychological and design research is restoration, which, in the ﬁrst place, guided our choice for the theoretical framework to structure the The third level concerns the instrument adopted for the process. Existing measurement tools for ART are mainly quantitative (e.g., Hartig et al., 1997; Pasini and Berto, 2007; Pasini et al., 2014), which implies the administration of scales to the participants involved in the process. Such an approach has both positive aspects (e.g., brief administration, if not associated with other scales; quantitative data; a more easily replicable data collection) and downsides (e.g., the need for a higher September 2021 \| Volume 12 \| Article 702525 14 Bofﬁet al. Nature Experiences of Older People FUNDING In our view, the applied methodology is not meant to be an automatic process for directly obtaining ﬁnal design solutions. It is, instead, conceived as a transcultural and trans-environmental approach to serve as a logical framework, the speciﬁc contents of which are meant to be adapted according to the context, i.e., sociocultural variables, environmental aspects, and design goals. Further developments of the method should also include the following phase of a post-occupancy evaluation to assess the actual impact of the natural solutions on experiences of peoples. Maintaining a consistent theoretical approach and instrument choice would allow a complete monitoring process during all the design phases. This work was supported by Fondazione Cariplo (Ageing and Social Research: People, Places, and Relation, 2018). AUTHOR CONTRIBUTIONS NF, GS, PI, and MB: conceptualisation and methodology. MB and LP: formal analysis and data curation. EF, MB, and LP: investigation. MB, EF, and LP: writing—original draft preparation. NF and MB: writing—review and editing. EF: visualisation. 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https://openalex.org/W1974191288	https://europepmc.org/articles/pmc3037922?pdf=render	English	null	Incidence of bone metastases and skeletal-related events in breast cancer patients: A population-based cohort study in Denmark	BMC cancer	2,011	cc-by	5,311	RESEARCH ARTICLE Open Access © 2011 Jensen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Breast cancer (BrCa) is the most commonly diagnosed cancer among women in the industrialized world. More than half of women presenting with metastatic BrCa develop bone metastases. Bone metastases increase the risk of skeletal-related events (SREs), defined as pathological fractures, spinal cord compression, bone pain requiring palliative radiotherapy, and orthopaedic surgery. Both bone metastases and SREs are associated with unfavorable prognosis and greatly affect quality of life. Few epidemiological data exist on SREs after primary diagnosis of BrCa and subsequent bone metastasis. We therefore estimated the incidence of bone metastases and SREs in newly-diagnosed BrCa patients in Denmark from 1999 through 2007. Methods: We estimated the overall and annual incidence of bone metastases and SREs in newly-diagnosed breast cancer patients in Denmark from January 1, 1999 to December 31, 2007 using the Danish National Patient Registry (DNPR), which covers all Danish hospitals. We estimated the cumulative incidence of bone metastases and SREs and associated 95% confidence intervals (CI) using the Kaplan-Meier method. Results: Of the 35,912 BrCa patients, 178 (0.5%) presented with bone metastases at the time of primary breast cancer diagnosis, and of these, 77 (43.2%) developed an SRE during follow up. A total of 1,272 of 35,690 (3.6%) BrCa patients without bone metastases at diagnosis developed bone metastases during a median follow-up time of 3.4 years. Among these patients, 590 (46.4%) subsequently developed an SRE during a median follow-up time of 0.7 years. Incidence rates of bone metastases were highest the first year after the primary BrCa diagnosis, particularly among patients with advanced BrCa at diagnosis. Similarly, incidence rates of a first SRE was highest the first year after first diagnosis of a bone metastasis. Conclusions: The high incidence of SREs following the first year after first diagnosis of a bone metastasis underscores the need for early BrCa detection and research on effective treatments to delay the onset of SREs. Incidence of bone metastases and skeletal-related events in breast cancer patients: A population- based cohort study in Denmark Annette Ø Jensen1†, Jacob B Jacobsen1†, Mette Nørgaard1†, Mellissa Yong2†, Jon P Fryzek2†, Henrik T Sørensen1† Jensen et al. BMC Cancer 2011, 11:29 http://www.biomedcentral.com/1471-2407/11/29 Jensen et al. BMC Cancer 2011, 11:29 http://www.biomedcentral.com/1471-2407/11/29 Correspondence: myong@amgen.com † Contributed equally 2Center for Observational Research, Amgen Inc., One Amgen Center Drive, MS 24-2-A, Thousands Oaks CA 91320, USA Full list of author information is available at the end of the article Background diagnosis, will develop bone metastases [5]. Bone metas- tases in BrCa patients are dominated by osteolytic lesions, which increase the risk for skeletal-related events (SREs), defined as pathological fractures, spinal cord compression, bone pain requiring palliative radio- therapy, and orthopaedic surgery [6]. Breast cancer (BrCa) is one of the most commonly diag- nosed cancers among women in the industrialized world [1], accounting for 28% of all new cancer cases in women in Denmark in 2008 [2]. At BrCa diagnosis, approximately 5%-6% of women present with distant spread [3,4], with bone representing the most common site of metastatic lesions. More than half of women, who present with metastatic breast cancer at primary Published data on incidence rates of bone metastases and SREs after primary diagnosis of BrCa and subsequent bone metastasis are few. One Canadian study evaluated the pattern of metastastic disease in 180 patients with tri- ple-negative (i.e., estrogen receptor-negative, progester- one receptor-negative and HER2/neu-negative) BrCa compared with other subgroups of BrCa patients (N = 1,428). The risk of developing bone metastases within * Correspondence: myong@amgen.com † Contributed equally 2Center for Observational Research, Amgen Inc., One Amgen Center Drive, MS 24-2-A, Thousands Oaks CA 91320, USA Full list of author information is available at the end of the article Page 2 of 6 Jensen et al. BMC Cancer 2011, 11:29 http://www.biomedcentral.com/1471-2407/11/29 Jensen et al. BMC Cancer 2011, 11:29 http://www.biomedcentral.com/1471-2407/11/29 Jensen et al. BMC Cancer 2011, 11:29 http://www.biomedcentral.com/1471-2407/11/29 time of diagnosis was recorded as local, regional, or dis- tant metastases (i.e., summary staging) in the DCR. From January 1, 2004 information on stage was recorded according to the “TNM Classification of Malig- nant Tumors” cancer staging system. Conversion of the TNM classification system to the summary staging sys- tem is presented in Table 1. 10 years after diagnosis was 7%-9% for all subgroups [7]. Hortobagyi et al. evaluated the efficacy of bisphospho- nates in reducing skeletal complications in patients with BrCa and bone metastases in a clinical trial setting [8,9]. They found that the median time to the first SRE was 13.9 months among bisphosphonate-treated women and 7.0 months in the placebo group (P = 0.001) [9]. The need remains for general population data on the incidence of bone metastases and SREs among unse- lected BrCa patients. Such data would allow further understanding of the clinical course of BrCa and related health care demand. Skeletal-related events The DNPR was used to identify SREs after BrCa diagnosis, with surgical procedures coded according to the Danish version of the Nordic Classification of Surgical Procedures (NCSP). For each patient, we recorded the following SREs: 1) radiation to the bone (NCSP-code: BWGC1 in addition to a bone metastasis diagnosis), 2) pathological or osteo- porotic fractures in addition to a bone metastasis diagnosis (see Table 2 for codes), 3) bone surgery (NCSP-code: KNAGxx in addition to a bone metastasis diagnosis), and 4) spinal cord compression in addition to a bone metasta- sis diagnosis (see Table 2 for codes). Statistical analysis We computed the cumulative incidence (%) and inci- dence rates of bone metastases during follow up among Table 1 Translation of AJCC groupings into summary staging for breast cancer Stage TNM categories Localized T1-4, N0, M0 Regional T1-4, N1-3, M0 Distant T1-4, N1-3, M1 0 Tis, N0, M0 I T1, N0, M0 IIA T0, N1, M0 T1, N1, M0 T2, N0, M0 IIB T2, N1, M0 T3, N0, M0 IIIA T0, N2, M0 T1, N2, M0 T2, N2, M0 T3, N1, M0 T3, N2, M0 IIIB T4, N0, M0 T4, N1, M0 T4, N2, M0 IIIC Any T, N3, M0 IV Any T, Any N, M1 Table 1 Translation of AJCC groupings into summary staging for breast cancer Breast cancer patients We identified all patients in the Danish National Patient Registry (DNPR) with a first primary diagnosis of BrCa (i.e., recurrent or relapsed cases were not included) recorded between January 1, 1999 and December 31, 2007 (during this period, there was no formal mammography screening program in Denmark). The DNPR collects elec- tronic data on inpatient, outpatient and emergency room visits. For each hospitalization, DNPR files include dates of admission and discharge, any surgical procedure per- formed, and up to 20 discharge diagnoses. Since 1994, information has been coded according to the International Classification of Diseases, 10th revision (ICD-10) [11]. BrCa patients were identified using ICD-10 code C50.x. Code C79.5 was used to identify bone metastases. We included both inpatient and outpatient diagnoses. Methods We conducted this population-based cohort study in Denmark (population ~ 5.4 million inhabitants). The entire population receives tax-supported health care from the Danish National Health Service, with free access to hospital care. All BrCa patients receive care in specialised oncology centres within public hospitals operating under the auspices of the Danish National Health Service. Since 1968, the Danish Civil Registration System has kept up-to-date electronic records on date of birth, gender, change of address, date of emigration, and changes in vital status for all Danish residents.10 From the Central Office of Civil Registration, each resi- dent in Denmark is assigned a unique 10-digit civil registration number, which allows unambiguous linkage among all of Denmark’s population-based registries [10]. Follow up Patients were followed from the date of their BrCa diagno- sis until the development of bone metastases or/and SREs, emigration, death, or April 1, 2008, whichever came first. Background We therefore estimated the inci- dence of bone metastases and SREs using a large popu- lation-based cohort of newly-diagnosed BrCA patients in Denmark from 1990 to 2007. Stage at diagnosis Information on stage at BrCa diagnosis was obtained from the Danish Cancer Registry (DCR) until December 31, 2007. The extent of spread of the tumour at the Page 3 of 6 Page 3 of 6 Jensen et al. BMC Cancer 2011, 11:29 http://www.biomedcentral.com/1471-2407/11/29 Table 2 Translation of fracture categories into discharge diagnoses in ICD-10 Fracture category ICD-10 codes: Fractures of the vertebrae, ribs and pelvis, femur and distal forearm M80.0, M84.4, M90.7, S12.0-12.9, S22.0, S22.1, S32.0-S32.8, S72.0- S72.9, S52.5-S.52.6 Spinal cord compression M43.9, M48.5, M54.5, M54.6, M54.9, G95.2, G95.8 Table 2 Translation of fracture categories into discharge diagnoses in ICD-10 Incidence of bone metastases and SREs Incidence of bone metastases and SREs Figure 2 shows the cumulative incidence of bone metas- tases among BrCa patients during follow up. The stee- pest increase appears in the first year after the primary diagnosis of BrCa [1-year cumulative incidence = 1.9% (95% CI: 1.7%-2.0%)], with the highest incidence observed among patients with an advanced stage at pri- mary diagnosis (Figure 3). Figure 4 shows the cumula- tive incidence of SREs among BrCa patients with bone metastases during follow up. The steepest increase in SREs is seen the first year after bone metastasis diagno- sis [1-year cumulative incidence = 38.5% (95% CI: 36.0- 41.0%)], with no difference by primary stage at BrCa diagnosis (data not shown). We found no difference in incidence of bone metastases and SREs by age at BrCa diagnosis (data not shown). patients diagnosed with BrCa, treating death as a com- peting risk [12], and plotted these estimates as a func- tion of time since BrCa diagnosis. Similarly, we calculated the cumulative incidence and incidence rates of SREs among BrCa patients diagnosed with bone metastases and plotted these estimates as a function of time since bone metastasis diagnosis. Table 3 presents the incidence rates of bone metas- tases among all BrCa patients, overall and by stage, from the first year following primary BrCa diagnosis up to five years afterwards. It also presents the incidence rates of SREs among BrCa patients with bone metastases from the first year following primary diagnosis of bone metastases up to five years afterwards. A total of 1,494 (4% of 35,912) BrCa patients were diagnosed with bone metastases either at the time of BrCa diagnosis or dur- ing follow up. Stage at diagnosis The incidence rate of bone metastases was highest the first year after the primary diagnosis of BrCa [incidence rate (IR) = 13.6 (95% CI: 12.4-14.9)] per 1,000 person-years (PY). This rate declined to 8.8 (95% CI: 8.2-9.5) per 1,000 PY from the second to the fifth year after the primary BrCa diagnosis. A total of 712 (47.6% of 1,494) BrCa patients with bone metastases were diagnosed with an SRE either at the time of pri- mary BrCa diagnosis or during follow up. Similar to the incidence of bone metastases after the primary BrCa diagnosis, the incidence rate of SREs was highest the first year after the primary diagnosis of bone metastases To evaluate age at BrCa diagnosis as a risk factor for bone metastases, we stratified the analyses according to the following age groups: =60 years, 60-69 years, and 70+ years. We also stratified incidence of bone metastases and SREs by summary stage at BrCa diagnosis in order to evaluate the impact of BrCa stage on these outcomes. This study was approved by the Danish Protection Agency (Record no. 2006-41-6387). The statistical soft- ware SAS, version 9.2 (SAS Institute Inc., Cary, NC), was used for all statistical analyses. Patient demographic characteristics BMC Cancer 2011, 11:29 Page 4 of 6 1-year incidence of bone metastasis (95% CI) 3-year incidence of bone metastasis (95% CI) 5-year incidence of bone metastasis (95% CI) 1.9 % (1.7 % - 2.0 %) 3.4 % (3.2 % - 3.6 %) 4.7 % (4.4 % - 4.9 %) Figure 2 Cumulative incidence of bone metastases among breast cancer patients (N = 35,912), Denmark, 1999-2007. 1- year incidence of SREs (95% CI) 3-year incidence of SREs (95% CI) 5-year incidence of SREs (95% CI) 38.5% (36.0% - 41.0%) 48.1% (45.4% - 50.8%) 51.7% (48.9% - 54.4%) Figure 4 Cumulative incidence of skeletal-related events among breast cancer patients with bone metastases (N = 1,272), Denmark, 1999-2007. 1-year incidence of bone metastasis (95% CI) 3-year incidence of bone metastasis (95% CI) 5-year incidence of bone metastasis (95% CI) 1.9 % (1.7 % - 2.0 %) 3.4 % (3.2 % - 3.6 %) 4.7 % (4.4 % - 4.9 %) Figure 2 Cumulative incidence of bone metastases among breast cancer patients (N = 35,912), Denmark, 1999-2007. 1- year incidence of SREs (95% CI) 3-year incidence of SREs (95% CI) 5-year incidence of SREs (95% CI) 38.5% (36.0% - 41.0%) 48.1% (45.4% - 50.8%) 51.7% (48.9% - 54.4%) Figure 4 Cumulative incidence of skeletal-related events among breast cancer patients with bone metastases (N = 1,272), Denmark, 1999-2007. 1-year incidence of bone metastasis (95% CI) 3-year incidence of bone metastasis (95% CI) 5-year incidence of bone metastasis (95% CI) 1.9 % (1.7 % - 2.0 %) 3.4 % (3.2 % - 3.6 %) 4.7 % (4.4 % - 4.9 %) Figure 2 Cumulative incidence of bone metastases among breast cancer patients (N = 35,912), Denmark, 1999-2007. 1- year incidence of SREs (95% CI) 3-year incidence of SREs (95% CI) 5-year incidence of SREs (95% CI) 38.5% (36.0% - 41.0%) 48.1% (45.4% - 50.8%) 51.7% (48.9% - 54.4%) Figure 4 Cumulative incidence of skeletal-related events among breast cancer patients with bone metastases (N = 1,272), Denmark, 1999-2007. Figure 2 Cumulative incidence of bone metastases among breast cancer patients (N = 35,912), Denmark, 1999-2007. Figure 4 Cumulative incidence of skeletal-related events among breast cancer patients with bone metastases (N = 1,272), Denmark, 1999-2007. [IR = 759.2 (95% CI: 662.0-870.5) per 1,000 PY]. This rate declined to 513.8 (95% CI: 465.4-567.3) per 1,000 PY from the second to the fifth year after diagnosis of bone metastases. Patient demographic characteristics the five-year incidence rate of bone metastases among BrCa patients to be 10 per 1,000 PY; the corresponding incidence rate of SREs was 578 per 1,000 PY. The inci- dence rate of bone metastases was highest the first year after primary diagnosis of BrCa and higher if the BrCa patient was diagnosed at a more advanced stage. The incidence rate of SREs was highest the first year after diagnosis of bone metastases and showed no differences across stage at BrCa diagnosis. The incidence of bone metastases was higher after the first year if the BrCa patient was diagnosed at a more advanced stage: localized spread: IR = 3.3 (95% CI: 2.5- 4.4) per 1,000 PY; regional spread: IR = 12.9 (95% CI: 11.1-15.1) per 1,000 PY, and metastatic spread: IR = 172.5 (95% CI: 147.9-201.4) per 1,000 PY (Table 3). However, there were no differences in the rates of SREs subsequent to bone metastases by stage of disease at BrCa diagnosis (Table 3). The lack of a formal mammography screening program in Denmark during the study period may explain the relatively high prevalence of BrCa patients with an advanced stage of BrCa at primary diagnosis. Since popu- lation-based mammography screening is an accurate tool for early BrCa detection [13], we would expect incidence rates of bone metastases to be lower in countries offering this screening. This was the case in a German observa- tional study that evaluated BrCa incidence rates and tumor characteristics (i.e., the distribution of invasive tumor size) before and after implementation of mammo- graphy screening [13]. The incidence rate of BrCa before implementation of the screening programme was 297.9 per 100,000 person-years. During the implementation of screening, this rate rose to 532.9 per 100,000 person- years. Of the 349 cancers detected with screening, 76% were invasive, compared to 90% of cases not detected with screening during the same period. Furthermore, there was a difference in nodal status between cancers detected with and without the screening program [13]. Patient demographic characteristics g p We identified 35,912 patients with a first primary BrCa in Denmark from 1999 through 2007. The median length of follow up was 3.5 years (range, 0-9.2 years). The median age at BrCa diagnosis was 62.3 years (range, 18-104 years). Among those who developed bone metastases, the median age at BrCa diagnosis was 62.2 years (range, 25-95 years), and among those with bone metastases who developed an SRE, the median age at BrCa diagnosis was 67.4 years (range, 25-95 years). A flowchart of the BrCa study cohort according to pre- sence of bone metastases and subsequent occurrence of SREs is presented in Figure 1. In the subset of BrCa patients (1999 through 2006) for whom we had informa- tion on primary stage of BrCa at diagnosis (n = 31,761), 13,515 (43%) patients presented with localized disease, 12,452 (39%) presented with regional disease, 1,557 (5%) presented with metastases, and 4,237 (13%) patients had missing stage data (data not shown). Patients diagnosed with breast cancer N= 35,912 Median age=62.3 years No bone metastases n=35,690 (99.4%) Median age=62.2 years Bone metastases present n= 176 (0.5%) Median age=68.7 years Bone metastases and SRE present n=46 (0.1%) Median age=67.4 years Developed bone metastases n=1,272 Median age=62.6 years Developed SREs n=76 Median age=67.3 years Developed SREs n=590 Median age=63.4 years Figure 1 Flowchart of the breast cancer study cohort (N = 35,912), according to presence of bone metastases and subsequent occurrence of skeletal-related events (SREs): Denmark, 1999-2007. The distribution of first-recorded SRE type among BrCa patients who developed an SRE (n = 712 patients) during the follow-up period was as follows: 394 (55%) had radiation to the bone, 133 (19%) had a pathological or osteoporotic fracture, 42 (6%) had bone surgery, and 143 (20%) had spinal cord compression as their first- recorded SRE type. Figure 1 Flowchart of the breast cancer study cohort (N = 35,912), according to presence of bone metastases and subsequent occurrence of skeletal-related events (SREs): Denmark, 1999-2007. Figure 1 Flowchart of the breast cancer study cohort (N = 35,912), according to presence of bone metastases and subsequent occurrence of skeletal-related events (SREs): Denmark, 1999-2007. Jensen et al. BMC Cancer 2011, 11:29 http://www.biomedcentral.com/1471-2407/11/29 Jensen et al. Discussion In this large population-based study conducted within a well-defined Northern European population, we found Stage 1-year incidence of bone metastasis (95% CI) 3-year incidence of bone metastasis (95% CI) 5-year incidence of bone metastasis (95% CI) Localized 0.4% (0.3% - 0.5%) 1.1% (0.9% - 1.3%) 1.8% (1.5% - 2.0%) Regional 1.3% (1.1% - 1.5%) 3.8% (3.5% - 4.1%) 5.7% (5.3% - 6.2%) Metastasis 18.6% (16.7% - 20.5%) 21.7% (19.6% - 23.8%) 23.7% (21.5% - 26.0%) Figure 3 Cumulative incidence of bone metastases among breast cancer patients (N = 35,912) by stage of disease at diagnosis, Denmark, 1999-2007. Stage 1-year incidence of bone metastasis (95% CI) 3-year incidence of bone metastasis (95% CI) 5-year incidence of bone metastasis (95% CI) Localized 0.4% (0.3% - 0.5%) 1.1% (0.9% - 1.3%) 1.8% (1.5% - 2.0%) Regional 1.3% (1.1% - 1.5%) 3.8% (3.5% - 4.1%) 5.7% (5.3% - 6.2%) Metastasis 18.6% (16.7% - 20.5%) 21.7% (19.6% - 23.8%) 23.7% (21.5% - 26.0%) Breast cancer patients have predominantly osteolytic bone metastases, which is characterised by increased bone degradation resulting from enhanced osteoclastic activity [14]. This activity in the bone causes pain, which is consistent with the most frequent SRE being ‘palliative radiation to the bone’ in our study. Bisphosphonates are Figure 3 Cumulative incidence of bone metastases among breast cancer patients (N = 35,912) by stage of disease at diagnosis, Denmark, 1999-2007. Figure 3 Cumulative incidence of bone metastases among breast cancer patients (N = 35,912) by stage of disease at diagnosis, Denmark, 1999-2007. Jensen et al. Discussion BMC Cancer 2011, 11:29 http://www.biomedcentral.com/1471-2407/11/29 Page 5 of 6 Page 5 of 6 Table 3 Incidence rates of bone metastases among breast cancer patients, and incidence rates of skeletal-related events (SREs) among breast cancer patients with bone metastases, from the first year after primary diagnosis of breast cancer and bone metastases, respectively, up to five years after diagnosis, Denmark, 1999-2007 Table 3 Incidence rates of bone metastases among breast cancer patients, and incidence rates of skeletal-related events (SREs) among breast cancer patients with bone metastases, from the first year after primary diagnosis of breast cancer and bone metastases, respectively, up to five years after diagnosis, Denmark, 1999-2007 events (SREs) among breast cancer patients with bone metastases, from the first year after primary diagnosis of breast cancer and bone metastases, respectively, up to five years after diagnosis, Denmark, 1999-2007 Event n Year 1 Year 2 - Year 5 Year 1 - Year 5 Per 1,000 person-years (95% CI) All patients Bone metastasis 1,272 13.6 (12.4 - 14.9) 8.8 (8.2 - 9.5) 10.2 (9.6 - 10.8) First SRE 712 759.2 (662.0 - 870.5) 513.8 (465.4 - 567.3) 578.0 (533.4 - 626.2) Localized Bone metastasis 237 3.3 (2.5 - 4.4) 3.9 (3.3 - 4.5) 3.7 (3.2 - 4.3) First SRE 117 503.1 (285.7 - 885.9) 574.7 (462.8 - 713.5) 564.4 (461.1 - 690.9) Regional Bone metastasis 663 12.9 (11.1 - 15.1) 13.5 (12.3 - 14.8) 13.3 (12.3 - 14.5) First SRE 310 774.6 (596.1 - 1006.5) 560.7 (490.7 - 640.6) 594.5 (527.8 - 669.5) Distant Bone metastasis 358 172.5 (147.9 - 201.4) 49.7 (38.8 - 63.6) 101.8 (89.3 - 116.0) First SRE 176 760.1 (620.9 - 930.4) 421.5 (329.9 - 538.5) 573.5 (490.7 - 670.2) risk of SREs in patients with bone metastases (i.e., bone metastases in combination with an SRE is more likely to be identified than bone metastases and no SRE). In addi- tion, we coded pathological and osteoporotic fractures under one code. This most likely overestimated the inci- dence of SREs among breast cancer patients because some of the fractures might have been osteoporotic. However, we do think that when a breast cancer patient is diagnosed with a bone metastasis and subsequently develops a fracture, it is most likely pathological. Thus, we anticipate a high proportion of fractures in this cate- gory of SREs as pathological. Discussion potent inhibitors of osteoclast-mediated bone resorption [15]; accordingly it has been shown that use of bispho- sphonates delay the time to an SRE among BrCa patients [9,16]. Data on bisphosphonate use has been recorded in the DNPR since 2002; however, the completeness of this registration is unknown. In our cohort of 35,912 breast cancer patients, only 1,090 (3%) had a record of bispho- sphonate use prior to a recorded bone metastasis diagno- sis, and an additional 245 women had a record of bisphosphonate use subsequent to a recorded bone metastasis diagnosis. Because the quality of data on bisphosphonate use is unknown in the DNPR, we did not include these data in our study. There are several potential explanations for the degree of under-coding of bone metastases. Diagnostic proce- dures used to screen for bone metastases in breast cancer patients may depend on the patient’s expected prognosis. For instance, if a patient’s overall status is deemed inap- propriate for radiation therapy or surgery (i.e., poor prog- nosis) then there may be little incentive to code bone metastases. Additionally, the numeric ICD-10 coding system is used to characterize obvious medical events; however, a bone metastasis may not be clinically obvious. Finally, reporting of bone metastases are not mandatory in Denmark, which may decrease the tendency for physicians and specialists to code these events. The study’s population-based cohort design enabled us to identify all hospital discharge and outpatient diagnoses of BrCa, bone metastases, and SREs over an 8-year per- iod, thereby minimizing the risk of referral and diagnostic bias. However, the validity of our findings depends on the accuracy of the coding of these events and completeness of reporting in the DNPR. In a previous study, we found that bone metastases and SREs secondary to BrCa were underestimated in the DNPR. Completeness of DNPR recording of bone metastases was 32%, and the positive predictive value was 86% compared with a medical record review. This may explain why the risk of bone metastases among BrCa patients in our study was lower than the one reported among BrCa patients in a previous study (4% vs. 9%, respectively) [7]. Completeness of DNPR recording of SREs was 75%, and the positive pre- dictive value was 75%, compared with a medical record review [17]. Given that we have a higher completeness of reporting of bone metastases and SREs compared to bone metastases alone, we may have overestimated the Author details 1 f 15. Fleisch H: Bisphosphonates: mechanisms of action. Endocr Rev 1998, 19:80-100. 15. Fleisch H: Bisphosphonates: mechanisms of action. Endocr Rev 1998, 19:80-100. 1Department of Clinical Epidemiology, Aarhus University Hospital, Ole Worms Alle 1150, DK-8000 Århus C., Denmark. 2Center for Observational Research, Amgen Inc., One Amgen Center Drive, MS 24-2-A, Thousands Oaks CA 91320, USA. 16. Aft R, Naughton M, Trinkaus K, Watson M, Ylagan L, Chavez-MacGregor M, Zhai J, Kuo S, Shannon W, Diemer K, Herrmann V, Dietz J, Ali A, Ellis M, Weiss P, Eberlein T, Ma C, Fracasso PM, Zoberi I, Taylor M, Gillanders W, Pluard T, Mortimer J, Weilbaecher K: Effect of zoledronic acid on disseminated tumour cells in women with locally advanced breast cancer: an open label, randomised, phase 2 trial. Lancet Oncol 2010, 11:421-428. Conclusions In conclusion, we found a high incidence rate of SREs among BrCa patients with bone metastases, particularly during the first year following a bone metastasis diagno- sis. This highlights the need for early detection of BrCa and use of existing effective treatments that can delay the onset of SREs. Page 6 of 6 Jensen et al. BMC Cancer 2011, 11:29 http://www.biomedcentral.com/1471-2407/11/29 Jensen et al. BMC Cancer 2011, 11:29 http://www.biomedcentral.com/1471-2407/11/29 Jensen et al. BMC Cancer 2011, 11:29 http://www.biomedcentral.com/1471-2407/11/29 Authors’ contributions AJ contributed to the conception and design, administrative support, provision of study material, data analysis and interpretation, and manuscript writing. JJ provided analytic support and interpretation and manuscript writing. MN contributed to the conception and design, data analysis and interpretation, and manuscript writing. MY contributed to data analysis and interpretation and manuscript writing. JF contributed to the conception and design, data analysis and interpretation, and manuscript writing. HS contributed to the conception and design, financial and administrative support, provision of study material, collection and assembly of data, and data analysis and interpretation. All authors read and approved the final manuscript. 17. Jensen AØ, Nørgaard M, Yong M, Fryzek JP, Sørensen HT: Validity of the recorded International Classification of Diseases, 10th edition diagnoses codes of bone metastases and skeletal-related events in breast and prostate cancer patients in the Danish National Registry of Patients. Clinical Epidemiology 2009, 1:101-108. Pre-publication history h bl h The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/11/29/prepub The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/11/29/prepub doi:10.1186/1471-2407-11-29 Cite this article as: Jensen et al.: Incidence of bone metastases and skeletal-related events in breast cancer patients: A population-based cohort study in Denmark. BMC Cancer 2011 11:29. Acknowledgements characteristics after implementation of population-based digital mammography screening. 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Louwman WJ, Voogd AC, van Dijck JA, Nieuwenhuijzen GA, Ribot J, Pruijt JF, Coebergh JW: On the rising trends of incidence and prognosis for breast cancer patients diagnosed 1975-2004: a long-term population-based study in southeastern Netherlands. Cancer Causes Control 2008, 19:97-106. 5. Coleman RE, Rubens RD: The clinical course of bone metastases from breast cancer. Br J Cancer 1987, 55:61-66. 6. Coleman RE: Skeletal complications of malignancy. Cancer 1997, 80:1588-1594. 7. Dent R, Hanna WM, Trudeau M, Rawlinson E, Sun P, Narod SA: Pattern of metastatic spread in triple-negative breast cancer. Breast Cancer Res Treat 2008, 115:423-428. 8. Hortobagyi GN, Theriault RL, Lipton A, Porter L, Blayney D, Sinoff C, Wheeler H, Simeone JF, Seaman JJ, Knight RD, Heffernan M, Mellars K, Reitsma DJ: Long-term prevention of skeletal complications of metastatic breast cancer with pamidronate. Protocol 19 Aredia Breast Cancer Study Group. J Clin Oncol 1998, 16:2038-2044. 8. 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Competing interests h d This study was supported in part by Amgen Inc, USA. Mellissa Yong is currently employed and has stock ownership in Amgen Inc. Jon Fryzek has stock ownership in Amgen Inc and, at the time this study was conducted, was an employee of Amgen Inc. References Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 10. Frank L: Epidemiology. When an entire country is a cohort. Science 2000, 287:2398-2399. 10. Frank L: Epidemiology. 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https://openalex.org/W2785376775	https://hal-pasteur.archives-ouvertes.fr/pasteur-02006305/document	English	null	Aedes Mosquitoes and Aedes-Borne Arboviruses in Africa: Current and Future Threats	International journal of environmental research and public health/International journal of environmental research and public health	2,018	cc-by	13,774	To cite this version: David Weetman, Basile Kamgang, Athanase Badolo, Catherine Moyes, Freya Shearer, et al.. Aedes Mosquitoes and Aedes-Borne Arboviruses in Africa: Current and Future Threats. International Jour- nal of Environmental Research and Public Health, 2018, 15 (2), pp.E220. 10.3390/ijerph15020220. pasteur-02006305 Aedes Mosquitoes and Aedes-Borne Arboviruses in Africa: Current and Future Threats David Weetman, Basile Kamgang, Athanase Badolo, Catherine Moyes, Freya Shearer, Mamadou Lamine Coulibaly, Joao Pinto, Louis A Lambrechts, Philip J Mccall To cite this version: David Weetman, Basile Kamgang, Athanase Badolo, Catherine Moyes, Freya Shearer, et al.. Aedes Mosquitoes and Aedes-Borne Arboviruses in Africa: Current and Future Threats. International Jour- nal of Environmental Research and Public Health, 2018, 15 (2), pp.E220. 10.3390/ijerph15020220. pasteur-02006305 David Weetman, Basile Kamgang, Athanase Badolo, Catherine Moyes, Freya Shearer, Mamadou Lamine Coulibaly, Joao Pinto, Louis A Lambrechts, Philip J Mccall Distributed under a Creative Commons Attribution 4.0 International License Review Aedes Mosquitoes and Aedes-Borne Arboviruses in Africa: Current and Future Threats McCall 1,* ID 1 Department of Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5Q UK; david.weetman@lstmed.ac.uk 2 Centre for Research in Infectious Diseases, Yaoundé PO Box 13501, Cameroon; kamgang_d@yahoo.fr 3 Laboratoire d’Entomologie Fondamentale et Appliquée (LEFA), Université Ouaga 1 Pr Joseph Ki-Zerbo, Ouagadougou 03 BP 7021, Burkina Faso; a.badolo@gmail.com 2 Centre for Research in Infectious Diseases, Yaoundé PO Box 13501, Cameroon; kamgang_d@yahoo.fr 3 Laboratoire d’Entomologie Fondamentale et Appliquée (LEFA), Université Ouaga 1 Pr Joseph Ki-Zerbo, Ouagadougou 03 BP 7021, Burkina Faso; a.badolo@gmail.com 4 Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford OX3 +7LF, UK; clmoyes@well.ox.ac.uk (C.L.M.); freya.m.shearer@gmail.com (F.M.S.) 5 University of Sciences, Techniques and Technologies of Bamako, Bamako BP 1805, Mali; 4 Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford OX3 +7LF, UK; clmoyes@well.ox.ac.uk (C.L.M.); freya.m.shearer@gmail.com (F.M.S.) 5 University of Sciences, Techniques and Technologies of Bamako, Bamako BP 1805, Mali; doudou@icermali.org 4 Big Data Institute, Li Ka Shing Centre for Health Information and Discovery, University of Oxford, Oxford OX3 +7LF, UK; clmoyes@well.ox.ac.uk (C.L.M.); freya.m.shearer@gmail.com (F.M.S.) 5 University of Sciences, Techniques and Technologies of Bamako, Bamako BP 1805, Mali; doudou@icermali.org 6 Global Health and Tropical Medicine (GHTM), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, 1349-008 Lisbon, Portugal; JPinto@ihm 7 6 Global Health and Tropical Medicine (GHTM), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, 1349-008 Lisbon, Portugal; JPinto@ihmt.unl.pt 7 Insect-Virus Interactions, Department of Genomes and Genetics, Institut Pasteur, 75015 Paris, France; louis.lambrechts@pasteur.fr 6 Global Health and Tropical Medicine (GHTM), Instituto de Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Rua da Junqueira 100, 1349-008 Lisbon, Portugal; JPinto@ihmt.unl.pt 7 Insect-Virus Interactions, Department of Genomes and Genetics, Institut Pasteur, 75015 Paris, France; louis.lambrechts@pasteur.fr q g p 7 Insect-Virus Interactions, Department of Genomes and Genetics, Institut Pasteur, 75015 Paris, France; louis.lambrechts@pasteur.fr 8 Centre National de la Recherche Scientiﬁque, Unité Mixte de Recherche 2000, 75015 Paris, Franc * C d hili ll@l d k T l 44 151 705 3132 8 Centre National de la Recherche Scientiﬁque, Unité Mixte de Recherche 2000, 75015 Paris, France * Correspondence: philip.mccall@lstmed.ac.uk; Tel.: +44-151-705-3132 Received: 12 December 2017; Accepted: 24 January 2018; Published: 28 January 2018 Abstract: The Zika crisis drew attention to the long-overlooked problem of arboviruses transmitted by Aedes mosquitoes in Africa. Review Aedes Mosquitoes and Aedes-Borne Arboviruses in Africa: Current and Future Threats Yellow fever, dengue, chikungunya and Zika are poorly controlled in Africa and often go unrecognized. However, to combat these diseases, both in Africa and worldwide, it is crucial that this situation changes. Here, we review available data on the distribution of each disease in Africa, their Aedes vectors, transmission potential, and challenges and opportunities for Aedes control. Data on disease and vector ranges are sparse, and consequently maps of risk are uncertain. Issues such as genetic and ecological diversity, and opportunities for integration with malaria control, are primarily African; others such as ever-increasing urbanization, insecticide resistance and lack of evidence for most control-interventions reﬂect problems throughout the tropics. We identify key knowledge gaps and future research areas, and in particular, highlight the need to improve knowledge of the distributions of disease and major vectors, insecticide resistance, and to develop speciﬁc plans and capacity for arboviral disease surveillance, prevention and outbreak responses. Keywords: Aedes aegypti; Aedes albopictus; Aedes formosus; Zika; dengue; chikungunya; yellow fever; vector HAL Id: pasteur-02006305 https://pasteur.hal.science/pasteur-02006305v1 Submitted on 4 Feb 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 International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Review Aedes Mosquitoes and Aedes-Borne Arboviruses in Africa: Current and Future Threats David Weetman 1, Basile Kamgang 2 ID , Athanase Badolo 3 ID , Catherine L. Moyes 4 ID , Freya M. Shearer 4, Mamadou Coulibaly 5, João Pinto 6 ID , Louis Lambrechts 7,8 ID and Philip J. McCall 1,* ID David Weetman 1, Basile Kamgang 2 ID , Athanase Badolo 3 ID , Catherine L. Moyes 4 ID , Freya M. Shearer 4, Mamadou Coulibaly 5, João Pinto 6 ID , Louis Lambrechts 7,8 ID and Philip J. www.mdpi.com/journal/ijerph 1. Introduction and Overview The crisis following the Zika epidemic across South America in 2015–2016 and the newly recognised severe neurological consequences of infection have raised global awareness of Aedes-borne arboviral diseases. Zika has also belatedly turned the scientiﬁc community’s attention to Africa, where this virus was originally identiﬁed [1] and where the other Aedes-transmitted arboviruses also occur. Despite its global importance [2], the extent of the dengue burden in Africa remains unknown [3], as does the true burden of yellow fever, though tens of thousands die annually in Africa and the threat Int. J. Environ. Res. Public Health 2018, 15, 220; doi:10.3390/ijerph15020220 www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2018, 15, 220 2 of 20 of outbreaks remains high [4,5], even with availability of an effective vaccine [6]. Moreover, the risk of exporting yellow fever from Africa remains a concern, particularly to immunologically naïve Asian populations [7,8]. of outbreaks remains high [4,5], even with availability of an effective vaccine [6]. Moreover, the risk of exporting yellow fever from Africa remains a concern, particularly to immunologically naïve Asian populations [7,8]. p p Why was so little attention paid to Aedes-borne arboviruses in Africa, until they led to outbreaks in locations outside of Africa? One explanation is that these viruses constitute greater public health threats for immunologically-naïve non-African human populations. A second is that the magnitude of the burden of malaria and many neglected tropical diseases in Africa detract attention from Aedes-borne infections. A third and related factor is that accurate identiﬁcation of arboviral infections in resource-poor settings is challenging, and in Africa, many fevers of unknown origin tend to be recorded as malaria [7]. Hence, the major arboviral infections remain neglected in Africa, while receiving signiﬁcant attention and resources for control elsewhere. In common with Zika [8], chikungunya [9], yellow fever [10], and perhaps also dengue [11], Aedes aegypti almost certainly originated in Africa [12–14]. Globally Ae. aegypti is the primary vector of all of these viruses, but a range of African Aedes species are competent and epidemiologically-signiﬁcant vectors. An important addition to this vector fauna is Aedes albopictus, introduced into Africa less than 30 years ago and still increasing its range [15]. Adaptation of arboviruses to novel vectors can occur rapidly and can have signiﬁcant consequences. A single amino acid substitution facilitated efﬁcient transmission of the chikungunya virus by Ae. 1. Introduction and Overview albopictus [16], permitting establishment of local transmission in many new regions beyond Africa, following the 2005–2006 outbreak on the island of La Réunion. Simple genetic changes can also affect the virus’s interaction with the vertebrate host, with uncertain but potentially severe consequences [17]. The Zika virus also can be transmitted through sexual intercourse, though the epidemiological importance of this mode of transmission remains to be determined [18]. Control of Zika and other Aedes-transmitted arboviruses is a major 21st century challenge for global public health, exacerbated by widespread insecticide resistance in the vectors [19] and a relentless growth in urban environments [20]. This is especially true in the resource-poor sub-Saharan nations, which have the lowest collective Gross Domestic Product (GDP) of any region [21]. A thorough exploration of the burden, transmission biology and control of Aedes-transmitted arboviruses is essential to deﬁne, understand and prepare for potential future threats in Africa and worldwide [22,23]. Here, we review current information on these key topics for Aedes vectors and the major arboviruses they transmit in Africa, highlighting knowledge gaps, and recommending priorities for further investigation. 2. The Burden of Aedes-Borne Viral Diseases in Africa Estimated population at risk of infection (PAR) by each arbovirus and by overlapping arboviruses in Africa, 2015. Population estimates were calculated using the methods and data sources as deﬁned and cited in Figure 1a,b, respectively. Table 1. Estimated population at risk of infection (PAR) by each arbovirus and by overlapping arboviruses in Africa, 2015. Population estimates were calculated using the methods and data sources as defined and cited in Figure 1a b respectively Infection Estimated Population at Risk Percentage of African Population Chikungunya 271 million 23% Dengue 750 million 63% Yellow fever * 21 million 2% Zika 406 million 34% At least one of the above 831 million 70% * The value for yellow fever has been adjusted to account for reductions in the population at risk following vaccination programmes. Infection Estimated Population at Risk Percentage of African Population Chikungunya 271 million 23% Dengue 750 million 63% Yellow fever * 21 million 2% Zika 406 million 34% At least one of the above 831 million 70% * The value for yellow fever has been adjusted to account for reductions in the population at risk following vaccination programmes. Infection Estimated Population at Risk Percentage of African Population Chikungunya 271 million 23% Dengue 750 million 63% Yellow fever * 21 million 2% Zika 406 million 34% At least one of the above 831 million 70% * The value for yellow fever has been adjusted to account for reductions in the population at risk following vaccination programmes. Infection Estimated Population at Risk Percentage of African Population Chikungunya 271 million 23% Dengue 750 million 63% Yellow fever * 21 million 2% Zika 406 million 34% At least one of the above 831 million 70% * The value for yellow fever has been adjusted to account for reductions in the population at risk following vaccination programmes. Infection Estimated Population at Risk Percentage of African Population Infection Estimated Population at Risk Percentage of African Population Ch k ll % * The value for yellow fever has been adjusted to account for reductions in the population at risk following vaccination programmes. * The value for yellow fever has been adjusted to account for reductions in the population at risk following vaccination programmes. Figure 1. The distributions of chikungunya, dengue, yellow fever and Zika virus infections in humans in Africa. 2. The Burden of Aedes-Borne Viral Diseases in Africa The extent of chikungunya, dengue and Zika virus distributions in Africa is largely unknown, and our knowledge of the variation in transmission risk within these zones, as well as within the yellow fever risk zone [6], is hampered by a lack of disease data. Ecological models have been used to estimate the spatial distributions of each virus [2,24–26], and we have used these maps to generate comparable estimates for the population at risk of infection (PAR) (Supplementary Materials). Table 1 shows the estimated African PAR for chikungunya, dengue, yellow fever and Zika [24–28]. The value for yellow fever was adjusted to take account of vaccination campaigns conducted in parts of Africa and shows the much lower PAR for this disease since vaccination began [29]. Individual disease distributions overlap (Figure 1), and an estimated 831 million people were living in an area at risk of at least one of these arbovirus infections in 2015 (Table 1). However, these estimates are based on predicted distributions within unknown disease ranges: actual PAR values cannot be estimated with certainty until the extent of each disease has been deﬁned and more case data are collected across the continent. Furthermore, the number of people living in an area of risk who are actually protected by life-long immunity conferred by previous infection with the chikungunya virus, Zika virus and each serotype of the dengue virus is unknown but could change the PAR ﬁgures dramatically [30–32]. Outbreaks of all four diseases are characterised by large spikes in the number of cases, often preceded 3 of 20 Int. J. Environ. Res. Public Health 2018, 15, 220 and followed by periods when no new cases are reported [33]. Therefore, the values shown (Table 1 and Figure 1) represent the presence of a transmission risk at some point in time over a number of years, rather than a year-round or seasonal risk. As the uncertainty in the PAR highlights, there is an urgent need to improve data availability and quality in order to map more accurately the disease burden in Africa. Int. J. Environ. Res. Public Health 2018, 15, x 3 of 19 over a number of years, rather than a year-round or seasonal risk. As the uncertainty in the PAR highlights, there is an urgent need to improve data availability and quality in order to map more accurately the disease burden in Africa Table 1. 2. The Burden of Aedes-Borne Viral Diseases in Africa (a) Areas at risk of one, two, three or all four infections; map generated as described in Supplementary Methods. (b) Locations of reported infections (symptomatic and non-symptomatic) of dengue, chikungunya, Zika and yellow fever [24–28]. Figure 1. The distributions of chikungunya, dengue, yellow fever and Zika virus infections in humans in Africa. (a) Areas at risk of one, two, three or all four infections; map generated as described in Supplementary Methods. (b) Locations of reported infections (symptomatic and non-symptomatic) of dengue, chikungunya, Zika and yellow fever [24–28]. Figure 1. The distributions of chikungunya, dengue, yellow fever and Zika virus infections in humans in Africa. (a) Areas at risk of one, two, three or all four infections; map generated as described in Supplementary Methods. (b) Locations of reported infections (symptomatic and non-symptomatic) of dengue, chikungunya, Zika and yellow fever [24–28]. Figure 1. The distributions of chikungunya, dengue, yellow fever and Zika virus infections in humans in Africa. (a) Areas at risk of one, two, three or all four infections; map generated as described in Supplementary Methods. (b) Locations of reported infections (symptomatic and non-symptomatic) of dengue, chikungunya, Zika and yellow fever [24–28]. Int. J. Environ. Res. Public Health 2018, 15, 220 Int. J. Environ. Res. Public Health 2018, 15, x 4 of 20 4 of 19 3. Range and Distribution of Aedes Vector Species in Africa g p African ranges are unknown, even for the two major A African ranges are unknown, even for the two major Aedes vector species, and there are insufﬁcient records to allow their deﬁnition either by expert opinion or any semi-quantitative method. Suitable environments for Ae. albopictus and Ae. aegypti within Africa have been predicted using ecological models (Figure 2) [34], and though the maps indicate extensive areas of suitability and a large potential for sympatric occurrence, these estimated distributions need to be treated with caution. Maps show locations where the species could potentially occur but not necessarily where they have been found. For example, much of sub-Saharan Africa is predicted to be suitable for Ae. albopictus, but records remain patchy [35–64]. Since the worldwide records of each species were compiled, new studies in Africa have reported Ae. aegypti in Ghana [51], Mozambique [55] and Namibia [59], and Ae. albopictus in Mali [57], Morocco [39], Mozambique [50] and São Tomé and Príncipe [62]. g j p insufficient records to allow their definition either by expert opinion or any semi-quantitative method. Suitable environments for Ae. albopictus and Ae. aegypti within Africa have been predicted using ecological models (Figure 2) [34], and though the maps indicate extensive areas of suitability and a large potential for sympatric occurrence, these estimated distributions need to be treated with caution. Maps show locations where the species could potentially occur but not necessarily where they have been found. For example, much of sub-Saharan Africa is predicted to be suitable for Ae. albopictus, but records remain patchy [35–64]. Since the worldwide records of each species were compiled, new studies in Africa have reported Ae. aegypti in Ghana [51], Mozambique [55] and Namibia [59], and Ae. albopictus in Mali [57], Morocco [39], Mozambique [50] and São Tomé and Príncipe [62]. Figure 2. Reports of mosquito occurrence and areas of predicted environmental suitability for Aedes albopictus and Ae. aegypti in Africa [36–64]: (a) Ae. aegypti; (b) Ae. albopictus. Figure 2. Reports of mosquito occurrence and areas of predicted environmental suitability for Aedes albopictus and Ae. aegypti in Africa [36–64]: (a) Ae. aegypti; (b) Ae. albopictus. Figure 2. Reports of mosquito occurrence and areas of predicted environmental suitability for Aedes albopictus and Ae. aegypti in Africa [36–64]: (a) Ae. aegypti; (b) Ae. albopictus. Figure 2. Reports of mosquito occurrence and areas of predicted environmental suitability for Aedes albopictus and Ae. aegypti in Africa [36–64]: (a) Ae. aegypti; (b) Ae. albopictus. Figure 2. 3. Range and Distribution of Aedes Vector Species in Africa g p African ranges are unknown, even for the two major A Reports of mosquito occurrence and areas of predicted environmental suitability for Aedes albopictus and Ae. aegypti in Africa [36–64]: (a) Ae. aegypti; (b) Ae. albopictus. Figure 2. Reports of mosquito occurrence and areas of predicted environmental suitability for Aedes albopictus and Ae. aegypti in Africa [36–64]: (a) Ae. aegypti; (b) Ae. albopictus. The areas of predicted suitability shown in Figure 2 are based on relationships with environmental and socio-economic variables. Studies using global records of each species found that temperature is the most influential predictor and that precipitation, vegetation indices and urban land cover also play a role [33,65]. Few studies have looked at these relationships in African populations, but temperature is still likely to be important [66,67] and rainfall is strongly linked to Aedes vector abundance in Kenyan populations [68]. Specific landscape factors are more likely to vary The areas of predicted suitability shown in Figure 2 are based on relationships with environmental and socio-economic variables. Studies using global records of each species found that temperature is the most inﬂuential predictor and that precipitation, vegetation indices and urban land cover also play a role [33,65]. Few studies have looked at these relationships in African populations, but temperature is still likely to be important [66,67] and rainfall is strongly linked to Aedes vector abundance in Kenyan 5 of 20 Int. J. Environ. Res. Public Health 2018, 15, 220 populations [68]. Speciﬁc landscape factors are more likely to vary between different regions of the world and the inﬂuence of some of these have been studied in African locations. In Central Africa, Ae. albopictus preferred man-made breeding sites (tyres and tanks) surrounded by vegetation whereas Ae. aegypti preferred man-made breeding sites in neighbourhoods with higher building density [69,70]. Similarly, in urban Cameroon, Ae. aegypti was more abundant in downtown environments in the dry season whereas Ae. albopictus was more prevalent in the suburbs in all seasons [71]. Studies in rural areas have found that within oil palm-dominated landscapes in Côte d’Ivoire Ae. aegypti was more abundant in polycultures than monocultures [72]. Large gaps in reporting of each species can be seen in Figure 2, but vector presence can be inferred from reports of locally acquired arbovirus infections (Figure 1b). These case reports show infections occurring in a number of countries where no vector data are available, implying that one of more of the arbovirus vectors is present. 4.1. Aedes aegypti vs. Aedes albopictus Aedes aegypti is considered the main vector of dengue viruses worldwide, largely attributable to its higher vector competence and stronger host preference for humans compared to Ae. albopictus [73]. However, Ae. albopictus has been a driving force in the worldwide emergence of chikungunya virus since 2004 [74], and in Central Africa it is considered to have played a key role in the 2007 emergence of dengue, chikungunya [75,76] and possibly Zika [77]. Moreover, its ongoing range expansion across Africa has the potential to increase the arbovirus transmission risk to areas far from urbanisation [78]. Improved knowledge of Ae. albopictus distribution and vectorial capacity in Africa is required to enhance arbovirus surveillance and prevention [79]. 3. Range and Distribution of Aedes Vector Species in Africa g p African ranges are unknown, even for the two major A Which vectors might be found in these locations can only be addressed by ﬁeld surveys, and priority should be given to the countries reporting arbovirus infections but no recent reports or records of vector species. Currently these are Guinea, Guinea Bissau, Sierra Leone, Liberia, Togo, Chad, South Sudan, Ethiopia, Eritrea, Djibouti, Somalia. 4.3. Other Aedes Species and Potential for Emergent Transmission Cycles Additional Aedes species play a critical role in arbovirus transmission cycles in Africa because (i) they are involved in sylvatic arbovirus transmission cycles and/or (ii) they bridge sylvatic and human transmission cycles. For example, Aedes africanus is considered the main sylvatic vector of yellow fever virus in Africa [100] and can also act as a bridge vector to humans, together with Aedes bromeliae, Aedes furcifer, Aedes taylori, Aedes luteocephalus, Aedes metallicus, Aedes opok, Aedes vittatus, and species of the Aedes simpsoni complex [101]. Sylvatic dengue viruses in Africa are transmitted among non-human primates by Ae. furcifer and Ae. luteocephalus, and usually cross over to humans through biting by Ae. furcifer [101]. Bridge vectors may initiate a human outbreak, but large epidemics typically occur only when virus transmission involves urban populations of Ae. aegypti or Ae. albopictus, though there may have been exceptions: Haddow [102] described Ae. simpsoni (=Ae. bromeliae [15]) as being “the principal vector in the greatest known African yellow fever epidemic”, which occurred in Ethiopia in the early 1960s. The majority of these Aedes vector species are found in rural or forest areas, and so are less likely to present a threat in the urban environments where Ae. aegypti populations thrive. Nonetheless, increasing erosion of their natural habitats could lead to greater contact with humans, and/or formerly obligate sylvatic species might adapt to new urban environments and hosts, potentially with a greater role as vectors [103]. Many readily feed on both domestic and wild animals including primates, as well as humans, hence their potential importance as bridging vectors [96,104–106]. Although a sylvatic dengue transmission cycle is known in restricted locations like eastern Senegal [107,108], it is unclear how important it is across the continent of Africa. All of the focal arboviral diseases circulate among non-human primates in Africa, though the true role of animal reservoirs in the epidemiology of human disease remains to be determined [109]. Less is known about the African vectors of Zika, and although multiple Aedes species from across Africa have been found naturally infected with the Zika virus, this is also true for non-Aedes species, and cannot be translated into vector competence [110,111]. In a rare demonstration, a small proportion of Ae. vittatus and Ae. luteocephalus were found to harbour the virus in their salivary glands following artiﬁcial infection with Zika strains, suggesting some potential for transmission [110]. 4.2. Sylvatic vs. Domestic Forms of Aedes aegypti Aedes aegypti is believed to have originated in Africa from a generalist, zoophilic tree-hole breeder [12]. Outside Africa, Ae. aegypti populations are exclusively found in close association with humans in the domestic environment, and this domestic form of Ae. aegypti may have originated from a population that adapted to (i) breeding in artiﬁcial water storage containers and (ii) biting humans [80]. Domestication events involving human-feeding specialization may have occurred both outside Africa as part of the species’ expansion, and within Africa [81], possibly, but perhaps not exclusively, on the West African coast [82]. African Ae. aegypti breed in both the domestic environment and in the ancestral sylvatic habitat. Whereas domestic Ae. aegypti larvae develop in artiﬁcial containers (e.g., tyres, discarded containers, jars, ﬂower pots, metal drums) within or in close proximity to human habitation, larvae of the sylvatic ecotype are found in natural breeding sites (e.g., rock pools, tree holes, plant axils, fruit husks) in forested areas [80]. Interestingly, larvae of the two Ae. aegypti ecotypes are exposed to different bacterial communities in their respective breeding sites, potentially resulting in differences in vectorial capacity [83]. Classically, two morphological subspecies were described in Africa that broadly correspond with these ecotypes: Ae. aegypti aegypti and Ae. aegypti formosus. However, there is evidence that Ae. aegypti formosus is increasingly found in urban environments [84], and the diagnostic morphological characters (presence/absence of white abdominal scaling patterns [85]) often form a continuum [86]. Similarly, clear genetic boundaries are absent, presumably as a result of extensive current or recent historical gene ﬂow [87,88]. The picture may be complicated further by introductions of domestic Ae. aegypti into Africa and interbreeding with local populations [89,90]. The result is a lack of clear correlation between morphology, genetics and ecology [81,90,91], which calls into question Int. J. Environ. Res. Public Health 2018, 15, 220 6 of 20 the utility of the simple division of the diverse African Ae. aegypti fauna into the aegypti and formosus subspecies [80,84]. African Ae. aegypti have traditionally been considered less competent for ﬂaviviruses than Ae. aegypti from the rest of the world [92,93]. A global survey of 28 populations found that sylvatic Ae. aegypti populations from West Africa were the least susceptible to yellow fever virus infection [94]. However, more recent observations indicated that vector competence of African Ae. aegypti is extremely variable and depends on speciﬁc pairings of mosquito population and viral isolate [76,95–98]. 4.2. Sylvatic vs. Domestic Forms of Aedes aegypti Such genotype-by-genotype speciﬁcity has been widely documented in other host-pathogen systems [99]. 4.3. Other Aedes Species and Potential for Emergent Transmission Cycles With a growing body of evidence from elsewhere showing the Zika virus’ capacity for development and transmission by species in addition to Ae. aegypti and Ae. albopictus [112], further studies to test competence in the African vectors from which it has been isolated should be prioritised. Better knowledge of African transmission cycles is important to determine the risk of viruses like Zika or chikungunya establishing similar sylvatic cycles in geographic regions where they have newly arrived [113,114]. Recent reports from Kenya suggest that human infections may be a source of ‘spillback’ infections in baboon populations [85], demonstrating the range of potential consequences following introduction of novel viruses or strains. Clearly, assessing the risk of human infection by arboviruses will require more in-depth studies on the biology of sylvatic Aedes spp. in Africa [104,107]. Int. J. Environ. Res. Public Health 2018, 15, 220 7 of 20 5. Vector Surveillance and Control of Aedes species in Africa The majority of Aedes spp. control studies have focused on dengue outbreaks in urban sites in Asia or Latin America, where transmission by Ae. aegypti and/or Ae. albopictus is exclusively human to human [115,116]. Indeed, a recent systematic review of the evidence for effectiveness of vector control in reducing dengue virus transmission included 41 trials, but none were from Africa [117]. The widespread use of the insecticide DDT (dichlorodiphenyltrichloroethane) in Ae. aegypti eradication programmes, that led to its near elimination from South America by the late 1960s, was never replicated in Africa [4,118]. Subsequently, factors such as increased international freight and travel, urbanization, and vector control strategies and tools poorly suited to urban environments or hampered by insecticide resistance [119] have resulted in the global explosion of dengue. Yellow fever outbreaks can be prevented or controlled by mass vaccine distribution [120], but no vaccines are available for chikungunya and Zika, and the licensed dengue vaccine offers incomplete and serotype-speciﬁc protection [121]. Consequently, outbreak prevention and response remain reliant on vector control. Preventing outbreaks, or mitigating their impact, is challenging even for those countries that have made considerable investment in capacity after ﬁghting against dengue outbreaks for decades [122]. This scenario and the associated challenges are exacerbated in African countries where recognition of dengue outbreaks has started relatively recently; dengue is often not a reported infection, even if correctly diagnosed, and where surveillance and response strategies may be absent or improperly developed [123–126]. Rolling out effective surveillance plans for Aedes-borne diseases, particularly dengue, is a global priority [127,128] but at present, early warning systems for Aedes-borne diseases like dengue are unreliable [129], and the standard indices used to monitor Ae. aegypti populations are inaccurate [130]. 5.1. Integrating Aedes Species Control with Malaria Vector Control: Uniquely African Opportunities? Malaria cases and deaths have declined in Africa since 2000 but sustaining control toward elimination remains a major focus for African healthcare [131]. Vector control has played a central role, primarily via long-lasting insecticide treated nets (LLINs) and indoor residual spraying (IRS) [132], and this is likely to continue. What opportunities might recognition of the importance of vector control for both malaria and Aedes-borne arboviruses offer in Africa? Some interventions targeting African malaria vectors are unlikely to have a signiﬁcant impact on Aedes-borne arboviral transmission because of differences in their biology. For example, LLINs primarily target nocturnal biters and zooprophylaxis targets domestic animal-biters, neither of which typically apply to Ae. aegypti [133,134], though the latter may be of some relevance to the more generalist feeder Ae. albopictus [135]. However, IRS targets indoor resting; a behaviour common not only to malaria vectors like Anopheles gambiae and An. funestus, but also to Ae. aegypti [133,136–138]. Results to date are promising [117,139–141], but to be effective across the target species, IRS will require careful consideration of available insecticides because in both An. gambiae and Ae. aegypti, resistance (see Section 5.2) can lead to operational IRS failure [140,142]. House screening [143] and other improvements which prevent mosquito entry [144,145] might be effective against both Anopheles malaria vectors and Ae. aegypti, although perhaps less so against the more exophilic Ae. albopictus. Larval source management can be effective individually for both malaria [146] and dengue [117] but its potential as a cross-cutting intervention is limited owing to the typically different breeding sites of Anopheles and Aedes. Larval stages of Anopheles are usually found in ground pools or irrigated sites, especially rice paddies, while Aedes proliferate in manmade containers or small natural sites such as rock pools and tree holes. Nevertheless, biological and chemical larviciding formulations, such as Bti and pyriproxyfen, represent important tools in the arsenal for control of Aedes larvae [147,148] and in some settings, they may also be appropriate for Anopheles control [149,150], potentially offering logistical and procurement synergies. 8 of 20 Int. J. Environ. Res. Public Health 2018, 15, 220 There is currently little evidence to suggest efﬁcacy of personal repellents for control of either dengue or malaria [117,151–153]. Though promising for both Anopheles and Aedes vector control [154,155], demonstration of the impact of volatile spatial insecticides on epidemiological indicators is currently lacking for malaria or arboviruses. 5.1. Integrating Aedes Species Control with Malaria Vector Control: Uniquely African Opportunities? Similarly, attractive toxic sugar baits (ATSB) might eventually prove to be effective against different diseases, although at present it is unclear whether the same bait will attract both Aedes and Anopheles species, whilst avoiding negative impacts of traps on economically-beneﬁcial insects [156]. The control of Aedes spp. in Africa could beneﬁt from association with already well-established national malaria control programmes. This is in line with the ﬁrst pillar of the global vector control response developed by the World Health Organisation (WHO) that calls for strengthening inter- and intra-sectoral action and collaboration [157]. Following identiﬁcation of disease overlaps [158] and control opportunities [61], provision of training will be a key issue to ensure that experienced malaria vector control practitioners become effective arboviral disease vector controllers. In addition, funding agreements will have to be reached to avoid diversion of resources from malaria programmes where the perceived current disease risk may be relatively low in elimination or pre-elimination settings, resources for which in many cases, are already inadequate to maintain a consistent downward malaria trajectory [137]. 5.2. Insecticide Resistance and Challenges to Control Blank cells indicate that the phenotype or mechanism was not investigated. secticide resistance in Aedes species (1990 onwards) from African mainland countries and islands. Resistance is classiﬁed according to WHO (R) < 90% mortality, suspected resistance (RS) 90–97% mortality, or susceptible (S) > 97% mortality in adult (a) tube or larval (l) bioassays. nce mechanisms are shown where reported. Studies are ordered by species, then from North-West toward South-East. Blank cells indicate that h i i i d Study Year Country Area Species DDT (a) Pyr I (a) Pyr II (a) Carb (a) OP (a) Temephos (l) Bti (l) Other (l) kdr Mutations Metabolic Resistance [162] 2013 Madeira Funchal, Paul do Mar Ae. aegypti R R R R, S F1534C; V1016I MFO, esterases (biochemistry); PBO, DEM signiﬁcant (synergist; metabolizing genes overexpressed) [43] 2009 Cape Verde Santiago Ae. aegypti R S S R S [163] 2012 Cape Verde Santiago, Praia Ae. aegypti R S S Not detected MFO, esterases, GSTs (biochemistry) [63] 2009 Senegal Dakar Ae. aegypti R S R, RS R S [52] 2010 Côte d’Ivoire Abidjan Ae. aegypti S RS, S R, RS [46] 2014 Côte d’Ivoire Abidjan Ae. aegypti R S RS RS S (DDT, Pyr) [164] 2012–2013 Ghana Accra Ae. aegypti R R R [51] 2013–2014 Ghana Widespread Ae. aegypti R R, RS, S F1534C; V1016I [51] 2013–2014 Ghana Widespread Aedes aedes formosus R R, RS, S F1534C [37] 2011–2012 Nigeria Lagos Ae. aegypti R S RS, S [60] 2013 Nigeria Kwara State Ae. aegypti S S R [49] 2007 Cameroon Widespread Ae. aegypti RS S S S S S [71] 2015–2016 Cameroon Yaoundé Ae. aegypti R S R R, S S Not detected Limited effect of synergist PBO [58] 2013 Central African Rep. Bangui Ae. aegypti R, RS RS, S S S S S Not detected MFO, esterases, GSTs (biochemistry) [49] 2007 Gabon Libreville Ae. aegypti R S S S S S [48] 2009, 2010 Sudan Port Sudan Ae. aegypti R RS, S S R, RS [165] 2015 Tanzania Dar es Salaam Ae. aegypti R, RS R, RS [166] 2010–2011 Mayotte Petit Terre Ae. aegypti S S S S (multiple) [49] 2007 Cameroon Widespread Ae. albopictus R RS, S S S S S [71] 2015–2016 Cameroon Yaounde Ae. albopictus R R, S R, RS R, RS S Not detected Variable effect of synergist PBO among locales [58] 2014 CAR Bangui Ae. 5.2. Insecticide Resistance and Challenges to Control Worldwide data on insecticide resistance in Ae. aegypti and Ae. albopictus are patchy, with by far the majority of data originating from Latin America and South-East Asia [19]. This paucity is readily evident in Africa, with just 18 published studies, of which three [44,64,159] were conducted over 30 years ago. The remainder involve WHO tube and larval bioassays on samples collected within the last ten years from 12 countries (Table 2). DDT resistance is widespread and has been reported from every country tested, and in both Ae. aegypti and Ae. albopictus. Resistance to pyrethroids (primarily permethrin and deltamethrin) appears more sporadic but there are conﬁrmed reports in Ae. aegypti from mainland West, Central and East Africa. It should also be noted that An. gambiae doses are almost always used for assessment of pyrethroids and may underestimate resistance because Ae. aegypti discriminant doses are lower [160]. Pyrethroid resistance may currently be less critical than in South East Asia and Latin America [19], but in Africa appears to be emergent, and is not limited to particular regions. Organophosphate adulticides (malathion or fenitrothion) have been less frequently tested but only in Madeira and Sudan has resistance been conﬁrmed in Ae. aegypti. All studies from West Africa and islands testing carbamates (usually propoxur) have reported resistance. Studies from other areas have typically reported susceptibility, but very recent testing in Yaoundé, Cameroon, detected bendiocarb resistance in both Ae. aegypti and Ae. albopictus [71], suggesting that apparent geographical patterns may be a consequence of the limited number of studies performed to date. Fortunately, tests of the ﬁrst-line biological and chemical larvicides, Bti and temephos, have not detected a problematic level of resistance. In dose-response assays the highest resistance ratios (calculated from the ratio of the lethal concentration killing 50% of the test, compared to a susceptible reference strain) are from Cape Verde and suggest little resistance [161]. Int. J. Environ. Res. Public Health 2018, 15, 220 9 of 20 Table 2. Records of insecticide resistance in Aedes species (1990 onwards) from African mainland countries and islands. Resistance is classiﬁed according to WHO standards as resistant (R) < 90% mortality, suspected resistance (RS) 90–97% mortality, or susceptible (S) > 97% mortality in adult (a) tube or larval (l) bioassays. Brief details of resistance mechanisms are shown where reported. Studies are ordered by species, then from North-West toward South-East. 5.2. Insecticide Resistance and Challenges to Control albopictus R, RS, S R, RS, S RS, S RS, S S S Not detected MFO, esterases, GSTs (biochemistry) [49] 2007 Gabon Libreville Ae. albopictus S [61] 2010–2011 Mayotte Kaweni Ae. albopictus S S S S (multiple) Abbreviations: DDT (dichlorodiphenyltrichloroethane); Pyr (pyrethroid); Carb (carbamate); OP (organophosphates); MFO (mixed function oxidases); PBO (piperonyl butoxide); DEM (diethyl maleate); GST (glutathione S-transferase). Int. J. Environ. Res. Public Health 2018, 15, 220 10 of 20 Susceptibility to Bti is expected based on its complex mode of toxicity and lack of any previous reports of resistance in Aedes ﬁeld populations [19]. In contrast, temephos resistance is extremely common in Latin America and is found throughout Asia [19], yet apparent full susceptibility in Africa allows temephos to be regarded as a viable option for water treatment. There is currently very limited information on the mechanisms of insecticide resistance in African populations of either key Aedes vector species. Knockdown resistance (kdr) mutations in the voltage-gated sodium channel, which can generate high levels of resistance when present in combinations [166,167], have recently been detected in West Africa, though not to date elsewhere, albeit from very limited investigations (Table 2). High frequencies of F1534C, the only kdr mutation known to show a worldwide distribution [19], have been found in samples from throughout Ghana [51], whilst the Latin American V1016I variant was detected in a single Ghanaian sample. In the absence of target site mutations, evidence for metabolic resistance via efﬁcacy of synergists in bioassays or elevated activity of mixed-function oxidases, esterases and glutathione-S-transferases in biochemical assays has been suggested as at least a partial explanation for some resistant phenotypes (Table 2). The source of resistance in African Aedes populations is less obvious than for areas of Latin America and Asia subjected to targeted control programmes. Widespread recent use of DDT and pyrethroids for IRS against malaria vectors might be one source; however, introductions, rather than simply in situ selection from local genetic variation, might be important. For example, the 1534C kdr mutation found in Africa is linked to a non-African, presumably immigrant, haplotype [34]. On Madeira, a multiple-insecticide resistant Ae. aegypti population has likely been introduced recently, possibly from Latin America [162], ready-equipped with two kdr mutations and overexpression of multiple pyrethroid metabolising genes [168,169]. In general, the picture of resistance suggests that viable insecticidal options remain available to target African Aedes populations. 5.2. Insecticide Resistance and Challenges to Control Yet, the potential for insecticide resistant Ae. aegypti and Ae. albopictus to be imported via human-aided movement of mosquitoes over long distances [170], rather than via slow natural migration, is a major concern, and a challenge to curbing the spread of resistance. 6. Conclusions Major Knowledge Gaps and Recommendations Major Knowledge Gaps and Recommendations Over 60 years ago, and with considerable prescience, Mahaffy [118] argued the case for eradication of Ae. aegypti from Africa, writing: “A successful eradication programme of this nature carries with it results of such profound importance, not only to Africa, but also to infectible territories outside Africa, that it is impossible to over-emphasize the necessity for its initiation at the earliest possible moment”. Most would agree that the prospect for elimination of Ae. aegypti from Africa is not a realistic one at present, but that the need to reverse decades of neglect of arboviral disease in Africa is long overdue. To build an appropriate evidence base on which disease prevention and control strategies and policies can be founded, we recommend the following topics be prioritized for investigation: distribution of cases of dengue, chikungunya, Zika and other arboviruses in humans; distributions of Ae. aegypti and Ae. albopictus in Africa; updated characterization and distribution of insecticide resistance in Ae. aegypti and Ae. albopictus, with broader geographical coverage using standardized methodologies; development of context-speciﬁc Aedes-borne arboviral disease surveillance plans, and of outbreak prevention and vector control response strategies. Integral to the success of the above topics is the need for capacity strengthening in biology, diagnosis and control of arboviral disease vectors in all African nations under threat from these infections. Additional topics or knowledge gaps that merit investigation should include the following: controlled trials of vector interventions in African settings; the pros and cons of developing Aedes control in liaison or integrated with malaria elimination programmes; vector competence, peridomestic behaviour and biology of African Ae. aegypti, especially the ecotypes, and of the other Aedes spp. 11 of 20 Int. J. Environ. Res. Public Health 2018, 15, 220 previously incriminated or suspected as potential vectors; and occurrence and distribution of dengue, chikungunya and Zika virus infections in non-human primates and other potential ‘reservoir’ hosts. previously incriminated or suspected as potential vectors; and occurrence and distribution of dengue, chikungunya and Zika virus infections in non-human primates and other potential ‘reservoir’ hosts. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Supplementary Materials (Methods and References). Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Supplementary Materials (Methods and References). Acknowledgments: B.K. is supported by the Wellcome Trust (204862/Z/16/Z). C.L.M. and F.M.S. are supported by the Wellcome Trust (108440/Z/15/Z). L.L. Major Knowledge Gaps and Recommendations receives support from the Agence Nationale de la Recherche (grants ANR-16-CE35-0004-01 and ANR-17-ERC2-0016-01), the French Government’s Investissement d’Avenir program Laboratoire d’Excellence Integrative Biology of Emerging Infectious Diseases (grant ANR-10-LABX-62-IBEID), the City of Paris Emergence(s) program in Biomedical Research, the European Union’s Horizon 2020 research and innovation programme under ZikaPLAN grant agreement No. 734584, and the Ecology of Health program of Centre National de la Recherche Scientiﬁque (grant AFRICAEDES). A.B. is supported by a WHO/TDR grant (RCS-KM 2015 ID235974). 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https://openalex.org/W2751838734	https://europepmc.org/articles/pmc5618361?pdf=render	English	null	Dynamic Response of Graphitic Flakes in Nematic Liquid Crystals: Confinement and Host Effect	Nanomaterials	2,017	cc-by	10,012	Dynamic Response of Graphitic Flakes in Nematic Liquid Crystals: Conﬁnement and Host Effect Tae Hyung Kim 2, MinSu Kim 2 ID , Joong Hee Lee 2 and Seung Hee Lee 2,* 1 Key Laboratory of Micro-Nano Materials for Energy Storage and Conversion of Henan Province, Institute of Surface Micro and Nano Materials, College of Advanced Materials and Energy, Xuchang University, Xuchang 461000, Henan, China; dwtie929@hotmail.com (W.T.); surjyasarathi@gmail.com (S.S.B.); gyh-2007@sohu.com (Y.G.); zhengzhi99999@gmail.com (Z.Z.) y g , , J g g 1 Key Laboratory of Micro-Nano Materials for Energy Storage and Conversion of Henan Province, Institute o Surface Micro and Nano Materials, College of Advanced Materials and Energy, Xuchang University, Xuchang 461000, Henan, China; dwtie929@hotmail.com (W.T.); surjyasarathi@gmail.com (S.S.B.); gyh-2007@sohu.com (Y.G.); zhengzhi99999@gmail.com (Z.Z.) 2 Applied Materials Institute for BIN Convergence, Department of BIN Convergence Technology and Graduate School of Printable and Flexible Electronics, Chonbuk National University, Jeonju 561-756, Jeonbuk, Korea; zxcvb823@naver.com (E.J.S.); kth1811@naver.com (T.H.K.); mkim12@kent.edu (M.K.); jhl@jbnu.ac.kr (J.H.L.) 2 Applied Materials Institute for BIN Convergence, Department of BIN Convergence Technology and Graduate School of Printable and Flexible Electronics, Chonbuk National University, Jeonju 561-756, Jeonbuk, Korea; zxcvb823@naver.com (E.J.S.); kth1811@naver.com (T.H.K.); mkim12@kent.edu (M.K.); jhl@jbnu.ac.kr (J.H.L.) j j 3 Asutosh College, 92, Shyamaprasad Mukherjee Road, Kolkata 700 026, West Bengal, India * C d l h1@ h b k k T l 82 63 270 2443 3 Asutosh College, 92, Shyamaprasad Mukherjee Road, Kolkata 700 026, West Bengal, India * Correspondence: lsh1@chonbuk.ac.kr; Tel.: +82-63-270-2443 * Correspondence: lsh1@chonbuk.ac.kr; Tel.: +82-63-270-2443 Received: 22 July 2017; Accepted: 30 August 2017; Published: 1 September 2017 Received: 22 July 2017; Accepted: 30 August 2017; Published: 1 September 2017 Abstract: Electric ﬁeld-induced reorientation of suspended graphitic (GP) ﬂakes and its relaxation back to the original state in a nematic liquid crystal (NLC) host are of interest not only in academia, but also in industrial applications, such as polarizer-free and optical ﬁlm-free displays, and electro-optic light modulators. As the phenomenon has been demonstrated by thorough observation, the detailed study of the physical properties of the host NLC (the magnitude of dielectric anisotropy, elastic constants, and rotational viscosity), the size of the GP ﬂakes, and cell thickness, are urgently required to be explored and investigated. Here, we demonstrate that the response time of GP ﬂakes reorientation associated with an NLC host can be effectively enhanced by controlling the physical properties. Dynamic Response of Graphitic Flakes in Nematic Liquid Crystals: Conﬁnement and Host Effect In a vertical ﬁeld-on state, higher dielectric anisotropy and higher elasticity of NLC give rise to quicker reorientation of the GP ﬂakes (switching from planar to vertical alignment) due to the ﬁeld-induced coupling effect of interfacial Maxwell-Wagner polarization and NLC reorientation. In a ﬁeld off-state, lower rotational viscosity of NLC and lower cell thickness can help to reduce the decay time of GP ﬂakes reoriented from vertical to planar alignment. This is mainly attributed to strong coupling between GP ﬂakes and NLC originating from the strong π-π interaction between benzene rings in the honeycomb-like graphene structure and in NLC molecules. The high-uniformity of reoriented GP ﬂakes exhibits a possibility of new light modulation with a relatively faster response time in the switching process and, thus, it can show potential application in ﬁeld-induced memory and modulation devices. Keywords: graphitic ﬂakes; liquid crystal; Maxwell-Wagner polarization; dynamic response nanomaterials nanomaterials nanomaterials 1. Introduction The importance of electric ﬁeld-induced reorientation of non-spherical particles has gradually emerged, such that polymer cholesteric liquid crystal ﬂakes (PCLC) for electro-optical devices and displays have been widely studied [1,2]. The PCLC ﬂakes in a ﬂuid opened a possibility to realize a particle-based and electric ﬁeld-driven technology into a wide variety of applications such as displays and electronic papers with no optical polarizers and ﬁlters. Despite of the immense possibility of various applications, the development in switching technology of PCLC ﬂakes is still in an early stage. For instance, the uniformity of reorientation needs to be improved and the relaxation time is required to be shortened. www.mdpi.com/journal/nanomaterials www.mdpi.com/journal/nanomaterials Nanomaterials 2017, 7, 250; doi:10.3390/nano7090250 2 of 10 Nanomaterials 2017, 7, 250 Recently, an optically-opaque two-dimensional (2D) graphitic (GP) ﬂake, which consists of multi-layered graphene sheets by π-π stacking, can provide an opportunity for a new light modulation technology [3–5]. Such technology can give rise to sufﬁcient darkness in a ﬁeld-off state due to the planar conﬁguration of GP ﬂakes in nematic liquid crystals (NLCs) with respect to the substrate. Under an applied electric ﬁeld as low as tens of millivolts per micrometer, GP ﬂakes reorient almost 90◦from a planar face-up conﬁguration to a perpendicular edge-up conﬁguration, and a transmitting bright state is realized [5]. This switching technology upon electric ﬁeld-induced reorientation of GP ﬂakes relies on a combined interaction of the NLC director reorientation and Maxwell-Wagner polarization. Recent investigation over graphene oxide/thermotropic NLC-dispersed systems [6] has revealed electro-optical properties, viz. the threshold voltage and splay elastic constant of such systems strongly increase with increasing graphene oxide concentration. However, we have realized much reliable and reproducible switching technology for practical application devoid of electric ﬁeld-induced ﬂake aggregation. Information displays or electronic papers assisted by GP ﬂake switching could realize low power consumption, high brightness, and multi-color capability without the use of ﬁlters and polarizers, which have great potential for both commercial and scientiﬁc applications. The response time for reorientation of NLC can be described as: The response time for reorientation of NLC can be described as: τdecay = γ1d2 π2K = γ1 ε0\|∆ε\|E2c (1) τdecay = γ1d2 π2K = γ1 ε0\|∆ε\|E2c (1 (1) and: τrise = γ1 ε0\|∆ε\|E2 −(π2 d2 )K = γ1 ε0\|∆ε\|(E2 −E2c) (2) (2) where γ1, d, K, ε0, and ∆ε denote physical properties of a liquid crystal, viz. 2. Materials and Methods The functionalized GP ﬂakes have been synthesized through room-temperature oxidation of precursor graphite to generate graphite oxide and subsequent low-temperature thermal exfoliation to obtain functionalized graphene. The synthesized functionalized graphene mostly consists of an exfoliated graphene layer and some portion of the stacked layer structures with an expanded interlayer spacing of 7.5 Å. The named functionalized graphitic ﬂake generated in the present synthesis scheme contains fewer defects over graphene sheets (relatively unbroken sp2 hybridization carbon atom over graphene plane) and less oxygen content over the graphene plane in comparison with commercial graphene oxide. The shape, uniformity, and internal structure of the ﬂakes have been characterized using optical microscopy (OM) and scanning electron microscopy (SEM). The synthesis and detailed physical characterization of the GP ﬂakes used in present investigation have been described elsewhere [3,14]. Test cells named as Case I, Case II and Case III shown in Table 1 were prepared in order to investigate the NLC host type and cell thickness-dependent reorientation and relaxation behavior of the GP ﬂakes in NLC by comparing with the previous results as in [5]. Indium-tin-oxide (ITO) is used as electrodes on transparent glass substrates. Homogeneous alignment layers on both substrates are made by spin-coating a polyimide solution, viz. SE-6514 (Japan Synthetic Rubber Co., Tokyo, Japan), and then the substrates are baked at 70 ◦C for 5 min and 200 ◦C for one hour. The baked alignment layers are uniformly rubbed by a rubbing machine. The GP ﬂakes and NLC suspension is sandwiched by the rubbed ITO substrates in anti-parallel direction and the uniform cell thickness is maintained at ~44 µm using a ﬁlm spacer between the substrates. The cell thickness has been measured by comparing the capacitance of an empty cell and a benzene-ﬁlled cell using a 4284A Inductance capacitance resistance (LCR-meter) (Agilent Technologies, Santa Clara, CA, USA) [3,5,15]. Two commercially-available positive dielectric anisotropic NLC mixtures with different physical properties (dielectric anisotropy ∆ε = 5.3, rotational viscosity γ = 132 mPa·s, splay elastic constant K11 = 13.2 × 10−12 N), viz. ZLI-4792, and (∆ε = 14.8, γ = 148 mPa·s, K11 = 9.3 × 10−12 N), viz. ZLI-4535 (Merck, Tokyo, Japan), have been used for present investigation. The GP ﬂake-NLC suspensions have been prepared by adding ~0.005 wt % of ﬂake powders directly into the NLC mixtures. 1. Introduction 3 of 10 Nanomaterials 2017, 7, 250 Nanomaterials 2017, 7, 250 The response time of such system is deﬁned in [5], and it can be further modiﬁed as: The response time of such system is deﬁned in [5], and it can be further modiﬁed as: τ ≈ 2π√ρaRg r 4π 3 liBiE2 + 2AK − γ2 1 4ρaRg2 , (4) (4) where notations represent that the mass per unit area of ﬂakes ρ, the projected area of ﬂakes a, radius of gyration Rg, dimension of ﬂakes in the axes (i = x, y, z) l, and a multiplicative constant A. The Clausius-Mosotti factor is deﬁned by: Bi = εp −εh εh + Li εp −εh , Bi = εp −εh εh + Li εp −εh , where Li is the geometrical depolarization factor. Therefore, variables in Equation (4), such as the physical properties of the NLC host, size, and aspect ratio of GP ﬂakes, will provide a detailed contribution to the response time of the reorientation, as well as the relaxation process of the GP ﬂakes. where Li is the geometrical depolarization factor. Therefore, variables in Equation (4), such as the physical properties of the NLC host, size, and aspect ratio of GP ﬂakes, will provide a detailed contribution to the response time of the reorientation, as well as the relaxation process of the GP ﬂakes. 2 M t i l d M th d 1. Introduction the rotational viscosity, the cell thickness, the elastic constant, the dielectric constant in vacuum, and the dielectric anisotropy, respectively, and Ec, and E denote the critical electric ﬁeld and an external electric ﬁeld, respectively [6–10]. This implies that reducing γ1 and d, and increasing K and ∆ε will contribute to a decrease in the response time of liquid crystals. GP ﬂakes, while suspended in liquid crystal materials, reorient and relax following the liquid crystal director [5,10]. The simpliﬁed model to describe such an inhomogeneous conﬁguration can be a double-layered structure with different dielectric permittivity ε and conductivity σ of each material, and the relaxation frequency can be described as [10]: τ = κε0 ε1 + ε2 σ1 + σ2 (3) (3) This interaction can be further explained as the electric ﬁeld-induced potential energy of GP ﬂakes coupling with the contribution of positive NLC reorientation to generate the rotational kinetic energy for ﬂake reorientation [5]. More importantly, the ﬁeld-induced reorientation of GP ﬂakes relaxes back to their initial planar face-up state for recovering the dark ﬁeld-off state aided by the NLC director reorientation. This reversible reorientation of GP ﬂakes allows optical modulation controlled by an electric ﬁeld signal. Although the total response time, i.e., the time to complete both the reorientation and relaxation process is faster than PCLC ﬂakes in general [1,2], the response time of the GP ﬂakes still needs to be further improved for practical applications. We have carefully studied the dynamic reorientation phenomenon of graphene/graphitic ﬂakes in NLCs under an electric ﬁeld [5]. In the ﬁeld-on state, we have found that the reorientation time of the GP device can be efﬁciently improved by optimizing several parameters, such as the electric ﬁeld strength, the ﬂake anisotropy, as well as the ﬂake size and, again, the reorientation depends on the ﬁeld-induced synergistic effect of interfacial Maxwell-Wagner polarization and NLC director reorientation. It is also found that π-π interaction between the GP surface and NLC molecule is so strong that the surface anchoring between the NLC molecules and GP ﬂakes will allow such GP ﬂakes following the NLC directors to reorient it collaboratively [7,8,11–13]. Thus, further investigation on the contribution of NLC director reorientation to the GP ﬂake reorientation and the relaxation process is urgently required. 2. Materials and Methods In order to achieve the homogeneity of the dispersion of ﬂakes with minimum damage, the suspensions are ultrasonicated for one hour, keeping the temperature at 30 ◦C (frequency, 28 kHz; input power, 60 W). 4 of 10 Nanomaterials 2017, 7, 250 Table 1. Prepared test cells. Samples LC Cell Thickness (µm) Case I ZLI-4792 44 Case II ZLI-4535 44 Case III (from reference [5]) ZLI-4792 70 The used GP ﬂake-NLC suspensions with ZLI-4792 and ZLI-4535 are ﬁlled into test cells and named as Case I, Case II, and Case III [5], respectively, as shown in Table 2. Samples are ﬁlled into the mentioned cells by capillary action at room temperature. Although most of the GP ﬂakes in the GP-NLC suspension are supposed to be single-layered, or composed of a few layers of graphene after exfoliation, some thicker graphitic stacks do remain suspended in the mixture, exhibiting the desired dark state. They are characterized by optical microscopy, having signiﬁcantly distinct thickness formed by several hundred layers forming an optically opaque state, and suspended in the planar face-up conﬁguration in NLCs with an area of 10–300 µm2 and, hence, a volume of 10–450 µm3. The sample textures are captured by a Panasonic 5100 digital camera with a time resolution of 100 ms attached with optical microscope (OM, Nikon DXM1200, Tokyo, Japan) while test cells are driven by an AC ﬁeld (0 to 200 mV/µm) with a constant driving frequency (60 Hz) using an arbitrary function generator (Tektronix AFG3022, Beaverton, OR, USA). The transmitted light intensity of the captured images has been analyzed with the help of IMT i-Solution software (Image and Microscope Technology Co.). Table 2. Measured GP ﬂakes of Case I, Case II and Case III. Samples Flake Area (µm2) Flake Aspect Ratio (µm) Case I Case 1: 35 Case 1: 2:1 Case 2: 85 Case 2: 1:1 Case II Case 1: 37 Case 2: 2:1 Case 2: 90 Case 2: 1:1 Case III (from reference [5]) Case 1: 65 Case 2: 2:1 Case 2: 85 Case 2: 2.6:1 Table 2. Measured GP ﬂakes of Case I, Case II and Case III. 3. Results and Discussion (Color online) Optical microphotographs exhibiting ﬁeld-induced reorientation of representative GP ﬂakes with similar ﬂake areas and aspect ratios in low (a,b) and high (c,d) dielectric anisotropy nematic liquid crystal. (a–d) Case I: ﬂake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: ﬂake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1. Figure 1. (Color online) Optical microphotographs exhibiting field-induced reorientation of representative GP flakes with similar flake areas and aspect ratios in low (a,b) and high (c,d) dielectric anisotropy nematic liquid crystal. (a–d) Case I: flake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: flake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1. 0 50 100 150 200 0 2 4 6 8 10 12 14 16 Field on state Field / mV / μm-1 Variation of light intensity / a.u. (a) (b) (c) (d) Figure 2. The measured reorientation-induced light intensity variation of representative GP flakes according to Figure 1. The applied electric fields for Case I and Case II are 0, 22, 44, 67, 90, 100, 112, 134, 156, 180, and 200 mV/µm, respectively (Case I: flake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: flake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1). The scattered points represent experimental data along with measurement error bars and the lines are guides for the eye. Error bars for respective data points are assigned considering standard deviations obtained in the repetitive observations 0 50 100 150 200 0 2 4 6 8 10 12 14 16 Field on state Field / mV / μm-1 Variation of light intensity / a.u. (a) (b) (c) (d) Figure 2. The measured reorientation-induced light intensity variation of representative GP flakes according to Figure 1. 3. Results and Discussion The scattered points represent experimental data along with measurement error bars and the lines are guides for the eye. Error bars for respective data points are assigned considering standard deviations obtained in the i i b i Figure 2. The measured reorientation-induced light intensity variation of representative GP flakes according to Figure 1. The applied electric fields for Case I and Case II are 0, 22, 44, 67, 90, 100, 112, 134, 156, 180, and 200 mV/µm, respectively (Case I: flake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: flake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1). The scattered points represent experimental data along with measurement error bars and the lines are guides for the eye. Error bars for respective data points are assigned considering standard deviations obtained in the repetitive observations. Figure 2. The measured reorientation-induced light intensity variation of representative GP ﬂakes according to Figure 1. The applied electric ﬁelds for Case I and Case II are 0, 22, 44, 67, 90, 100, 112, 134, 156, 180, and 200 mV/µm, respectively (Case I: ﬂake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: ﬂake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1). The scattered points represent experimental data along with measurement error bars and the lines are guides for the eye. Error bars for respective data points are assigned considering standard deviations obtained in the repetitive observations. Figure 3 shows variation of transmitted light intensity as a function of relaxation time of functionalized graphene flakes in the LC matrix. It is interesting to mention here that the transmission intensity around GP flakes is found to be independent of the electric field. The flakes gradually recover to their initial state and, hence, the corresponding transmittance decreases after Figure 3 shows variation of transmitted light intensity as a function of relaxation time of functionalized graphene flakes in the LC matrix. It is interesting to mention here that the transmission intensity around GP flakes is found to be independent of the electric field. 3. Results and Discussion y p q g p consideration of the field strength dependent reorientation time previously discussed in detail [5], we have investigated the influence of cell confinement on the response time of GP flakes in the next section. Figure 1. (Color online) Optical microphotographs exhibiting field-induced reorientation of representative GP flakes with similar flake areas and aspect ratios in low (a,b) and high (c,d) dielectric anisotropy nematic liquid crystal. (a–d) Case I: flake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: flake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1. Figure 1. (Color online) Optical microphotographs exhibiting ﬁeld-induced reorientation of representative GP ﬂakes with similar ﬂake areas and aspect ratios in low (a,b) and high (c,d) dielectric anisotropy nematic liquid crystal. (a–d) Case I: ﬂake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: ﬂake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1. Figure 1. (Color online) Optical microphotographs exhibiting field-induced reorientation of representative GP flakes with similar flake areas and aspect ratios in low (a,b) and high (c,d) dielectric anisotropy nematic liquid crystal. (a–d) Case I: flake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: flake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1. Figure 1. (Color online) Optical microphotographs exhibiting field-induced reorientation of representative GP flakes with similar flake areas and aspect ratios in low (a,b) and high (c,d) dielectric anisotropy nematic liquid crystal. (a–d) Case I: flake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: flake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1. Figure 1. 3. Results and Discussion The micrometer-sized GP ﬂakes dispersed in the homogeneously-aligned NLC host in the mentioned cell geometry have been observed under an optical microscope. Figure 1 shows the representative case study of arbitrarily-shaped GP ﬂakes’ reorientation for comparable morphology at ﬁeld-off and ﬁeld-on states with a ﬁxed AC electric ﬁeld strength of 90.0 mV/µm. Areas of suspended GP ﬂakes in the ﬁeld-off planar face-up conﬁguration are microscopically measured to be 35 and 85 µm2 in the NLC host of ZIL4792 (a,b) (Case I) and those for the GP ﬂake counterpart are 37 and 90 µm2 in the NLC host of ZIL4535 (c,d) (Case II), respectively. Figure 1 clearly displays all GP ﬂakes reorient from face-up to almost edge-up conﬁguration around their long axis parallel to the applied ﬁeld direction at ﬁeld-on state. Once the driving ﬁeld is removed, the GP ﬂakes relax back to their planar initial state. This observed reorientation behavior is reliable and reproducible under hundreds of trials. Figure 2 depicts the ﬁeld-dependent variation of transmitted light intensity surrounding GP ﬂakes. The optical transmittance has been measured locally on regions surrounding the GP ﬂake in an area ~20 µm × 20 µm from optical microscopic images using i-Solution image analysis software and data points are plotted while the solid line represents a guide for the eye to the experimental data. Initially, transmission intensity is low as most of the transmission is blocked by the considerably large, effectively exposed area of the opaque ﬂake in the direction of propagation of light. As the ﬂakes reorient with the increasing applied ﬁeld, the transmission gradually increases and, ﬁnally, reach 5 of 10 creases eases mum Nanomaterials 2017, 7, 250 light. As the flakes re light. As the flakes reo d fi ll h th their maximum transmittance for each case. Normally, a different maximum transmission intensity is ascribed to the different thicknesses of opaque graphitic ﬂakes. In consideration of the ﬁeld strength dependent reorientation time previously discussed in detail [5], we have investigated the inﬂuence of cell conﬁnement on the response time of GP ﬂakes in the next section. transmission intensity is ascribed to the different thicknesses of opaque graphitic flakes. In consideration of the field strength dependent reorientation time previously discussed in detail [5], we have investigated the influence of cell confinement on the response time of GP flakes in the next section. 3. Results and Discussion The applied electric fields for Case I and Case II are 0, 22, 44, 67, 90, 100, 112, 134, 156, 180, and 200 mV/µm, respectively (Case I: flake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: flake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1). The scattered points represent experimental data along with measurement error bars and the lines are guides for the eye. Error bars for respective data points are assigned considering standard deviations obtained in the repetitive observations. Figure 2. The measured reorientation-induced light intensity variation of representative GP ﬂakes according to Figure 1. The applied electric ﬁelds for Case I and Case II are 0, 22, 44, 67, 90, 100, 112, 134, 156, 180, and 200 mV/µm, respectively (Case I: ﬂake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: ﬂake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1). The scattered points represent experimental data along with measurement error bars and the lines are guides for the eye. Error bars for respective data points are assigned considering standard deviations obtained in the repetitive observations. 0 50 100 150 200 0 2 4 6 8 10 12 14 16 Field on state Fi ld / V / -1 Variation of light intensity / a.u. (a) (b) (c) (d) 0 50 100 150 200 0 2 4 6 8 10 12 14 16 Field on state Field / mV / μm-1 Variation of light intensity / a.u. (a) (b) (c) (d) Figure 2. The measured reorientation-induced light intensity variation of representative GP flakes according to Figure 1. The applied electric fields for Case I and Case II are 0, 22, 44, 67, 90, 100, 112, 134, 156, 180, and 200 mV/µm, respectively (Case I: flake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: flake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1). 3. Results and Discussion Error bars for respective data points are assigned considering standard deviations obtained in repetitive observations. Figure 3. The measured relaxation-induced light intensity variation of representative GP ﬂakes according to Figure 1. The ﬁeld off state for Case I is 0 s, 0.5 s, 1 s, 1.5 s, and 2 s, and for Case II is 0 s, 1.0 s, 2.0 s, 3.0 s, and 4.0 s, respectively (Case I: ﬂake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: ﬂake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1). The scattered points represent the experimental data along with measurement error bars, and the lines are guides for the eye. Error bars for respective data points are assigned considering standard deviations obtained in repetitive observations. Figure 4 shows the NLC host-type and cell thickness-dependent reorientation time of GP flakes with comparable flake morphology under gradually-increasing AC electric field strength from 0 mV/µm to 200.0 mV/µm. It is worth mentioning here that the reported reorientation time has been measured from hundreds of case studies for flakes of several different shapes with aspect ratios from 1:1 to 1:3 and flake area in the range 20 µm2 to 90 µm2 while suspended in different NLC materials viz. ZIL4792 and ZIL4535, and variable cell confinement, i.e., cell thicknesses of 44 µm and 70 µm. Hence, we have classified numerous case studies for the reorientation time of GP flakes based on widely different dielectric anisotropy of the NLC host and cell confinement, i.e., the thickness of the used cells. Case I, Case II, and Case III of Figure 4 exhibit the reorientation time results in representative groups obtained under different experimental conditions with low dielectric anisotropy of the NLC host and low cell thickness, high dielectric anisotropy of the NLC host, and low cell thickness, low dielectric anisotropy of the host NLC, and high cell thickness, respectively. Reasonable differences obtained in the reorientation time of GP flakes in the NLC matrix for unique magnitudes of applied electric fields are represented by vertical error bars. 3. Results and Discussion The flakes gradually recover to their initial state and, hence, the corresponding transmittance decreases after Figure 3 shows variation of transmitted light intensity as a function of relaxation time of functionalized graphene ﬂakes in the LC matrix. It is interesting to mention here that the transmission intensity around GP ﬂakes is found to be independent of the electric ﬁeld. The ﬂakes gradually recover to their initial state and, hence, the corresponding transmittance decreases after ﬁeld removal. Experimental data is represented as scattered points with the solid line as a guide for the eye. However, 6 of 10 6 of 10 Nanomaterials 2017, 7, 250 Nanomaterials 2017, 7, 250 it is observed that the ﬂakes suspended in different NLC hosts recover to their initial state with different relaxation times, which will be mainly discussed in the following section. The mentioned observation conﬁrms the reversible reorientation of GP ﬂakes according to the optical images of Figure 1. Hence, the light transmission modulation is evidently controlled by GP ﬂake reorientation and relaxation. Thus, electric ﬁeld-controlled uniform reorientation and relaxation of GP ﬂakes and the subsequent modulation of transmission intensity provide us a promising method for making new polarizer-independent electro-optic ﬂake devices. the eye. However, it is observed that the flakes suspended in different NLC hosts recover to their initial state with different relaxation times, which will be mainly discussed in the following section. The mentioned observation confirms the reversible reorientation of GP flakes according to the optical images of Figure 1. Hence, the light transmission modulation is evidently controlled by GP flake reorientation and relaxation. Thus, electric field-controlled uniform reorientation and relaxation of GP flakes and the subsequent modulation of transmission intensity provide us a promising method for making new polarizer-independent electro-optic flake devices. 0 1 2 3 4 -2 0 2 4 6 8 10 12 14 16 Variation of Light intensity / a.u. Relaxing time / sec Field off state (a) (b) (c) (d) Figure 3. The measured relaxation-induced light intensity variation of representative GP flakes according to Figure 1. 3. Results and Discussion The field off state for Case I is 0 s, 0.5 s, 1 s, 1.5 s, and 2 s, and for Case II is 0 s, 1.0 s, 2.0 s, 3.0 s, and 4.0 s, respectively (Case I: flake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: flake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1). The scattered points represent the experimental data along with measurement error bars, and the lines are guides for the eye. Error bars for respective data points are assigned considering standard deviations obtained in repetitive observations. Figure 3. The measured relaxation-induced light intensity variation of representative GP ﬂakes according to Figure 1. The ﬁeld off state for Case I is 0 s, 0.5 s, 1 s, 1.5 s, and 2 s, and for Case II is 0 s, 1.0 s, 2.0 s, 3.0 s, and 4.0 s, respectively (Case I: ﬂake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: ﬂake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1). The scattered points represent the experimental data along with measurement error bars, and the lines are guides for the eye. Error bars for respective data points are assigned considering standard deviations obtained in repetitive observations. Figure 3. The measured relaxation-induced light intensity variation of representative GP flakes according to Figure 1. The field off state for Case I is 0 s, 0.5 s, 1 s, 1.5 s, and 2 s, and for Case II is 0 s, 1.0 s, 2.0 s, 3.0 s, and 4.0 s, respectively (Case I: flake area and aspect ratio in (a,b): 35 µm2 with an aspect ratio of 2:1 and 85 µm2 with an aspect ratio of 1:1; Case II: flake area and aspect ratio in (c,d): 37 µm2 with an aspect ratio of 2:1 and 90 µm2 with an aspect ratio of 1:1). The scattered points represent the experimental data along with measurement error bars, and the lines are guides for the eye. 3. Results and Discussion 40 80 120 160 200 0 1 2 3 4 5 Case I: Experimental Data Low γ / low d Model Fitted Case I Case II: Experimental Data High γ / low d Model Fitted Case II Case III: Experimental Data Low γ / High d Model Fitted Case III Electric field / mVμm-1 Reorientation time / sec Figure 4. The averaged reorientation time of representative GP flakes with different NLC host-types and cell thicknesses as a function of the electric field (Case I: low γ, low d; Case II: high γ, low d; Case III: low γ, high d). The scattered points represents experimental data, along with measurement error bars and the lines, are model fitted with Y = P1/(X2 + P2)0.5 following Equation (4), where P1 is the numerator of Equation (4), and P2 is the constant part in the denominator of Equation (4) divided by coefficient of E2. Error bars for respective data points are assigned considering the standard deviations obtained of repetitive observations. Figure 4. The averaged reorientation time of representative GP ﬂakes with different NLC host-types and cell thicknesses as a function of the electric ﬁeld (Case I: low γ, low d; Case II: high γ, low d; Case III: low γ, high d). The scattered points represents experimental data, along with measurement error bars and the lines, are model ﬁtted with Y = P1/(X2 + P2)0.5 following Equation (4), where P1 is the numerator of Equation (4), and P2 is the constant part in the denominator of Equation (4) divided by coefﬁcient of E2. Error bars for respective data points are assigned considering the standard deviations obtained of repetitive observations. As the reorientation time of GP flakes in NLC with the field switched on is described in Equation (2), in Case I and Case II it is observed that strong dependence of the NLC host-types in identical cell confinement conditions exists. It is also found that GP flakes in NLC, viz. ZLI-4792 (Case I), having lower dielectric anisotropy (Δε = 5.3), lower rotational viscosity (γ = 132 mPa·s), and higher splay elastic constant (K11 = 13.2 × 10−12 N) reorient quicker than that in NLC, viz. ZLI-4535 (Case II), having higher dielectric anisotropy (Δε = 14.8), higher rotational viscosity (γ = 148 mPa·s), and lower (K11 = 9.3 × 10−12 N). 3. Results and Discussion The positive dielectric anisotropic NLC host experiences dielectric torque proportionate with the dielectric anisotropy of the NLC host under an oscillatory AC field. As the π-π interaction between the GP surface and NLC molecules is well known [5–8,11–13], NLC molecules adsorbed over the GP surface should induce a proportionate dielectric torque induced over the NLC host by the oscillating field to the suspended GP flakes. In addition, a comparable Maxwell-Wagner polarization is assumed to be induced over the GP surface under similar experimental conditions. Hence, GP flakes suspended in NLC, viz. ZLI-4535, should experience stronger dielectric torque in comparison with GP flakes suspended in NLC, viz. ZLI-4792, and should reorient faster. However, the larger rotational viscosity of ZLI-4535 hinders the reorientation process and GP flakes suspended in ZLI-4792 exhibit faster reorientation time, which agrees with our previous model that the rotational viscosity is one important factor for GP flakes’ reorientation As the reorientation time of GP ﬂakes in NLC with the ﬁeld switched on is described in Equation (2), in Case I and Case II it is observed that strong dependence of the NLC host-types in identical cell conﬁnement conditions exists. It is also found that GP ﬂakes in NLC, viz. ZLI-4792 (Case I), having lower dielectric anisotropy (∆ε = 5.3), lower rotational viscosity (γ = 132 mPa·s), and higher splay elastic constant (K11 = 13.2 × 10−12 N) reorient quicker than that in NLC, viz. ZLI-4535 (Case II), having higher dielectric anisotropy (∆ε = 14.8), higher rotational viscosity (γ = 148 mPa·s), and lower (K11 = 9.3 × 10−12 N). The positive dielectric anisotropic NLC host experiences dielectric torque proportionate with the dielectric anisotropy of the NLC host under an oscillatory AC ﬁeld. As the π-π interaction between the GP surface and NLC molecules is well known [5–8,11–13], NLC molecules adsorbed over the GP surface should induce a proportionate dielectric torque induced over the NLC host by the oscillating ﬁeld to the suspended GP ﬂakes. In addition, a comparable Maxwell-Wagner polarization is assumed to be induced over the GP surface under similar experimental conditions. Hence, GP ﬂakes suspended in NLC, viz. ZLI-4535, should experience stronger dielectric torque in comparison with GP ﬂakes suspended in NLC, viz. ZLI-4792, and should reorient faster. 3. Results and Discussion Such variations are attributed to the differences in shape, area, and aspect ratio of the GP flakes suspended in the NLC matrix within the abovementioned range, as discussed in our earlier publication [5]. The electric field-dependent reorientation time of GP flakes shows a wide range of variation from 6.5 s to 1.5 s. Reorientation time of GP flakes in the NLC matrix clearly exhibits a decreasing trend with increasing Figure 4 shows the NLC host-type and cell thickness-dependent reorientation time of GP ﬂakes with comparable ﬂake morphology under gradually-increasing AC electric ﬁeld strength from 0 mV/µm to 200.0 mV/µm. It is worth mentioning here that the reported reorientation time has been measured from hundreds of case studies for ﬂakes of several different shapes with aspect ratios from 1:1 to 1:3 and ﬂake area in the range 20 µm2 to 90 µm2 while suspended in different NLC materials viz. ZIL4792 and ZIL4535, and variable cell conﬁnement, i.e., cell thicknesses of 44 µm and 70 µm. Hence, we have classiﬁed numerous case studies for the reorientation time of GP ﬂakes based on widely different dielectric anisotropy of the NLC host and cell conﬁnement, i.e., the thickness of the used cells. Case I, Case II, and Case III of Figure 4 exhibit the reorientation time results in representative groups obtained under different experimental conditions with low dielectric anisotropy of the NLC host and low cell thickness, high dielectric anisotropy of the NLC host, and low cell thickness, low dielectric anisotropy of the host NLC, and high cell thickness, respectively. Reasonable differences obtained in the reorientation time of GP ﬂakes in the NLC matrix for unique magnitudes of applied electric ﬁelds are represented by vertical error bars. Such variations are attributed to the differences in shape, area, and aspect ratio of the GP ﬂakes suspended in the NLC matrix within the abovementioned range, as discussed in our earlier publication [5]. The electric ﬁeld-dependent reorientation time of GP ﬂakes shows a wide range of variation from 6.5 s to 1.5 s. Reorientation time of GP ﬂakes in the NLC matrix clearly exhibits a decreasing trend with increasing applied electric ﬁeld strength until saturation for all 7 of 10 7 of 10 Nanomaterials 2017, 7, 250 Nanomaterials 2017, 7, 250 three cases. 3. Results and Discussion The vertical error bars for respective data points are assigned considering the standard deviations obtained through repetitive observations are also found to be signiﬁcantly suppressed due to the high reproducibility of the recorded reorientation time at signiﬁcantly higher ﬁelds. data points are assigned considering the standard deviations obtained through repetitive observations are also found to be significantly suppressed due to the high reproducibility of the recorded reorientation time at significantly higher fields. 40 80 120 160 200 0 1 2 3 4 5 Case I: Experimental Data Low γ / low d Model Fitted Case I Case II: Experimental Data High γ / low d Model Fitted Case II Case III: Experimental Data Low γ / High d Model Fitted Case III Electric field / mVμm-1 Reorientation time / sec Figure 4. The averaged reorientation time of representative GP flakes with different NLC host-types and cell thicknesses as a function of the electric field (Case I: low γ, low d; Case II: high γ, low d; Case III: low γ, high d). The scattered points represents experimental data, along with measurement error bars and the lines, are model fitted with Y = P1/(X2 + P2)0.5 following Equation (4), where P1 is the numerator of Equation (4), and P2 is the constant part in the denominator of Equation (4) divided by coefficient of E2. Error bars for respective data points are assigned considering the standard deviations obtained of repetitive observations. Figure 4. The averaged reorientation time of representative GP ﬂakes with different NLC host-types and cell thicknesses as a function of the electric ﬁeld (Case I: low γ, low d; Case II: high γ, low d; Case III: low γ, high d). The scattered points represents experimental data, along with measurement error bars and the lines, are model ﬁtted with Y = P1/(X2 + P2)0.5 following Equation (4), where P1 is the numerator of Equation (4), and P2 is the constant part in the denominator of Equation (4) divided by coefﬁcient of E2. Error bars for respective data points are assigned considering the standard deviations obtained of repetitive observations. 3. Results and Discussion However, the larger rotational viscosity of ZLI-4535 hinders the reorientation process and GP ﬂakes suspended in ZLI-4792 exhibit faster reorientation time, which agrees with our previous model that the rotational viscosity is one important factor for GP ﬂakes’ reorientation time [5]. time [5]. In order to determine the cell confinement conditions necessary for GP device application we have further examined the reorientation time of GP flakes in NLC, viz. ZLI-4792, with widely different cell thicknesses of ~44 µm (Case I) and 70 µm (Case III). The experimental results clearly suggest GP flakes suspended in thinner cells reorient faster than in thicker cells. The phenomenon In order to determine the cell conﬁnement conditions necessary for GP device application we have further examined the reorientation time of GP ﬂakes in NLC, viz. ZLI-4792, with widely different cell thicknesses of ~44 µm (Case I) and 70 µm (Case III). The experimental results clearly suggest GP ﬂakes suspended in thinner cells reorient faster than in thicker cells. The phenomenon might be anticipated from the considerable difference in the anchoring conditions of the NLC molecules 8 of 10 8 of 10 Nanomaterials 2017, 7, 250 Nanomaterials 2017, 7, 250 in widely different cell thicknesses. The thicker cell (Case III) imposes weaker anchoring over NLC molecules, and dielectric torque-induced reorientation, and subsequent secondary-induced torque over suspended GP ﬂakes remain weaker than its thinner counterpart (Case I and Case II). Hence, dependence of the reorientation time over the nature of NLC host properties is evident. q q secondary-induced torque over suspended GP flakes remain weaker than its thinner counterpart (Case I and Case II). Hence, dependence of the reorientation time over the nature of NLC host properties is evident. For practical display applications the relaxation time of the GP device is also indispensable. The For practical display applications the relaxation time of the GP device is also indispensable. The relaxation process of GP flakes in the NLC matrix from the field-aligned edge-up state to the planar face-up state after removing the electric field is recorded under an optical microscope. We have defined the relaxation time as the time taken by GP flakes to relax from the edge-up to the face-up configuration. Figure 5 shows the NLC host-type and cell thickness-dependent relaxation time of GP flakes with comparable flake morphology as a function of reorienting the AC electric field strength from 40 mV/µm to 200 mV/µm. 3. Results and Discussion Every case study of GP flakes’ field-off relaxation is followed by the abovementioned electric field-induced GP flake reorientation. Thus, an equivalent number of case studies in different NLC matrices and cell confinement with identical experimental conditions to the investigation of reorientation time is guaranteed. We have further regrouped the experimental relaxation time results similar to earlier demonstrated reorientation time cases. Hence, Case I, Case II, and Case III of Figure 5 exhibit the relaxation time results in representative groups obtained under different experimental conditions with low rotational viscosity of the NLC host and low cell thickness, high rotational viscosity of the NLC host, and low cell thickness, low rotational viscosity of the NLC host and high cell thickness, respectively. Reasonable differences are obtained in the relaxation time of GP flakes in the NLC matrix for distinctive magnitudes of the applied electric field, represented by vertical error bars derived from the standard deviations obtained in repetitive observations. Such variations are attributed to the differences in shape, area, and aspect ratio of GP flakes suspended in the NLC matrix within the previously-mentioned range, as discussed in our earlier publication [5]. The relaxation time of GP flakes is found to be independent of the applied reorienting field strength, as is the uncertainty in the relaxation time computation. However, relaxation time strongly depends on the confinement condition of the GP flakes in the NLC matrix, as well as the nature of the NLC host material. relaxation process of GP flakes in the NLC matrix from the field-aligned edge-up state to the planar face-up state after removing the electric field is recorded under an optical microscope. We have defined the relaxation time as the time taken by GP flakes to relax from the edge-up to the face-up configuration. Figure 5 shows the NLC host-type and cell thickness-dependent relaxation time of GP flakes with comparable flake morphology as a function of reorienting the AC electric field strength from 40 mV/µm to 200 mV/µm. Every case study of GP flakes’ field-off relaxation is followed by the abovementioned electric field-induced GP flake reorientation. Thus, an equivalent number of case studies in different NLC matrices and cell confinement with identical experimental conditions to the investigation of reorientation time is guaranteed. We have further regrouped the experimental relaxation time results similar to earlier demonstrated reorientation time cases. 3. Results and Discussion Hence, Case I, Case II, and Case III of Figure 5 exhibit the relaxation time results in representative groups obtained under different experimental conditions with low rotational viscosity of the NLC host and low cell thickness, high rotational viscosity of the NLC host, and low cell thickness, low rotational viscosity of the NLC host and high cell thickness, respectively. Reasonable differences are obtained in the relaxation time of GP flakes in the NLC matrix for distinctive magnitudes of the applied electric field, represented by vertical error bars derived from the standard deviations obtained in repetitive observations. Such variations are attributed to the differences in shape, area, and aspect ratio of GP flakes suspended in the NLC matrix within the previously-mentioned range, as discussed in our earlier publication [5]. The relaxation time of GP flakes is found to be independent of the applied reorienting field strength, as is the uncertainty in the relaxation time computation. However, relaxation time strongly depends on the confinement condition of the GP flakes in the NLC matrix, as well as the nature of the NLC host material. 40 60 80 100 120 140 160 180 200 220 0 1 2 3 4 5 6 7 Electric field / mVμm-1 Relaxation time / sec Case I Low γ / low d Case II High γ / low d Case III Low γ / high d Figure 5. The relationship of the averaged relaxation time of representative GP flakes with different NLC host-types and cell thicknesses after removing the electric field (Case I: low γ, low d; Case II: high γ, low d; and Case III: low γ, high d). The scattered points represent the experimental data along with the measurement error bars, and the lines are guides for the eye. Error bars for respective data points are assigned considering the standard deviations obtained in repetitive observations. The GP flakes in NLC viz ZLI-4792 (Case I) having lower rotational viscosity (γ = 132 mPa Figure 5. The relationship of the averaged relaxation time of representative GP ﬂakes with different NLC host-types and cell thicknesses after removing the electric ﬁeld (Case I: low γ, low d; Case II: high γ, low d; and Case III: low γ, high d). The scattered points represent the experimental data along with the measurement error bars, and the lines are guides for the eye. 3. Results and Discussion Error bars for respective data points are assigned considering the standard deviations obtained in repetitive observations. Electric field / mVμm-1 Figure 5. The relationship of the averaged relaxation time of representative GP flakes with different NLC host-types and cell thicknesses after removing the electric field (Case I: low γ, low d; Case II: high γ, low d; and Case III: low γ, high d). The scattered points represent the experimental data along with the measurement error bars, and the lines are guides for the eye. Error bars for respective data points are assigned considering the standard deviations obtained in repetitive observations. Figure 5. The relationship of the averaged relaxation time of representative GP ﬂakes with different NLC host-types and cell thicknesses after removing the electric ﬁeld (Case I: low γ, low d; Case II: high γ, low d; and Case III: low γ, high d). The scattered points represent the experimental data along with the measurement error bars, and the lines are guides for the eye. Error bars for respective data points are assigned considering the standard deviations obtained in repetitive observations. Figure 5. The relationship of the averaged relaxation time of representative GP flakes with different NLC host-types and cell thicknesses after removing the electric field (Case I: low γ, low d; Case II: high γ, low d; and Case III: low γ, high d). The scattered points represent the experimental data along with the measurement error bars, and the lines are guides for the eye. Error bars for respective data points are assigned considering the standard deviations obtained in repetitive observations. Figure 5. The relationship of the averaged relaxation time of representative GP ﬂakes with different NLC host-types and cell thicknesses after removing the electric ﬁeld (Case I: low γ, low d; Case II: high γ, low d; and Case III: low γ, high d). The scattered points represent the experimental data along with the measurement error bars, and the lines are guides for the eye. Error bars for respective data points are assigned considering the standard deviations obtained in repetitive observations. 9 of 10 Nanomaterials 2017, 7, 250 The GP ﬂakes in NLC, viz. ZLI-4792 (Case I), having lower rotational viscosity (γ = 132 mPa·s) and higher splay elastic constant (K11 = 13.2 × 10−12 N) relax faster than that in NLC, viz. 3. Results and Discussion ZLI-4535 (Case II), having higher rotational viscosity (γ = 148 mPa·s) and lower (K11 = 9.3 × 10−12 N) in a similar conﬁnement condition. The relaxation process of GP ﬂakes in the NLC matrix is attributed to the relaxation of the NLC host. The π-π electron stacking between the graphene’s honeycomb structure and the NLC’s benzene rings results in a considerable amount of charge transfer and strong binding of NLC molecules over graphene surface with a binding energy of Uanchoring = −2 eV [7,8,13]. Density functional calculations further support the said facts [16]. In relaxation, this strong anchoring energy between NLC molecules and the graphene surface drives the GP ﬂake to rotate following relaxation of the NLC director. However, the rotational viscosity of the NLC matrix hinders the relaxation process and, hence, faster relaxation time has been realized in the NLC host having lower rotational viscosity, viz. ZLI-4792 (Case I). In addition, the cell thickness-dependent relaxation time of GP ﬂakes with the same NLC host (Case I and Case III) has also been reported in Figure 5. Here, GP ﬂakes’ relaxation time in NLC, viz. ZLI-4792, with 44 µm (Case I) has been compared to that in NLC, viz. ZLI-4792, with 70 µm (Case III), where the GP ﬂakes with similar morphology have been tested [5]. The averaged relaxation time in Case I with lower d is found to be quicker than that in Case III. As mentioned earlier, the thinner cell imposes a stronger surface-induced anchoring over conﬁned NLC molecules. Hence, NLC molecules adsorbed over the GP surface will induce stronger relaxing torque onto the GP ﬂake due to the strong coupling of graphene/NLC than its thicker counterpart. Hence, dependence of the relaxation time over cell conﬁnement conditions is evident. This observation is in compliance with the relaxation mechanism of NLC as described elsewhere [9,17–19]. References 1. Kosc, T.Z.; Marshall, K.L.; Trajkovska-Petkoska, A.; Kimball, E.; Jacobs, S.D. Progress in the development of polymer cholesteric liquid crystal ﬂakes for display applications. Displays 2004, 25, 171–176. [CrossRef] 1. Kosc, T.Z.; Marshall, K.L.; Trajkovska-Petkoska, A.; Kimball, E.; Jacobs, S.D. Progress in the development of polymer cholesteric liquid crystal ﬂakes for display applications. Displays 2004, 25, 171–176. [CrossRef] 2. Trajkovska-Petkoska, A.; Varshneya, R.; Kosc, T.Z.; Marshall, K.L.; Jacobs, S.D. Enhanced electro-optic behavior for shaped polymer cholesteric liquid-crystal ﬂakes made using soft lithography. Adv. Funct. Mater. 2005, 15, 217–222. [CrossRef] 2. Trajkovska-Petkoska, A.; Varshneya, R.; Kosc, T.Z.; Marshall, K.L.; Jacobs, S.D. Enhanced electro-optic behavior for shaped polymer cholesteric liquid-crystal ﬂakes made using soft lithography. Adv. Funct. Mater. 2005, 15, 217–222. [CrossRef] [ ] 3. Novoselov, K.S.; Geim, A.K.; Morozov, S.V.; Jiang, D.; Zhang, Y.; Dubonos, S.V.; Grigorieva, I.V.; Firsov, A.A. 3. Novoselov, K.S.; Geim, A.K.; Morozov, S.V.; Jiang, D.; Zhang, Y.; Dubonos, S.V.; Grigorieva, I.V.; Firsov, A.A. Electric ﬁeld effect in atomically thin carbon ﬁlms. Science 2004, 306, 666–669. [CrossRef] [PubMed] 3. Novoselov, K.S.; Geim, A.K.; Morozov, S.V.; Jiang, D.; Zhang, Y.; Dubonos, S.V.; Grigorieva, I.V.; Firsov, A.A. Electric ﬁeld effect in atomically thin carbon ﬁlms. Science 2004, 306, 666–669. [CrossRef] [PubMed] ovoselov, K.S.; Geim, A.K.; Morozov, S.V.; Jiang, D.; Zhang, Y.; Dubonos, S.V.; Grigorieva, I.V.; Firsov, A.A. ctric ﬁeld effect in atomically thin carbon ﬁlms. Science 2004, 306, 666–669. [CrossRef] [PubMed] 4. Meyer, J.C.; Geim, A.K.; Katsnelson, M.I.; Novoselov, K.S.; Booth, T.J.; Roth, S. The structure of suspended graphene sheets. Nature 2007, 446, 60–63. [CrossRef] [PubMed] 4. Meyer, J.C.; Geim, A.K.; Katsnelson, M.I.; Novoselov, K.S.; Booth, T.J.; Roth, S. The structure of suspended graphene sheets. Nature 2007, 446, 60–63. [CrossRef] [PubMed] 5. Tie, W.W.; Bhattacharyya, S.S.; Lim, Y.J.; Lee, S.W.; Lee, T.H.; Lee, Y.H.; Lee, S.H. Dynamic electro-optic response of graphene/graphitic ﬂakes in nematic liquid crystals. Opt. Express 2013, 21, 19867–19879. [CrossRef] [PubMed] 5. Tie, W.W.; Bhattacharyya, S.S.; Lim, Y.J.; Lee, S.W.; Lee, T.H.; Lee, Y.H.; Lee, S.H. Dynamic electro-optic response of graphene/graphitic ﬂakes in nematic liquid crystals. Opt. 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Kim, J.H.; Kang, W.S.; Choi, H.S.; Park, K.; Lee, J.H.; Yoon, S.; Yoon, S.; Lee, G.D.; Lee, S.H. Effect of surface anchoring energy on electro-optic characteristics of a fringe-ﬁeld switching liquid crystal cell. J. Phys. D Appl. Phys. 2015, 48, 465506. 4. Conclusions We have demonstrated the electric ﬁeld-induced dynamic reorientation and relaxation processes of GP ﬂakes in an NLC host. The ﬁeld-induced reorientation time of GP ﬂakes has been effectively reduced by using the suitable physical properties of an NLC host. Our investigation reveals quicker reorientation time of GP ﬂakes in the NLC host with higher K11 and lower γ1. Considering the ﬂake reorientation phenomenon in positive dielectric anisotropy NLCs, the reorientation process depends not only on NLC director reorientation, but also the interfacial Maxwell-Wagner model. At the interface between GP ﬂakes and NLC molecules, strong π-π interaction exists, and the relaxation process is owing to the elasticity-driven rotation of NLC director. The higher K11 and lower γ1 are responsible for higher elastic torque, which matches with the modiﬁed rising time equation well in conformity with our previous reported reorientation model. In a ﬁeld-off state, the relaxation of GP ﬂakes mainly follows the nematic NLC director that mechanically rotates after the ﬁeld switches off. The GP ﬂakes relax quicker in the NLC host with higher K11 and lower γ1 than its higher counterpart, and with lower d than its thicker counterpart, where the anchoring effect over the NLC director and subsequent secondary induced torque over suspended GP ﬂakes are mainly responsible for this behavior. The reversible reorientation behavior of GP ﬂakes in NLC and the improved response open up an efﬁcient way of dynamically and quickly controlling transmittance properties of electro-optic devices, which makes them interesting candidates for potential applications. Acknowledgments: This research was supported by Basic Research Laboratory Program (2014R1A4A1008140) through the Ministry of Science, ICT & Future Planning and Polymer Materials Fusion Research Center. W. Tie also thanks to the support from the National Nature Science Foundation of China (61605167), the Science and Technology Research Fund of Henan Provence (162102410093, 172102310478) and XuChang City, and the Key Scientiﬁc Research Project of Universities and Colleges in Henan Province (17A430028). Author Contributions: Seung Hee Lee conceived and designed the experiments; Weiwei Tie, Eun Jeong Shin, Tae Hyung Kim, and MinSu Kim performed the experiments; Seung Hee Lee, Weiwei Tie, Surjya Sarathi Bhattacharyya, and Joong Hee Lee analyzed the data; Seung Hee Lee, Yuanhao Gao and Zhi Zheng contributed reagents/materials/analysis tools; and Weiwei Tie, Surjya Sarathi Bhattacharyya, Seung Hee Lee, and MinSu Kim wrote the paper. 10 of 10 Nanomaterials 2017, 7, 250 Conﬂicts of Interest: The authors declare no conﬂict of interest. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References [CrossRef] © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2807883727	https://edoc.unibas.ch/69243/1/20180718141825_5b4f301135764.pdf	English	null	Colorectal cancer-associated Streptococcus infantarius subsp. infantarius differ from a major dairy lineage providing evidence for pathogenic, pathobiont and food-grade lineages	Scientific reports	2,018	cc-by	10,929	Colorectal cancer-associated Streptococcus infantarius subsp. infantarius differ from a major dairy lineage providing evidence for pathogenic, pathobiont and food-grade lineages Received: 18 November 2017 Accepted: 25 May 2018 Published: xx xx xxxx Dasel Wambua Mulwa Kaindi1, Wambui Kogi-Makau1, Godfrey Nsereko Lule2, Bernd Kreikemeyer3, Pierre Renault4, Bassirou Bonfoh5,6,7, Nize Otaru8, Thomas Schmid8, Leo Meile8, Jan Hattendorf6,7 & Christoph Jans 8 Streptococcus infantarius subsp. infantarius (Sii), a member of the Streptococcus bovis/Streptococcus equinus complex (SBSEC), predominates as dairy-adapted and non-adapted variants in fermented dairy products (FDP) in East and West Africa. Epidemiologic data suggest an association with colorectal cancer for most SBSEC members, including Sii from Kenyan patients. Phylogenetic relationships of East African human (EAH) isolates to those of dairy and pathogenic origin were analysed to better estimate potential health implications via FDP consumption. The MLST-derived population structure was also evaluated to provide host, disease, geography and dairy adaptation associations for 157 SBSEC isolates, including 83 novel Sii/SBSEC isolates of which 40 originated from Kenyan colonoscopy patients. Clonal complex (CC) 90 was delineated as potential pathogenic CC for Sii. Single EAH, West African dairy (WAD), food and animal Sii isolates clustered within CC-90, suggesting a potential link to pathogenic traits for CC-90. The majority of EAH and WAD Sii were clustered in a shared clade distinct from CC-90 and East African dairy (EAD) isolates. This indicates shared ancestry for the EAH and WAD clade and limitations to translate disease associations of EAH and CC-90 to EAD Sii, which could support the separation of pathogenic, pathobiont/commensal and food lineages. Streptococcus infantarius subsp. infantarius (Sii) belongs to the Streptococcus bovis/Streptococcus equinus com- plex (SBSEC). SBSEC is a diverse group of bacteria including commensal inhabitants of the human and animal gastrointestinal tract, opportunistic pathogens and variants in food1,2. The SBSEC is composed of seven sub- species: Streptococcus gallolyticus subsp. gallolyticus (Sgg), Streptococcus gallolyticus subsp. macedonicus (Sgm), Streptococcus gallolyticus subsp. pasteurianus (Sgp), Streptococcus equinus, Streptococcus infantarius subsp. infan- tarius (Sii), Streptococcus lutetiensis and Streptococcus alactolyticus. SBSEC provides enhanced discrimination power in contrast to the former Streptococcus bovis designation that is still used for its wide recognition2,3. SBSEC group members are associated with bacteraemia, infective endocarditis, urinary tract infections, men- ingitis, sepsis, gastroenteritis, endophthalmitis and carcinoma of the colon4–6. Sgg feature a strong association with colorectal cancer, particularly in patients affected by Sgg-related bacteraemia or infective endocarditis7. To a lesser 1Department of Food Science, Nutrition and Technology, University of Nairobi, Nairobi, Kenya. 2School of Medicine, University of Nairobi, Nairobi, Kenya. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Received: 18 November 2017 Accepted: 25 May 2018 Published: xx xx xxxx www.nature.com/scientificreports/ www.nature.com/scientificreports/ extent, this association is also observed for S. lutetiensis, Sgp and possibly even Sii and Sgm. However, the causality of SBSEC members in colorectal cancer is still questioned and rather explained by a bacterial driver-passenger model7–10. In contrast to Sgg, Sgp and S. lutetiensis, Sii is a rare isolate in clinical specimens11,12, partially because Sii was only recently separated from S. lutetiensis (former S. infantarius subsp. coli) within the S. infantarius branch2,3. This separation provides clarity regarding the involvement of subspecies of S. infantarius in disease that was rarely available in studies conducted prior to this new taxonomy. Sii is implicated in human infections including infective endocarditis13, biliary tract infection, cirrhotic bacteraemia and non-colorectal cancer8,10,14–18.h g y The habitat of Sii as well as most other SBSEC members is mainly in the gastrointestinal tract of humans as well as animals such as ruminants, sea otters and birds2,3, where they are considered to be commensals but also fit as putative representatives of pathobionts19. Furthermore, Sii and Sgm are regularly found as predominant organ- isms in fermented dairy products (FDP)2. Particularly, Sii is highly prevalent as a predominant bacterium in tra- ditional FDP in sub-Saharan Africa20–23. Thus, live Sii are ingested by millions of FDP consumers in sub-Saharan Africa at levels over 108 CFU mL−1. However, East African and many West African dairy Sii are clearly different from the human Sii analysed to date24–26. African dairy Sii feature dairy adaptations such as a modified lactose metabolism via a lacS and lacZ encoded lactose uptake instead of the SBSEC-typical lactose phosphotransferase system24,25. Phylogenetic analysis further supports the differentiation of specific African dairy lineages by multi locus sequence typing (MLST)24. Several of these Sii lineages seem to be clearly separating from human commen- sal and human pathogenic lineages while other dairy isolates share a closer relationship with potentially harmful strains24. Knowledge of phylogeny of human commensal and pathogenic lineages is currently limited to strains iso- lated from humans in Europe and Asia; analogous knowledge of African Sii is limited to dairy strains only24. Representative human Sii isolates from African countries such as Kenya, Somalia or Côte d’Ivoire with a docu- mented presence of dairy Sii were missing and therefore did not allow for a more comprehensive phylogenetic evaluation of lineages, strains and their relationships. Results MLSTana MLST analysis of novel SBSEC isolates and update on key characteristics of the SBSEC MLST scheme. This study marks the first comparison of human SBSEC and Sii isolates of African origin to those of human commensal, human pathogen, animal commensal and dairy origin of a global collection. A total of 83 SBSEC and Sii isolates were integrated into the SBSEC multi locus sequence typing (MLST) scheme to yield 157 isolates for the subsequent assessment of evolutionary and phylogenetic relationships (Fig. 1). All isolates originating from human, animal and food collections were previously identified as SBSEC members using SBSEC-specific PCR assays targeting the 16S rRNA gene and to species level using partial groEL sequencing6,20,21,24,27,29–33.h y g g g p g p g q g The SBSEC MLST scheme currently contains 120 STs out of these 157 isolates (Table 1).i h y ( ) MLST analysis confirmed the subspecies of all 83 isolates, including 75 Sii, three Sgm, four Sgp, and one S. lutetiensis previously determined by groEL sequencing (Table 1). The new isolates were responsible for a total of 54 new STs of which 47 were due to Sii isolates in addition to one S. lutetiensis, three Sgm and three Sgp isolates as a result of four to 30 new alleles for the 10 different loci (Table 1). IA and IA S were calculated to be 0.534–2.784 and 0.059–0.309, respectively, indicating significant linkage disequilibrium for most species except Sgp (Table 1). The values of dN/dS < 1 indicate no selective pressure for most alleles except for gki in Sgm (Table 1). However, while overall SBSEC (n = 157) and Sii (n = 114) provided sufficient data for calculations, the number of alleles for S. lutetiensis, Sgm and Sgp is still comparably low for reliable calculations. Phylogenetic analysis of Sii by MLST in relation to host, source, geography and genetic markers. A total of 114 Sii isolates were included in this analysis, of which 41 originated from human faecal samples and rectal swabs obtained in Kenya, referred hereafter as East African human (EAH) isolates (colonoscopy patient Sii isolates n = 34, infant faecal sample Sii isolates n = 7). Four out of the 33 Kenyatta National Hospital (KNH) patients delivered Sii isolate pairs. www.nature.com/scientificreports/ In a recent hospital-based study on patients undergo- ing colonoscopy at Kenyatta National Hospital in Nairobi, Kenya, strains to investigate this missing link were obtained27. Among these colonoscopy patients, Sii and other SBSEC members were isolated from faecal samples and rectal swabs, and in conjunction, the socioeconomic aspects and dietary habits of the patients were assessed. The result was a set of isolates that included comprehensive descriptions of the patients27. In that study, SBSEC and Sii carriage rate among 273 participants was about 20%27. Furthermore, Sii isolates among these study partic- ipants indicated associations with colorectal cancer and haemorrhoids27. However, the population structure and relationships to human and dairy strains is unknown, leaving an important knowledge gap in the epidemiology of Sii-associated diseases and the role of dairy Sii in Africa, particularly in the example of Kenya. Given the large number of daily consumers of FDP and thus Sii consumers in Kenya, closing this knowledge gap of dairy vs human Sii in Kenya also has a significant public health impact.hi y gi p p Therefore, the objective of this study was to evaluate the phylogenetic relationships among the first African human Sii and other SBSEC members in comparison with dairy, commensal and pathogenic Eurasian strains through the SBSEC-specific MLST approach. Thereby, we investigated the evolution of Sii and SBSEC with a global and an African perspective. Particularly for Africa, a thorough safety assessment of dairy Sii and their rela- tionship to potentially pathogenic and human commensal lineages is of high priority, given the role of Sii in the daily diet of millions of pastoralists in sub-Saharan Africa28. This study represents the first phylogenetic analysis of African human Sii and SBSEC isolates. Colorectal cancer-associated Streptococcus infantarius subsp. infantarius differ from a major dairy lineage providing evidence for pathogenic, pathobiont and food-grade lineages 3Institute of Medical Microbiology, Virology, and Hygiene, Rostock University Medical Centre Rostock, Rostock, Germany. 4Institut National de la Recherche Agronomique, UMR 1319 MICALIS, Jouy-en-Josas, France. 5Centre Suisse de Recherches Scientifiques en Côte d’Ivoire, Adiopodoume, Ivory Coast. 6Department of Epidemiology and Public Health, Swiss Tropical and Public Health Institute, Basel, Switzerland. 7University of Basel, Basel, Switzerland. 8Laboratory of Food Biotechnology, ETH Zurich, Zurich, Switzerland. Correspondence and requests for materials should be addressed to C.J. (email: christoph.jans@hest.ethz.ch) SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 1 SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 Results MLSTana WAD and EAD Sii isolates analysed in this study clustered predominantly in the clades (i) and (vii), respectively. In contrast, EAH isolates clustered in clade (i) with the exception of isolate D1266, which clustered in clade (ii). EAH Sii isolates obtained from colonoscopy patients at KNH, Nairobi, Kenya (33 isolates) and from infant faecal samples in Msambweni, Kenya (7 isolates), clustered in clade (i) shared with WAD/Asian Sii iso- lates (19 isolates), whereas one EAH Sii isolate (D1266) clustered in clade (ii) of human pathogens (Fig. 1 and Supplementary Fig. S1). Subdivisions of individual clades were not further followed due to limited bootstrap reliability. y Analysis of alleles and loci revealed that clustering of WAD/Asian Sii (n = 19) with EAH Sii (n = 40) in clade (i) was due to shared or highly similar alleles for glnA, mutS2, proS, thrS and tpi, despite different geographical origins and biological sources. The consensus allele array (ddl, gki, glnA, mutS, mutS2, pheS, proS, pyrE, thrS, tpi) for WAD and EAH was (2,2,2,6,2,2,2,3,2,2) and (3,3,2,1,2,3,2,41,2,2), respectively. This consensus is clearly dif- ferent from that of EAD isolates and human commensal isolates of clade (iii) featuring the consensus allele array (1,1,1,4,6,1,1,1,4,1). DNA sequence analysis of all shared alleles (glnA, mutS2, proS, thrS and tpi) among WAD and EAH isolates in clade (i) displayed highest sequence identity to other Sii isolates for glnA, mutS2, proS and tpi. In contrast, thrS allele sequences of all Sii isolates of clade (i) featured highest sequence identity to S. thermophilus. This highest sequence identity to S. thermophilus was observed for all thrS alleles (2, 8, 18, 32 and 34) comprised in isolates of clade (i), and confirms previous indications of potential horizontal gene transfer (HGT) in this clade for thrS24. In addition, thrS displayed also characteristics of HGT among other SBSEC members. thrS alleles 23 and 31, as well as 33, shared highest sequence identity with S. salivarius and S. thermophilus, respectively. Alleles 23 and 33 were observed in Kenyan Sgm isolates of human origin and Sgm DSM15879T from Greek cheese. Allele 31 was present in S. alactolyticus DSM 20728 T.i p y Clonal Complex (CC) calculations, with a CC definition based on STs sharing seven or more out of 10 alleles24, revealed six CCs for Sii, two for S. equinus/S. bovis, two for Sgp and one each for S. lutetiensis and Sgm (Fig. 2). Results MLSTana The isolate pairs of three patients were determined as potential isolate dupli- cates per patient (D261/D933, D1201/D1202, D1396/D1437), whereas one patient delivered two unique iso- lates D1095 and D1098 (Supplementary Fig. S1).h pp y g The Sii were separated into eight distinct clades with the major clusters comprised of (i) isolates of W African Dairy (WAD) and EAH, (ii) isolates of human blood (pathogenic), animals, EAH, WAD and pozol; SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 2 www.nature.com/scientificreports/ Figure 1. Phylogenetic MLST tree of 157 SBSEC isolates including 83 new isolates. Maximum likelihood phylogenetic tree calculated from the concatenated sequences of the 10 MLST loci of 157 SBSEC isolates including a total of 83 new isolates of East African (EA) human, EA dairy, West African (WA) dairy and global (G) isolates of human, dairy, food or animal origin. The tree is rooted to S. alactolyticus DSM20728T. Validation was performed using 200 bootstrap replications for which the percentage of clustered trees is given next to the branches. The horizontal bar at the bottom indicates the evolutionary distance in the same units as used for branch length. * includes one isolate of animal origin; §marks strains with species designations according to groEL sequence but tree position not within the expected subspecies clades. Figure 1. Phylogenetic MLST tree of 157 SBSEC isolates including 83 new isolates. Maximum likelihood phylogenetic tree calculated from the concatenated sequences of the 10 MLST loci of 157 SBSEC isolates including a total of 83 new isolates of East African (EA) human, EA dairy, West African (WA) dairy and global (G) isolates of human, dairy, food or animal origin. The tree is rooted to S. alactolyticus DSM20728T. Validation was performed using 200 bootstrap replications for which the percentage of clustered trees is given next to the branches. The horizontal bar at the bottom indicates the evolutionary distance in the same units as used for branch length. * includes one isolate of animal origin; §marks strains with species designations according to groEL sequence but tree position not within the expected subspecies clades. vii) isolates of East Africa dairy (EAD) origin as well as the Sii type strain and presumptive commensal Sii (Fig. 1 nd Supplementary Fig. S1). Sii strains added in this study were clustered in all Sii clades except clade (v). Geography and sample origin were observed as important aspects for clade structure in relation to dairy isolates. www.nature.com/scientificreports/ IA: index of association; IA S: standardized index of association; dN/dS: ratio of non-synonymous sites over synonymous sites. Table 1. Key characteristics of the SBSEC-MLST scheme for 157 SBSEC isolates calculated for the overall complex and subspecies with newly incorporated alleles. New alleles include all new alleles incorporated into the SBSEC MLST scheme. For individual species, the number corresponds to new alleles assigned to a species, which includes also existing SBSEC alleles that were previously not assigned to a specific. IA: index of association; IA S: standardized index of association; dN/dS: ratio of non-synonymous sites over synonymous sites. Origin and source were strongly associated with Sii CCs (Fig. 2). CC-90 encompassed all Sii human blood iso- lates of potential pathogenic backgrounds from worldwide sources, human clinical isolates of unknown body sites as well as several EAH, WAD, European dairy and plant isolates, raising safety concerns for members of CC-90. CC-71 was predominantly comprised of EAD Sii isolates. EAD Sii isolates and STs were all comprised within CC-71 with the exception of two singletons. CC-101 was a mix of WAD and EAH Sii isolates, including a Spanish hospital isolate (ST88). In contrast to the focused clustering of EAD Sii isolates in CC-71, WAD and EAH Sii isolates displayed wider distribution among three different CC-s and numerous singletons. This higher diversity of WAD vs. EAH and EAD Sii isolates was supported by SID analysis of the Sii STs for EAD, EAH and WAD of 0.940, 0.970 and 0.975, respectively, and also for the individual ten alleles (Supplementary Table S1). The higher SID of EAH and WAD Sii was mainly due to isolates being spread over different clades. Focusing only on clade (i), SID indicated a lower diversity of 0.968 and 0.961 for EAH and WAD Sii, respectively (Supplementary Table S1). Similarly, sequence identity analysis of the concatenated sequences of the 10 alleles of all EAH Sii isolates featured an average identity of 99.6% vs. 99.8 for EAD and 98.7 for WAD Sii (Supplementary Table S2). Within clade (i), EAH and WAD Sii featured 99.7% identity, showing that within a clade, sequence conservation was rather high (Supplementary Table S2).h pp y The presence of the dairy adaptation marker genes lacS/lacZ was strongly associated with Sii CCs and MLST clades (Supplementary Fig. S1 and Fig. 2). Marker genes were only detected in EAD and WAD Sii isolates. Results MLSTana Among these, five CCs were comprised of more than two STs and were labelled CC-101, CC-71, CC-90 and CC-161 for Sii and CC-37 for Sgm. Out of the Sii CCs, CC-101 was comprised of 32 isolates yielding 21 different sequence types (STs) (Fig. 2 and Supplementary Fig. S1). The predicted founder of CC-101 was ST 101. The other Sii CC-71, CC-90 and CC-161 were comprised of 17, 12 and 4 STs representing 31, 13 and 10 isolates that were centred around ST71, ST90 and ST161, respectively. However, a founding ST prediction was only possible for ST161 in CC-161, while for the CCs comprising ST71 or ST90, no founding ST could be calculated. SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ SBSEC n = 157 Sii n = 114 S. lutetiensis n = 9 Sgm n = 10 Sgp n = 7 IA = 2.784 dN/dS IA = 1.864 dN/dS IA = 1.959 dN/dS IA = 2.195 dN/dS IA = 0.534 dN/dS IA S = 0.309 IA S = 0.207 IA S = 0.218 IA S = 0.244 IA S = 0.059 ST and alleles ST and alleles ST and alleles ST and alleles ST and alleles New Total New Total New Total New Total New Total ST 54 120 47 78 1 9 3 10 3 6 ddl 10 38 0.080 8 13 0.100 0 6 0.149 2 4 0.121 1 3 0.072 gki 7 34 0.057 4 9 0.125 0 5 0.089 3 6 1.368 1 2 0.328 glnA 6 27 0.038 4 11 0.129 0 6 0.080 1 2 0.141 0 3 0 mutS 7 37 0.028 4 9 0.213 0 5 0.600 2 6 0.065 1 3 0.146 mutS2 11 47 0.030 8 18 0.179 0 5 0 3 5 0 1 3 0.036 pheS 8 36 0.023 5 15 0.011 0 4 0.046 1 3 0.017 0 3 0 proS 12 40 0.063 9 16 0.322 1 6 1.089 2 3 0.398 2 2 0 pyrE 30 66 0.022 22 38 0.043 1 6 0 1 4 0.058 1 2 0 thrS 4 35 0.082 3 14 0.107 0 5 0.074 2 4 0.099 1 2 0.140 tpi 7 24 0.148 4 8 0.761 0 5 0 2 4 0.575 0 2 0 Table 1. Key characteristics of the SBSEC-MLST scheme for 157 SBSEC isolates calculated for the overall complex and subspecies with newly incorporated alleles. New alleles include all new alleles incorporated into the SBSEC MLST scheme. For individual species, the number corresponds to new alleles assigned to a species, which includes also existing SBSEC alleles that were previously not assigned to a specific. IA: index of association; IA S: standardized index of association; dN/dS: ratio of non-synonymous sites over synonymous sites. Table 1. Key characteristics of the SBSEC-MLST scheme for 157 SBSEC isolates calculated for the overall complex and subspecies with newly incorporated alleles. New alleles include all new alleles incorporated into the SBSEC MLST scheme. For individual species, the number corresponds to new alleles assigned to a species, which includes also existing SBSEC alleles that were previously not assigned to a specific. Table 1. Key characteristics of the SBSEC-MLST scheme for 157 SBSEC isolates calculated for the overall complex and subspecies with newly incorporated alleles. New alleles include all new alleles incorporated into the SBSEC MLST scheme. For individual species, the number corresponds to new alleles assigned to a species, which includes also existing SBSEC alleles that were previously not assigned to a specific. IA: index of association; IA S: standardized index of association; dN/dS: ratio of non-synonymous sites over synonymous sites. www.nature.com/scientificreports/ Schematic relationships of SBSEC isolates by MLST sequence types coloured according to isolate metadata. Philoviz/eBURST clustering of SBSEC MLST sequence types (STs) of human, animal and dairy sources calculated from ST profiles. ST bullet points were coloured according to schemes for (A) species an subspecies tree including dotted lines in the same colour for graphs A-C for species and subspecies; (B) ori Figure 2. Schematic relationships of SBSEC isolates by MLST sequence types coloured according to isolate metadata. Philoviz/eBURST clustering of SBSEC MLST sequence types (STs) of human, animal and dairy sources calculated from ST profiles. ST bullet points were coloured according to schemes for (A) species and subspecies tree including dotted lines in the same colour for graphs A-C for species and subspecies; (B) origin and source of isolates and STs; and (C) presence of dairy adaptation marker genes lacS and lacZ. Clustering was initially calculated using eBURST algorithm set to fullMST specifications in Phyloviz 2.0 and subsequently reduced to clonal complex (CC) definition of 7 or more alleles shared with at least one other member of the CC to yield this figure. For CC with >2 STs and predicted founder, the CC is labelled by underlined font. ST bullet point positions and cluster layout correspond to nearest phylogenetic relationships with the exception of S. alactolyticus DSM 20728T (ST66), which was placed outside of other species for improved visibility. ST bullet point size corresponds to the number of isolates for each specific ST with the smallest bullet points representing single isolates. Figure 2. Schematic relationships of SBSEC isolates by MLST sequence types coloured according to isolate metadata. Philoviz/eBURST clustering of SBSEC MLST sequence types (STs) of human, animal and dairy sources calculated from ST profiles. ST bullet points were coloured according to schemes for (A) species and subspecies tree including dotted lines in the same colour for graphs A-C for species and subspecies; (B) origin and source of isolates and STs; and (C) presence of dairy adaptation marker genes lacS and lacZ. Clustering was initially calculated using eBURST algorithm set to fullMST specifications in Phyloviz 2.0 and subsequently reduced to clonal complex (CC) definition of 7 or more alleles shared with at least one other member of the CC to yield this figure. For CC with >2 STs and predicted founder, the CC is labelled by underlined font. www.nature.com/scientificreports/ None were detected in EAH Sii isolates (Supplementary Fig. S1). EAD Sii isolates shared 96.9% prevalence of lacS/lacZ genes among 32 isolates. In contrast, WAD Sii isolates (n = 24) only presented 54.2% lacS/lacZ prevalence that was focused on isolates clustered in four small clades and two single branches. In relation to CCs, CC-71 was mainly comprised of EAD Sii isolates featuring lacS/lacZ adaptation in 15 out of 17 STs, with Sii type strain (ST1, human faecal isolate) and Sii 150A (ST7, EAD isolate) being the exceptions. CC-101 including WAD Sii isolates only presented lacS/lacZ in six out of 11 WAD STs, but not in any of the EAH STs. Similarly, no lacS/lacZ adapta- tion was detected in human blood isolates or any STs of CC-90 and CC-161. Phylogenetic analysis of S. gallolyticus subspecies. A total of seven new S. gallolyticus isolates and STs were added to the SBSEC MLST scheme (Figs 1 and 3). The three Sgm isolates comprised of one WAD and two EAH isolates clustered clearly distinct. They did not match existing ST or CC definitions and therefore formed singletons. Sgm PV1(1) (WAD) branched as a single isolate and separated from the main S. gallolyticus clade. Within the main Sgm clade, a clear division between the Sgm type strain clade and the majority of African Sgm isolates was observed using sequence-based analysis (Fig. 3). However, profile-based analysis assigned the major- ity of EAD and WAD Sgm to CC-37, together with the Sgm type strain. Sgm CC-37 was defined around ST37, although no direct founder was predicted. CC-37 was comprised of six isolates each representing a unique ST, but all members of CC-37 shared a dairy origin suggesting CC-37 as a main dairy CC for Sgm.i y g gg g y g EAH Sgp isolates formed subclades with Sgp ATCC700338 and Sgp DSM15351T. CC definition among Sgp was only possible for two pairs while the other STs remained singletons. Sgg represented only singletons without any CCs. This finding of a majority of singleton isolates was also reflected in highest SID values of 1 for Sgm, Sgg and Sgp indicating absolute diversity (Supplementary Table S1). DNA sequence identity across the 10 alleles showed high conservation of 99.2 and 99.5% for Sgg and Sgp, respectively, whereas Sgm featured 98.4% (Supplementary Table S2). SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 4 www.nature.com/scientificreports/ entificreports/ Figure 2. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 3. Phylogenetic MLST tree of the S. gallolyticus branch. Maximum likelihood phylogenetic tree of the S. gallolyticus branch extracted from the overall rooted SBSEC MLST tree of the concatenated sequences of 10 MLST loci. New isolate additions were: Sgm PV1(1), Sgm SP49-0307-1.3, Sgm D1143, Sgp D1017, Sgp D1186, Sgp D1238 and Sgp D1243. The horizontal bar at the bottom indicates the evolutionary distance in the same units as used for branch length. Isolate information is given for host/source of animal (A), dairy (D), or human (H) origin followed by participant ID for isolates obtained from colonoscopy patients of this study. “n/a” indicates that this information is not available for this strain. Participant information was only filed for human samples. Sequence type (ST) and clonal complex (CC) assignment was indicated based on MLST profile analysis. Figure 3. Phylogenetic MLST tree of the S. gallolyticus branch. Maximum likelihood phylogenetic tree of the S. gallolyticus branch extracted from the overall rooted SBSEC MLST tree of the concatenated sequences of 10 MLST loci. New isolate additions were: Sgm PV1(1), Sgm SP49-0307-1.3, Sgm D1143, Sgp D1017, Sgp D1186, Sgp D1238 and Sgp D1243. The horizontal bar at the bottom indicates the evolutionary distance in the same units as used for branch length. Isolate information is given for host/source of animal (A), dairy (D), or human (H) origin followed by participant ID for isolates obtained from colonoscopy patients of this study. “n/a” indicates that this information is not available for this strain. Participant information was only filed for human samples. Sequence type (ST) and clonal complex (CC) assignment was indicated based on MLST profile analysis. EAH Sii were distributed among clade (i), with the exception of Sii D1266 clustering in clade (ii) yielding a clear separation for most EAH Sii isolates from those of blood origin of clade (ii)24 and EAD Sii of clade (vii) (Supplementary Fig. S1). There was no apparent allocation of diagnoses as well as normal colon types or patient origin to specific subclades within clade (i) due to a high degree of sequence identity (Supplementary Fig. S1). Isolate D1266 of a patient suffering from haemorrhoids clustered among these presumptive pathogens of clade (ii). f Consumption of FDP was a rare trait among participants and only recorded for seven patients (Fig. 3 and Supplementary Fig. S1). Discussion Sub-Saharan Africa has unique lineages of dairy adapted Sii isolated from traditional FDP that are clearly dis- tinct from human and animal isolates obtained from Europe or Asia24. Previous epidemiologic data of patients undergoing colonoscopy at KNH suggested an association between CRC and Sii as well as haemorrhoids and Sii. Although no causality is proven, this finding raises concerns considering the large population consuming Sii on a daily basis via FDP. However, before this study, no African human Sii isolates had been analysed to reveal the population structure, phylogenetic relationships and potential evolution of African human Sii vs other human and particularly dairy Sii. In addition, this study reports the first human Sgm and Sgp isolates obtained in Africa to give a more comprehensive insight into SBSEC in Africa. g p g A total of 83 new SBSEC isolates including 75 Sii isolates of human, dairy and animal origins were therefore included in this study for a total of 157 SBSEC isolates in the MLST scheme. Of these 75 Sii, 34 Sii isolates orig- inated from human patients undergoing colonoscopy at KNH, Kenya while others were obtained from Kenyan infants as well as food and human sources in Africa, Europe, Asia and America. Among the Sii, CC-90 was clearly delineated from all other CCs, and encompassed all human blood isolates and thus potential human pathogens. CC-90 could therefore be considered as a key pathogen CC of Sii. The presence of single EAH and WAD Sii among CC-90 raises concerns for human carriers and food products containing these strains. However, the clear delineation of CC-90 from the EAD CC-71 and EAH clade (vii) as well as most other WAD/EAH CCs and clades supports further in-depth functional and genomic comparisons between key representative isolates of these CCs. The unexpectedly close relationship between EAH Sii and WAD Sii raised further concerns and questions. The EAH Sii isolates were, with the exception of one isolate, closest related to WAD Sii isolates and clearly distinct from EAD Sii isolates. The majority of EAH and WAD Sii isolates clustered in shared CC-101 and CC-161 instead of CC-71 of EAD. This phylogenetic division was related to highly similar or shared alleles in five out of ten loci, suggesting a common ancestor for most EAH and WAD Sii. www.nature.com/scientificreports/ This included two exclusive Sii carriers, three exclusive Sgp carriers and two carriers of either Sii and Sgp or Sii and S. lutetiensis. None of these isolates of FDP consumers were found to harbour dairy adaptation markers lacS or lacZ. Furthermore, MLST clustering of Sii and Sgm of these EAH isolates was clearly distinct from the corresponding species among the dairy adapted EAD isolates, indicating that EAH Sii and pos- sibly also Sgm represent different lineages from EAD Sii and Sgm, respectively. y g pf g g p y For Sgm, Sgp and S. lutetiensis, none of the patients reported to carry Sgm (one patient, one isolate), Sgp (four patients, four isolates) or S. lutetiensis (one patient, one isolate) were diagnosed with CRC, whereas haemorrhoids was the most common finding in three patients, followed by normal colon in two patients (Fig. 3). www.nature.com/scientificreports/ ST bullet point positions and cluster layout correspond to nearest phylogenetic relationships with the exception of S. alactolyticus DSM 20728T (ST66), which was placed outside of other species for improved visibility. ST bullet point size corresponds to the number of isolates for each specific ST with the smallest bullet points representing single isolates. Distribution of Sii, Sgm and Sgp isolates in relation to colonic disorders and consumption of traditionally fermented dairy products. A total of 40 SBSEC isolates originating from 33 colonos- copy patients were analysed. Of these, EAH Sii isolates (n = 34) from 30 different colonoscopy patients were integrated into the MLST scheme (Supplementary Fig. S1). These 34 Sii isolates were obtained from eight partic- ipants with colorectal cancer (CRC) (8/34 isolates), 12 participants with haemorrhoids (14/34 isolates) and nine participants with normal colons (11/34 isolates). SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 5 www.nature.com/scientificreports/ This will of course require further functional comparisons between isolates and addi- tional field-based studies to directly link dairy product analysis with consumer stool analysis, as well as compar- isons between East and West Africa.hff This East-West comparison seems crucial due to the different dairy Sii lineages and potentially different human Sii lineages. It also has important safety implications, as the clear division between the EAH/WAD Sii lineages of clade (i), the potential pathobiont human Sii lineage of clade (ii) and the dairy adapted EAD line- age of clade (vii) might enable better definition of possible virulence factors and dairy marker genes for a thor- ough safety assessment. However, this also means that food isolates (WAD, Asian and Mexican Sii) clustered in clades (i) and (ii) will require even more detailed examinations to provide recommendations for fermented food production.h p The causality of SBSEC to CRC is an ongoing investigation9. In this study, no apparent link between EAH Sii isolates, geographic patient origin and CRC or haemorrhoids could be established (Supplementary Fig. S1). This was certainly limited by the available number of strains for analysis. It is however an important step towards establishing better epidemiologic data for Sii. Sii showed an association with CRC and haemorrhoids among colo- noscopy patients at KNH, Nairobi, Kenya27. This finding therefore supports the recommendation that the detec- tion of any SBSEC member justifies colorectal examination8. However, these EAH Sii were not related to EAD Sii, and thus disease associations found for EAH in colonoscopy patients might not apply to EAD Sii. Further epidemiologic and comparative investigations are required to determine the role of EAD Sii lineage strains in relation to EAH and WAD, particularly among larger sample numbers of FDP consumers. These assessments will also need to consider recent findings relating to rotavirus vaccine responses among Ghanaian infants, which showed positive correlation with SBSEC titres in gut microbiota35, and could thus further highlight a special role of SBSEC members in sub-Saharan Africa. Within the S. gallolyticus branch, Sgm lineages provided several indications for unexpected phylogenetic dif- ferentiations. Sgm PV1(1) isolated from WAD branched off early from all other S. gallolyticus members. This find- ing will require in-depth analysis via whole genome sequencing to determine the reasons for this branching and assist in species classification. www.nature.com/scientificreports/ www.nature.com/scientificreports/ unique feature of thrS alleles with highest sequence identity to S. thermophilus/S. salivarius. This could be a result of genetic exchange at the base of this clade to define this new lineage. As observed for Sgp and Sgm in compar- ison to Sgg, this might further suggest a common ancestral Sii, possibly of human or animal origin, from which these lineages diverged and undergo niche-adaptation, particularly for EAD Sii7,34.h unique feature of thrS alleles with highest sequence identity to S. thermophilus/S. salivarius. This could be a result of genetic exchange at the base of this clade to define this new lineage. As observed for Sgp and Sgm in compar- ison to Sgg, this might further suggest a common ancestral Sii, possibly of human or animal origin, from which these lineages diverged and undergo niche-adaptation, particularly for EAD Sii7,34.h g g g p p y The combined clustering of EAH Sii and WAD Sii isolates also raised questions regarding the original res- ervoir of these lineages as well as the possibility for regular exchange of isolates between humans and dairy products. EAH isolates were obtained from several participants consuming traditional FDP. Kenyan FDP were observed to contain dairy adapted Sii in over 90% of samples, of which strains clustered exclusively in the EAD Sii clade (vii)21,24. The absence of the dairy adaptation marker genes lacS/lacZ in all EAH Sii isolates as well as a clear MLST separation from EAD Sii isolates strongly suggests that none of the EAH Sii isolates originated from FDP. This also suggests that the observed association between EAH Sii27, haemorrhoids and CRC does not directly apply to the EAD Sii lineage without further strain-to-strain evaluation. pp y g Whether the reason for the absence of EAD Sii in FDP consumers is related to the ability of EAD Sii to com- pete against intestinal Sii and then colonize the gastrointestinal tract remains to be investigated. Colonization is often related to an initial adhesion step. Adhesion to extracellular matrix proteins of the gut such as mucin was limited among EAD in contrast to human isolates24, and could be an indication of reduced competitiveness within the gut microbiota. www.nature.com/scientificreports/ Within the Sgm main clade, only one EAH isolate (SP49-0307-1.3) clustered close to the Sgm type strain, whereas all others formed a clearly-separated African lineage when using sequence-based analysis. However, profile-based analysis indicated the formation of a possible dairy CC-37 together with the Sgm type strain while EAH Sgm and PV1(1) represented singletons, suggesting possible diverging human and dairy lineages as for Sii but on a more global scale. However, the differentiation between EAH, EAD and WAD Sgm is less established compared to Sii and too early for further interpretation. In contrast, Sgp displayed a single African lineage clearly integrated into a separated branch and CC-99/106 within the Sgp clade. The number of available strains and the presence of mostly singletons for Sgp as well as Sgg are too little for further interpretation at this stage. g These different observations in MLST trees calculated from profiles (Fig. 2) vs concatenated sequences (Fig. 3 and Supplementary Fig. S1) relate to the effect of recombination as a major driver for intrastrain variations within the SBSEC24,36. A profile-based approach in the case of a recombinant population provides equal weight for any kind of mutation and thus reduces the impact of genetic exchange among lineages36. In recombinant populations, this can lead to discrepancies between CC definitions according to profiles and concatenated sequence phylog- enies as observed in this study (Fig. 3 and Supplementary Fig. S1). Often, species do not evolve as strictly clonal or recombinant populations but rather feature a combination of clonal and recombinant evolution36. Profile and sequence-based analysis in parallel helped to reveal the thrS-allele influenced delineation of clade (i) among Sii, but also the clear definition of key CCs for EAD Sii (CC-71), EAH/WAD Sii (CC-101 and CC-161), potential human pathogens Sii (CC-90) as well as potential Sgm dairy lineage (CC-37) (Fig. 3 and Supplementary Fig. S1).i p g p g y g g pp y g Conclusively, MLST analysis of 157 SBSEC isolates including the first set of Sii, Sgm and Sgp isolates obtained from patients undergoing colonoscopy at KNH, Nairobi, Kenya, revealed unexpected insights into the phylog- eny and population structure of the SBSEC including multiple recombination events. Africa-specific lineages were obtained for Sii, Sgm and Sgp requiring in-depth comparisons to described pathogens. Discussion Furthermore, EAH and WAD Sii isolates shared the g p g A total of 83 new SBSEC isolates including 75 Sii isolates of human, dairy and animal origins were therefore included in this study for a total of 157 SBSEC isolates in the MLST scheme. Of these 75 Sii, 34 Sii isolates orig- inated from human patients undergoing colonoscopy at KNH, Kenya while others were obtained from Kenyan infants as well as food and human sources in Africa, Europe, Asia and America. Among the Sii, CC-90 was clearly delineated from all other CCs, and encompassed all human blood isolates and thus potential human pathogens. CC-90 could therefore be considered as a key pathogen CC of Sii. The presence of single EAH and WAD Sii among CC-90 raises concerns for human carriers and food products containing these strains. However, the clear delineation of CC-90 from the EAD CC-71 and EAH clade (vii) as well as most other WAD/EAH CCs and clades supports further in-depth functional and genomic comparisons between key representative isolates of these CCs.h pp p g p y p The unexpectedly close relationship between EAH Sii and WAD Sii raised further concerns and questions. The EAH Sii isolates were, with the exception of one isolate, closest related to WAD Sii isolates and clearly distinct from EAD Sii isolates. The majority of EAH and WAD Sii isolates clustered in shared CC-101 and CC-161 instead of CC-71 of EAD. This phylogenetic division was related to highly similar or shared alleles in five out of ten loci, suggesting a common ancestor for most EAH and WAD Sii. Furthermore, EAH and WAD Sii isolates shared the SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 6 Methods hi l Ethical approval of the study. This study was approved in Kenya by the Kenyatta National Hospital/ University of Nairobi, Ethics and Research Committee (KNH/UoN ERC)-approval number P389/07/2012. In Switzerland, the Ethics Committees of ETH Zurich and Kantonale Ethik Kommission Zurich (KEK) approved the study under decision numbers EK 2013-N-78 and KEK-StV-Nr. 47/14, respectively. Strains originat- ing from Msambweni, Kenya, were obtained from human faecal samples covered by KNH-ERC/A/337, the University of KwaZulu-Natal (BF121/08), the ETH Zurich (EK 2009-N-53) and registered at clinicaltrials.gov (NCT01111864)29. Informed consent was obtained from all study participants and/or their legal guardians. The study was conducted in accordance with the Declaration of Helsinki37. Origin and overview of bacterial strains and criteria for isolate selection. This study was based on the pool of 130 SBSEC isolates obtained during the colonoscopy study performed at Kenyatta National Hospital (KNH), Nairobi, Kenya involving 273 participants recruited from 2013 until 201527. The 130 isolates were obtained from 56 out of 228 unique study participants. These isolates represent the first human SBSEC isolates of African origin to be analysed for their phylogeny and evolutionary relationships. For each isolate, cor- responding participant hospital diagnosis of colon disorders and responses from a guided interview on lifestyle, socio-demographics and dietary habits is available27. g p y The criteria for isolate selection among KNH human isolates for this study were based on patients’ colonic disorder, FDP consumption status, residence (urban or rural) as well as isolate rep-PCR fingerprint and presence of lacS/lacZ marker gene. This detailed selection was performed to achieve a wide variety of isolates with different patient backgrounds. A total of 40 SBSEC and Sii from 33 different patients were thus selected. Species and sub- species status of these isolates was previously determined using a 16S rRNA gene-based assay and partial groEL sequencing21,24,27. The species distribution was 34 Sii (30 patients), one Sgm (one patient), four Sgp (four patients) and one S. lutetiensis (one patient) of which one patient each was determined as combined carrier of Sii and Sgm, Sii and Sgp or Sii and S. lutetiensis. gp As an outgroup to the hospital-collected samples, Sii (n = 7) and Sgm (n = 1) isolates from faecal samples of eight infants participating in a gut microbiota study in Mswambeni, Kenya were incorporated29. In addition, further SBSEC and Sii isolates were incorporated to increase the diversity for subsequent comparisons. www.nature.com/scientificreports/ Among Sii, CC-90 was delineated as a potential main pathogen lineage comprised of all human blood isolates but also single EAH, WAD, animal and food isolates, indicating health risks for these food and faecal isolates. Unexpectedly, most EAH Sii isolates shared closest phylogeny, allele sequences and absence of dairy adaption markers with most WAD Sii isolates to form one major clade comprised of two mixed CCs and multiple singletons. This clade struc- ture indicates a shared ancestor for EAH and WAD Sii isolates that is clearly distinct from the EAD Sii. In East SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 7 www.nature.com/scientificreports/ Africa, this finding seems to suggest the evolution of a unique Sii dairy lineage, which is significantly separated from any African human Sii isolate, even those obtained from East African FDP consumers and colonoscopy patients. The association of EAH Sii with CRC and haemorrhoids should therefore not be directly linked to EAD Sii without further functional and genomic comparison to potentially separate pathogenic, pathobiont and food lineages. Consequently, clade- and CC-specific marker genes will need to be identified to enable a thorough safety assessment per lineage. Furthermore, detailed epidemiological data on human SBSEC isolates paired with dairy product analysis from the regular population in Africa, particularly West Africa due to the EAH-WAD Sii link, and other continents will be required for a more comprehensive picture beyond the hospital setting. The daily consumption of Sii-containing food products by approximately 200 million people in sub-Saharan Africa demands such actions given the general recent pathogenicity implication postulated for all SBSEC members8, but under consideration of the novel population structure findings of this study. www.nature.com/scientificreports/ www.nature.com/scientificreports/ purified DNA amplicons was performed at GATC (Konstanz, Germany) and Microsynth AG (Switzerland) using the same primers as for PCR amplification. purified DNA amplicons was performed at GATC (Konstanz, Germany) and Microsynth AG (Switzerland) using the same primers as for PCR amplification. Screening for dairy adaptation marker genes lacS and lacZ. All strains were subjected to lacS and lacZ-specific PCR assays to determine the presence of a lacS/lacZ-mediated lactose uptake system using lacS and lacZ as marker genes for dairy adaption24. SBSEC MLST assay. SBSEC and Sii phylogeny was assessed using the SBSEC MLST assay24. A total of 83 SBSEC and Sii strains were processed and submitted to the public repository on pubmlst.org. Processing of DNA sequencing chromatograms was performed in CLC Genomic workbench 7.5 (Qiagen Aarhus A/S, Denmark). Sequence quality trimming was performed with a parameter setting of 0.1 followed by paired-read assembly per strain and loci. Curation, allele and ST assignment was performed as previously described using MEGA7.0 and START224. Analysis of phylogeny was performed in MEGA7.0 and START2 based on the Maximum likelihood algorithm (200 bootstrap replications) to construct phylogenetic trees using the concatenated sequence-based MLST data of all 10 alleles for all 157 SBSEC isolates. To investigate the genetic diversity of the ten housekeeping genes and to elucidate the impact of the newly added strains, key performance indices such as dN/dS ratio, index of association (IA and IA S for the standard- ized index) were calculated using START224. For dN/dS, N is the number of non-synonymous sites (nucleotide substitutions change the amino acid) and S is the number of synonymous sites (nucleotide substitutions do not change the amino acid). dN is the proportion of non-synonymous sites and dS the proportion of synonymous sites. Investigating the dN/dS ratio provides information about the degree of selection; in our case the degree of selection within the ten housekeeping genes. A ratio with a value less than 1 indicates that the respective gene is under stabilizing selective pressure, which means that the population mean stabilizes and the genetic diversity decreases41. IA and IA S quantify the amount of linkage disequilibrium between the alleles of the ten loci. The index of association has an expected value of 0, which means that no association between the different loci exists and indicates free recombination42. IA and IA S were calculated using a single isolate per ST to avoid bias. www.nature.com/scientificreports/ Clonal com- plex (CC) calculations were performed in Phyloviz 2.0 and eBURSTv3 for groups of STs sharing 7 or more out of 10 alleles with at least one other member of this group24. Simpson’s Index of Diversity (SID) was calculated for allele profiles to indicate infinite diversity for values of 1 and no diversity for values of 043. Data availability. All data generated or analysed during this study are included in this published article and pubmlst.org. Additional information and access to raw data is available from the corresponding author on reasonable request. Methods hi l Among these additional isolates, Sii and Sgm dairy isolates were previously collected from traditionally fermented dairy products in Kenya (Sii n = 18), Somalia (Sii n = 2) and Côte d’Ivoire (Sii n = 8, Sgm n = 1)20,21. Further diversity among the isolate pool was achieved through SBSEC isolates kindly donated by other research collections includ- ing single isolates from external culture collections from hospitals in Spain (n = 2; Sii P-9 and JIM9345)32, Taiwan (n = 1, Sii NTUH-8)6, animal bovine milk isolate from France (n = 1; Sii ANSES 6953)33, Italian Grana cheese (n = 1; Sii 42)31 and Mexican fermented maize (n = 1, Sii pozol)30. Overall, 83 SBSEC isolates were processed in this study and included in the SBSEC MLST scheme. y For validation of assays, the following reference strains were obtained for this study from the Culture Collection of the Laboratory of Food Biotechnology (FBT) of ETH Zurich, Zurich, Switzerland, Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (Braunschweig, Germany) and the Culture Collection of the University of Gothenburg (CCUG) (Gothenburg, Sweden): Sii CJ1825, Streptococcus thermo- philus DSM20259 (yoghurt isolate, DSMZ), Sgg DSM16831T 38, Enterococcus faecium DSM20477T39 and Sii CCUG43820T 3. Growth media and growth conditions. Cultivation of SBSEC strains was performed overnight in Brain Heart Infusion broth (BHI, Biolife, Milan, Italy) at 37 oC under aerobic conditions. The purity of strains was evaluated by streak plating onto Mitis Salivarius agar medium (Becton Dickinson, Allschwil, Switzerland) under aerobic incubation for 1–2 days at 37 °C. The stock cultures were stored at −80 °C in BHI broth supplemented with 33% (v/v) glycerol. Molecular characterization of SBSEC isolates. General DNA isolation procedures, PCR conditions, downstream processing and sequencing of amplified DNA fragments. DNA from single colonies was extracted by lysis and storage in an EDTA, Trizma-base, Triton-X 100-based buffer24,40. All PCR assays were performed using 2× concentrated PCR Master Mix (Thermo Scientific, St. Leon-Rot, Germany), 1 µM primer concentration and sterile ddH2O to a final volume of 20 µL21. All primers were obtained from Microsynth (Balgach, Switzerland). Purification of DNA amplicons was performed using the GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK) or direct filtration by Microsynth AG (Switzerland). Sanger sequencing of 8 SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 References 1. Booijink, C. C. et al. 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Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitut Mol. Biol. Evol. 3, 418–426 (1986). ( ) 42. Smith, J. M., Smith, N. H., O’Rourke, M. & Spratt, B. G. How clonal are bacteria? Proc. Natl. Acad. Sci. USA 90, 4384–4388 (1993). Acknowledgementsh g This study was funded by the ERAfrica program theme “New Ideas” project No ERAfrica_NI_021 (Swiss Partners funded by the Swiss National Science Foundation Project No IZEAZ0_154308). Further fundings were received from the UBS Optimus Foundation, Switzerland. This paper is partially based also on work supported by the DELTAS Africa Initiative [Afrique One-ASPIRE /DEL-15-008]. Afrique One-ASPIRE is funded by a consortium of donor including the African Academy of Sciences (AAS) Alliance for Accelerating Excellence in Science in Africa (AESA), the New Partnership for Africa’s Development Planning and Coordinating (NEPAD) Agency, the Wellcome Trust [107753/A/15/Z] and the UK government. The funders had no role in study design, data collection, analysis, interpretation, decision to publish, or preparation of the manuscript. The authors would like to acknowledge invaluable support by Jonathan Oloo, Mercy Kathio John, Ms. Stella Kibet, Ms. Miriam Arandi, Geoffrey Nzili, and Kenyatta National Hospital staff; Jane Mwangi, Edward Kanata, Ann Salo and Jackie Ngige (Nurses), Dr. Edna Kamau, Dr. Stephen Onyango and Prof. Elly Ogutu (medical doctors/Gastroenterologists); and Prof. Christophe Lacroix, PD Dr. Esther Schelling and Prof. Jakob Zinsstag for contributing to this study. The authors greatly acknowledge strains or DNA received for analysis from Carmen Wacher (Departamento de Alimentos y Biotecnología, Facultad de Química, Mexico), Diego Mora (Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, sezione Microbiologia Industriale Universita degli Studi di Milano, Milano, Italy), Marisa Haenni/Jean-Yves Madec (Anses - Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail, France), Po-Ren Hsueh (Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan) and Rosa del Campo/ Beatriz Romero Hernández (Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Madrid, Spain). SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 10 www.nature.com/scientificreports/ Author Contributions D.W.M.K., N.O. and T.S. performed the experiments. C.J. and L.M. designed and supervised the experiments. All authors contributed to data analysis and interpretation under the lead of C.J., J.H., D.W.M.K. and L.M. The main draft of the manuscript was written by D.W.M.K. Critical review and major revisions of the manuscript were performed by D.W.M.K., W.K.M., G.N.L., B.K., P.R., B.B., L.M., J.H. and C.J. under the coordination of C.J. All authors read and approved the final manuscript. SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-27383-4 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. 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) 2018 SCIENTIFIC RePOrts \| (2018) 8:9181 \| DOI:10.1038/s41598-018-27383-4 11
https://openalex.org/W2803568844	http://sedici.unlp.edu.ar/bitstream/handle/10915/118155/Documento_completo.pdf?sequence=1	English	null	What happened when the Pleistocene megafauna became extinct?	Quaternary international	2,018	cc-by	17,587	a b s t r a c t Article history: Received 26 October 2016 Received in revised form 27 December 2017 Accepted 6 January 2018 Article history: Received 26 October 2016 Received in revised form 27 December 2017 Accepted 6 January 2018 Keywords: End Pleistocene Massive extinctions Pampa-Patagonia Human agency This work presents an updated revision of the information about Pleistocene fauna records in archeo- logical sites of the Pampa and Patagonian regions of South America. The purpose is to assess the role played by humans within the extinction process of Pleistocene mammals in the South Cone and the effects that the disappearance of Pleistocene large mammals had in human populations which colonized both regions. This is based on the theory of “Broken Zig-Zag”, which considers that the process was gradual in time and in different species, taking place between 15 Ka BP and 8.5 Ka BP in Patagonia and during a longer period, until ca. 7.5 Ka BP in Pampa. For this aim it was considered all those sites with accurate chronological and taxonomic information about the presence of extinct species of mega and large mammals of xenarthrans, camelids, equids and carnivores. Thus, the work is focused on three analytical lines: regional analysis of radiocarbon records of ﬁrst and last taxonomic appearance, ecology and etiology of species with archeological record and variability of associations of the archaeofauna with material culture. We discuss how the ﬁrst humans took possession of Pampa and Patagonian regions during and after the last part of the continental extinction process. Thus, there is a contribution with new hypotheses about the differential use of the extinct fauna in both regions. This interdisciplinary approach of social and environmental agency has not been considered in the specialized bibliography so far; therefore novel information is given for interpreting the way in which humans took possession of the fauna, not only as subsistence resources but also as other important agents in the socio-economic and symbolic relationship of humans with the landscape. On the whole, the ﬁnal result is that the gregarious species of large herbivores (camelids) were the most important resources for hunter-gatherers from the beginning of human colonization. The extinct fauna inﬂuenced subsistence complementarily, though it played an important role in the social and symbolic spheres. Keywords: End Pleistocene Massive extinctions Pampa-Patagonia Human agency © 2018 Elsevier Ltd and INQUA. All rights reserved. © 2018 Elsevier Ltd and INQUA. All rights reserved. a b s t r a c t that period of time introduces new questions about the level of human-fauna interaction in such a complex process that began operating at the end of the Pleistocene and that in some regions like Pampa, lasted until mid Holocene (Martínez and Gutierrez, 2011; Martínez et al., 2016). The main role given to humans in the extinction process was due to ﬁndings of large sites of mammoth and bison slaughter that, since the ﬁrst decades of the 20th century, have been carried out in North America. Thus, the scenario and role of the ﬁrst settlers in the continent may have been magniﬁed as regards the excessive hunting of mega and large mammals, with the minimization of the small species hunting and recollection of animal and vegetable products (Sellards et al., 1947; Wormington, 1957). In this sense, and together with the progression of a pro- gramme of systematic archaeological excavations in sites of ﬁrst Americans from the great North America plateaus, in the 1970s the main ideas about human-megafauna interactions were stemmed in three hypothesis lines: 1) That of an intensive human exploitation What happened when the Pleistocene megafauna became extinct? Laura Miotti a, , Eduardo Tonni b, Laura Marchionni a a CONICET/ Division Arqueología, Facultad de Ciencias Naturales y Museo-UNLP, FCNyM-UNLP, Argentina b Museo de La Plata, Facultad de Ciencias Naturales y Museo- UNLP, FCNyM-UNLP, Argentina a CONICET/ Division Arqueología, Facultad de Ciencias Naturales y Museo-UNLP, FCNyM-UNLP, Argentina b Museo de La Plata, Facultad de Ciencias Naturales y Museo- UNLP, FCNyM-UNLP, Argentina Corresponding author. E-mail address: laura.miotti2@gmail.com (L. Miotti). https://doi.org/10.1016/j.quaint.2018.01.004 1040-6182/© 2018 Elsevier Ltd and INQUA. All rights reserved. Quaternary International 473 (2018) 173e189 Quaternary International 473 (2018) 173e189 Contents lists available at ScienceDirect 2. Background and methodology ) This is based on the special fauna structure of South America, stemmed partly from the impact produced by the Great American Biotic Interchange (GABI); its conﬁrmation was obtained from Pampa archeological sites towards Pleistocene and beginning of Holocene, which present a prevalence of xenarthrans in the megafauna; the scarce representation of the total diversity of the extinct mega-mammals and large mammals in the archeological sites, and in short the extinct mega-mammals and large mammals are part of GABI in South America. In this background Cione (Cione et al., 2009) consider that humans participated in the GABI, but they were not the main actors. In this sense, this hypothesis is in concordance with our works in Pampa and Patagonia (Miotti, 1998; Miotti and Salemme, 1999, 2004; Miotti and Marchionni, 2012; Salemme, 2014). The selected sites are grouped in four time blocks (Fig. 1 and Tables 1 a, b and 2) that involve the contexts of hunter-gatherer societies populating both regions during the “Colonization” phase (13e8.5 Ka BP), which includes “scouting” and choice of new en- vironments for inhabiting (Meltzer, 2003; Rockman, 2003; Miotti, 2008; Miotti et al., 2015), and the “Territorial consolidation” phase (8.5e4.5 Ka BP) sensuMiotti (2003) and Miotti and Salemme (1999, 2004); or colonization phase sensuBorrero (1990). ) In those works, we proposed that the ﬁrst settlers of Pampa and Patagonia could have had a generalized or broad spectrum hunting- gathering strategy, and that at the end of the early Holocene and at the beginning of Holocene they could have started to change it towards a specialized one, mainly based on the exploitation of the species Lama guanicoe (guanaco) (Miotti and Salemme, 1999). In the light of the information that is presented herein, it is perceived that such change could have happened previously, when Pampa and Patagonia peopling was still in early development. Therefore, in this work we carry out an updated and re-assessed summary of the available information about the record of Pleistocene species, whose survival until early Holocene allows suggesting a longer use of some species in both regions aforementioned. In this case, even though the environmental changes must have been of great impact in each region for the disappearance of mega-mammals, the pur- pose of this work is to approach new ideas about the kind of appropriation performed by human groups and as a consequence, to bring up for discussion the human impact about extinctions. 2. Background and methodology The reference framework used here is that of zooarcheology, taphonomy and biogeography of updating basis and regional scale. These parameters are the most suitable for the data analysis necessary for drafting the relationship trends between the ﬁrst humans and faunas, as well as the concept of changes of human appropriation in different paleo-environmental scenarios. Both in Pampa and Patagonia the advances produced in the paleo- environmental reconstruction, either from the study of archae- ofaunas or from several environmental proxy, and from the use that humans made of the fauna resources, have been outstanding in the last decades. However, in order to delve into the ways hunter- gatherers developed for appropriating new resources, it has also been considered the ethology and ecology of species with archeo- logical record and corresponding to Lujanian fauna (40 Ka1 BP to 7.5 Ka BP). ) More thorough studies and in more extended regions in South America allow highlighting another observation, presented by Alan Bryan (1986, 1995) but dodged by many, which lies on the principle of the huge environmental and cultural variability of the end of the Pleistocene in North and South America. This key idea made us redirect our exploration to the southern portion of the continent and thus formulate new ideas about the ways in which the ﬁrst humans explored, got in contact with the great variety of envi- ronments in South America and ﬁnally, colonized the continent (Miotti, 2003; Miotti and Marchionni, 2012). Now, in the light of new paleo-environmental and archeological information, the most moderated hypothesis to explain the scenario of mega-mammal and large mammal extinction in South America, close to the Pleistocene-Holocene transition, seems to be that summarized in the “Broken Zig-Zag” theory (Cione et al., 2003, 2009, 2015; Prado et al., 2015; Metcalf et al., 2016; Politis et al., 2016). The study of the recorded species behavior is an important tool for relating the trends of the use of different weapons and hunting strategies (collective or solitary; by encounter or by lurking). In that sense, the assumption is that the most recurrent species in the archeological sites were those that ﬁtted most the hunting tech- niques. For deﬁning the use or human coexistence with different species throughout the transition Pleistocene-Holocene and early Holocene, the dates of the ﬁrst and last appearance of the main Pleistocene mammals with archeological record were considered. 1. Introduction The extinction of Pleistocene megafauna and other large and small mammals in America is a topic that has already been dis- cussed academically for more than a century regarding the factor(s) which produced such process. The main ideas were being outlined dissimilarly about the role the ﬁrst humans played in such extinc- tions when they entered the new world. In the last years, the most widely accepted hypothesis is that the ﬁnipleistocenic extinctions are considered as a process of deep changes, temporarily long and environmentally heterogeneous, where the human impact about the faunas was collateral. On the other hand, every new archaeological ﬁnding referred to https://doi.org/10.1016/j.quaint.2018.01.004 1040-6182/© 2018 Elsevier Ltd and INQUA. All rights reserved. L. Miotti et al. / Quaternary International 473 (2018) 173e189 174 of the Pleistocene fauna which provoked the fast extinction by “Overkill or blitzkrieg” (Martin,1973, 2005), here the humans could have been the main cause of extinctions; 2) a scenario of scarce interaction of humans hunting those mega faunas, and where it is understood that the deep postglacial environmental changes were mainly responsible for the fauna extinctions (Bryan, 1978; Guthrie, 1984). Finally, 3) where the humans may have been another factor in the extinction process but not the main one. Their role could have been that of a catalyst or “coup de grace” that boosted the extinction of animal populations that, due to environmental changes at the end of Pleistocene, were already in clear numerical regression (Guilday, 1984; Miotti and Salemme, 1999; Cione et al., 2009; Metcalf et al., 2016). colonized Pampa and Patagonia. In that sense, this new information contributes to re-assess the hypothesis about the megafauna extinction and its environmental and social consequences in Pampa and Patagonia, to support the variability of forms that this process had in different latitudes, and ﬁnally, to evaluate human- megafauna interactions and the impact that its disappearance had in different regions for the ﬁrst settlers, not only in terms of economic resources but also social and symbolic resources. 1 Ka BP is considered in 14C years before present. 2. Background and methodology Cueva del Medio; 9.Monte Verde; 10, 33.Los Toldos-Cave 3; 11, 23.Piedra Museo-AEP-1; 12. Tres Arroyos; 13.Cerro Tres Tetas; 17.Casa del Minero-1; 18, 38.Fell; 19, 28.Cerro Casa de Piedra-7; 20.La Gruta-1; 21.El Trebol; 22.Túnel; 24.Mariﬁlo; 25.Epullan Grande; 26.Cuyín Manzano; 27.Chorrillo Malo-2; 29.Marazzi; 32, 42.Maripe; 34.El Ceibo-7; 35.Traful-1; 36 Arroyo Feo; 37.Cueva de las Manos; 39.La Mesada; 40.La Martita; 41.El Verano; 43.Las Buitreras; 44.Cerro Bombero; 45.Fitz Roy; 46.Puerto Deseado; 47.Bahía Sanguineto; 48, 49, 63. Arroyo Seco-2; 50.Paso Otero-5; 51.Cerro La China; 52.Tixi; 53.Los Pinos; 54.Amalia; 55.Burucuya; 56.Paso Otero-4; 57.Campo Laborde; 58.Lobería-1; 59.Los Helechos; 60.El Abra; 61. La Brava; 62, 65.El Guanaco; 64.La Moderna; 66.Laguna de los Pampas. Holocene, early Holocene and mid Holocene (Fig.1 and Tables 1 and 2), and where remains of Pleistocene fauna have been found. Holocene, early Holocene and mid Holocene (Fig.1 and Tables 1 and 2), and where remains of Pleistocene fauna have been found. 2015), the last stage of the great biotic interchange between North American and South American subcontinents took place (GABI). During this interchange, numerous mammal families of the Hol- arctic Region emigrated to South America. The immigrant mega mammals (heavier than 1 ton) were only the Gomphoterids, while the large mammals (heavier than 44 kg) included ursids, tayas- suids, equids, camelids and cervids, among other felids (Cione et al., 2009). Regarding the native fauna from South America, xenarthrans 2. Background and methodology To achieve these objectives, the contextual archaeofauna and archeological information has been brought into play. This infor- mation allows better understanding of the social and symbolic impact, beyond that ecological and economic of the humans who Calculations of taxonomic diversity/abundance with the pur- pose at evaluate the use of fauna in both regions was carried out based on a Ntaxa of each site. This is due to the fact that NISP values are only available for some of the researched contexts, and there- fore, even though the production of more speciﬁc abundance in- dexes such as diversity (d1), and richness (H0) indexes (Grayson, 1984) would provide a better deﬁnition to tendencies, data quan- tiﬁcation in this parameter becomes very heterogeneous in different sites of both regions. Finally, to evaluate the social and symbolic impact that the disappearance of large mammals and mega-mammals could have had among the ﬁrst settlers, contextual archaeological markers were taken beyond the zooarchaeological data. These are rock art as well as specimens of special anatomic portions of certain animals, such as paws and teeth of large carni- vores, scutes of glyptodonts and leather of mylodonts (Taçon et al., 1996; Politis and Saunders, 2002; Miotti and Carden, 2007). For this work it has been chosen 45 zooarcheological contexts of Pampa and Patagonian (Chilean and Argentinian) regions, whose human settlements correspond to the transition Pleistocene- L. Miotti et al. / Quaternary International 473 (2018) 173e189 175 phical distribution of the archaeological and paleontological sites: 1, 30. Huenul; 2, 15.Milodon cave; 3.Lago Sofía-4; 4, 31.Ba~no Nuevo-1; 5.Las Guana Chingues; 8, 16. Cueva del Medio; 9.Monte Verde; 10, 33.Los Toldos-Cave 3; 11, 23.Piedra Museo-AEP-1; 12. Tres Arroyos; 13.Cerro Tres Tetas; 17.Casa del Cerro Casa de Piedra-7; 20.La Gruta-1; 21.El Trebol; 22.Túnel; 24.Mariﬁlo; 25.Epullan Grande; 26.Cuyín Manzano; 27.Chorrillo Malo-2; 29.Marazzi; 3 35.Traful-1; 36 Arroyo Feo; 37.Cueva de las Manos; 39.La Mesada; 40.La Martita; 41.El Verano; 43.Las Buitreras; 44.Cerro Bombero; 45.Fitz Roy; 46.Pu uineto; 48, 49, 63. Arroyo Seco-2; 50.Paso Otero-5; 51.Cerro La China; 52.Tixi; 53.Los Pinos; 54.Amalia; 55.Burucuya; 56.Paso Otero-4; 57.Campo Laborde; os; 60.El Abra; 61. La Brava; 62, 65.El Guanaco; 64.La Moderna; 66.Laguna de los Pampas. / Q y ( ) Fig. 1. Geographical distribution of the archaeological and paleontological sites: 1, 30. Huenul; 2, 15.Milodon cave; 3.Lago Sofía-4; 4, 31.Ba~no Nuevo-1; 5.Las Guanacas; 6, 14.Lago Sofía-1; 7.Los Chingues; 8, 16. Table 1 Table 1 Frequencies of extinct and living mammals at archaeological sites in Patagonia region. PATAGONIA sites showed in Fig. 1 14 C dating Frequencies of species References # Name living extinct total Nsp 1 Huenul cave 13,840 ± 56e11,841 ± 56 1 1 2 Barberena et al., 2015, Pompei et al., 2012 2 Milodon cave 13,630 ± 50e12,000± 1 5 6 Martin, 2013 3 Lago Sofía 4 cave 13,400 ± 90e11,590 ± 100 8 5 13 Martin, 2013 4 Ba~no Nuevo 1 cave 13,480 ± 35e11,240 ± 40 1 2 3 Mena and Reyes, 2001 5 Las Guanacas rockshelter 13,275 ± 50 3 5 8 Labarca et al., 2008; 6 Lago Sofía 1 cave 12,990 þ 490 1 2 3 Borrero et al., 1997 7 Los Chingues cave 12,165 þ 80 1 3 4 Martin, 2013 8 Cueva del Medio 12,720 ± 300 1 1 2 Nami and Nakamura, 1995 9 Monte Verde 12,500± 11,800± 2 3 5 Dillehay, 1997 10 Los Toldos cave 3 layer 11 12,600 ± 650 e ca. Table 1 11,000 3 2 5 Cardich, 1987 11 Piedra Museo AEP-1 layer 6 12,890 ± 60e11,000 ± 50 3 4 7 Miotti et al., 1999; 2003 12 Tres Arroyos rockshelter 11,880 ± 250e10,280 ± 110 4 3 7 Massone, 2003 13 Cerro Tres Tetas cave 11,575 ± 140e10,260 ± 110 1 0 1 Paunero, 2000, 2003 14 Lago Sofía 1 cave 11,560 ± 70e10,300 ± 40 4 3 7 Martin, 2013 15 Milodon cave 11,330± -10,200± 1 2 3 Borrero and Martin, 2012 16 Cueva del Medio 11,120 ± 130e10,310 ± 70 5 3 8 Nami and Nakamura, 1995 17 Casa del Minero I cave 11,000 ± 55e10,970 ± 55 1 3 4 Paunero et al., 2007 18 Fell cave 11,000 ± 160e10.720 ± 300 5 3 8 Bird, 1988 19 Cerro Casa de Piedra 7 (CCP 7) 10,690± e 10,530 ± 620 1 1 2 De Nigris, 2004 20 La Gruta 1 cave 10,845 ± 61e10,400 ± 50 1 0 1 Franco et al., 2010 21 El Trebol rockshelter 10,600 ± 100e10, 570 ± 130 15 2 17 Hajduk et al., 2012 22 La María, Túnel cave 10,510± 10,400 ± 100 6 4 10 Paunero et al., 2007, 2010 23 AEP1- layer 4/5 10.470 ± 65e9230 ± 105 3 2 5 Miotti et al., 2003 24 Mariﬁlo rockshelter 1 10,410 ± 70e8420 ± 40 2 0 2 Mera and García 2004 25 Epullan Grande cave 9970 ± 100e7550 ± 70 1 7 8 Cordero, 2011 26 Cuyín Manzano cave 9920 ± 240 3 0 3 Cordero, 2011 27 Chorrillo Malo-2 9740 ± 50e9690 ± 80 1 0 1 Franco and Borrero, 2003 28 Cerro Casa de Piedra 7 (CCP7) rockshelter 9730 ± 100e8300 ± 115 7 0 7 De Nigris, 2004 29 Marazzi rockshelter 9590 ± 210 1 1 2 Morello et al., 1999 30 Huenul cave unit IV 10,155 ± 98e9261 ± 66 2 0 2 Barberena et al., 2015 31 Ba~no Nuevo 1 cave 9530 ± 25e8530 ± 160 2 0 2 Mena et al., 2003 32 Maripe cave 9518 ± 64e7153 ± 50 3 0 3 Miotti et al., 2007, 2014 33 Los Toldos cave 3, layers 9 and 8 ca. 9500e8750 ± 480 5 2 7 Cardich, 1987 34 El Ceibo 7 rockshelter, layer 12 ca. Table 1 9500 5 1 6 Miotti and Salemme, 1999 35 Traful 1 cave 9430 ± 230e9285 ± 105 1 0 1 Cordero, 2011 36 Arroyo Feo cave 9410 ± 70e8410 ± 70 8 0 8 Silveira, 1979 37 Cueva de Las Manos 9320 ± 90e9300 ± 90 7 0 7 Mengoni Go~nalons and Silveira, 1976 38 Fell Cave 10,080 ± 160e9100 ± 70 1 0 1 Bird, 1988 39 La Mesada cave 9090 ± 40 2 0 2 Paunero et al., 2007 40 La Martita cave 8050 ± 90e7940 ± 260 2 0 2 Aguerre, 2003 41 El Verano cave 8960 ± 140e7500 ± 250 3 3 6 Duran, 1986/1987 42 Maripe cave, layer 4 88270 ± 87e8012 ± 80 2 2 Miotti et al., 2014 43 Las Buitreras cave layer VII CA. 9000 1 2 3 Sanguinetti, 1976 44 Cerro Bombero ca. 8850 1 1 Paunero, 2010 45 Fitz Roy n/d 2 2 Carlini, pers. com. 46 Puerto Deseado n/d 1 1 Carlini, pers. com. 47 Bahía Sanguineto (Lujanian stock) n/d x x x Ameghino, 1880 Table 1 Frequencies of extinct and living mammals at archaeological sites in Patagonia region. Table 2 Frequencies of extinct and living mammals at archaeological sites in Pampa region. # Pampa sites in Fig. 1 14 C dating Extinct Living N species References 48 Arroyo Seco 2. Fauna 12,240 ± 110e11,190 ± 110 12 15 27 Politis et al., 2014 49 Arroyo Seco 2. 3. The great biotic interchange. Implications of the holartic species in Pleistocene extinctions in South America Around three million years ago, when the terrestrial bridge between North America and South America was linked (Cione et al., L. Miotti et al. / Quaternary International 473 (2018) 173e189 176 Table 1 Human occupation 11,320 ± 110e11,000 ± 100 10 13 23 Politis et al., 2014 50 Paso Otero 5 10,440 ± 100e10,210 ± 50 10 2 12 Martínez and Gutierrez, 2011 51 Cerro La China 10,804 ± 75e10,525 ± 74 1 0 1 Mazzia, 2011 52 Tixi cave 10,375 ± 90e10,045 ± 95 2 13 15 Mazzanti and Quintana 2001 53 Los Pinos 10,465 ± 65e8750 ± 160 2 13 15 Mazzanti et al., 2013 54 Amalia site 2 10,425 ± 75 2 13 15 Martínez and Osterrieth, 2003 55 Cueva Burucuya 10,000 ± 120 1 14 15 Mazzanti et al., 2013 56 Paso Otero 4 9283 ± 83e7314 ± 73 2 16 19 Alvarez et al., 2013 57 Campo Laborde 8090 ± 190e7750 ± 250 3 6 9 Politis and Messineo, 2008 58 Lobería 1 sitio 1 9878 ± 81 2 28 30 Mazzanti et al., 2013 59 Los Helechos 9640 ± 40 0 8 8 Mazzia, 2011 60 El Abra cave 9834 ± 65 1 14 15 Quintana et al., 2003 61 La Brava cave 9670 ± 120 0 9 9 Mazzanti, 1999 62 El Guanaco 1 9250 ± 40e7494 ± 74 3 6 9 Frontini, 2012 63 Arroyo Seco 2. Human burials 8980 ± 410e7043 ± 82 5 18 23 Politis et al., 2014 64 La Moderna 8356 ± 65e7448 ± 109 3 6 9 Politis and Gutierrez 1998 65 El Guanaco 2 9140 ± 120e8123 ± 82 1 2 3 Frontini, 2012 66 Laguna de Los Pampas without human 8971 ± 77e8835 ± 83 3 7 10 Politis et al., 2012 5. Zooarchaeological results The results of the relative taxonomic abundance expressed in Ntaxa obtained for both regions have resulted from the quantitative analyses of each site (Tables 1e4; Figs. 3 and 4). In this context, it is worth highlighting that if humans only hunted some of the mega and large mammal species of the Luja- nian stock, this action did not cause the extinction ﬁrsthand, but rather a kind of chain reaction in the highly unstable ecosystems of the end of Pleistocene, which were producing a numerical reduc- tion of these animal populations. In that sense, the numerical decline of large and mega mammals that had already been sub- jected to re-accommodation due to profound postglacial environ- mental changes was accelerated by human presence since the selective hunting managed to break the weakened food chains, and this way favored the eventual extinction of different species. In this respect, it cannot be established a sequence of extinctions between mega, large and small mammals due to the focus of human interest for certain preys. These hunts, when reducing selectively some populations of mega and large mammals, managed to break the ecological balance between faunas and their environments, being therefore an extremely disturbing factor in the latter. An example of y ( g ) In all sites of both regions, the common factor is the Lama guanicoe species, which, as it was seen above, it is characterized by the high environmental adaptability, even in very eremic condi- tions. On the other hand, it is the species that even today inhabits in some sectors of the south and west portion of Pampa region and all Patagonian region, and exceeding its geographic dispersion, rea- ches the Andean territories of Argentina, Chile and Peru, and the Bolivian plateau. The zooarcheological remains indicate a high level of human consumption in both regions, moreover, its gregarious ethology and the preference for open steppe environments, together with the associated weapons in the archeological sites, allow inferring that these camelids were the favourite preys of the ﬁrst hunter-gatherers, even until the post-Hispanic times (Borrero, 1990; Miotti, 1998; Mengoni Go~nalons, 1999; Miotti and Salemme, 1999, 2004; Politis and Madrid, 2001; Miotti, 2012; Salemme and Miotti, 2008; among others). 4. Zooarchaeological database All archeological sites with accurate chronological and taxo- nomic information about the presence of Lujanian fauna species were considered for this work. Moreover, the paleontological in- formation whose temporal dispersion coincides, at least in part, with the human presence in both regions was also taken into ac- count. In some cases, they are penne contemporary and in others, the taxa date is available specifying even more the presence of determined taxa in an area. Thus, we analyzed 18 sets for Pampa region and 44 sets for Patagonia, having both present and extinct fauna and with taxa of large and mega mammals. Also, those taxa of medium mammals were taken into account, but only the presence/ absence of those of small and micro mammals was reported since their information is very heterogeneous in the publications. Tables 1a and 1b, 2 and 3 show these data summarized and the other taxa recorded in Pampa and Patagonia sites. These data are considered indispensable for the analysis of biodiversity and fauna use in different environments of both regions. Even though the biodiversity was greater during the period of late Pleistocene and early Holocene, when the ﬁrst signals of hu- man settlement in the south of South America were recorded (ca.13 Ka BP), it was lower in Patagonia than that recorded in Pampa re- gion (Miotti and Salemme, 1999). Of the 14 extinct species of large mammals,12 are Holarctic type and two are native from South America, of these, those corre- sponding to mega fauna were fully extinct (Alberdi et al., 1995; Cione et al., 2009, pp. 135). The biotic interchange did not produce an irreversible declination of biodiversity, which even increased in South America towards the end of Pleistocene, especially at the expense of the immigrants. Some mega mammals (glyptodonts) managed to survive the dramatic postglacial changes, until ca. of 7.5 Ka BP, date of the last appearance in Pampa region (Politis and Gutierrez, 1998; Miotti and Salemme, 1999; Politis et al., 2004; Cione et al., 2015). Table 2 L. Miotti et al. / Quaternary International 473 (2018) 173e189 177 (hairy and cingulates), and notoungulates emigrated to the North, (Cione et al., 2009). this is the case of guanacos that were, both in Pampa and in Pata- gonia, the species mostly used by hunter-gatherers, and they have reached the present time, in spite of the fact that hunting was sustained by humans in both regions throughout all Holocene. Of the 12 Holarctic families (excluding Hominidae) which emigrated to South America from the limit Pliocene-Pleistocene, felids, ursids, camelids and equids (Equus and Hippidion), along with the South American native fauna, made up the Lujanian fauna that at the end of the Pleistocene was found by hunter-gatherer pioneers in Pampa and Patagonia. Of the 122 Lujanian species, only 16 have archeological record between both regions. On the other hand, if we only register those corresponding to mega mammals and large mammals, the use could have been much lower as regards selected species (Fig. 2). 5. Zooarchaeological results We found out that in the time period previous to 12.6 Ka BP only archeological sites with Pleistocene fauna were recorded in Pata- gonia (Tables 1a, 1b, and 2), and only after that date, these taxa appeared represented in archeological sites of both regions, what indicates an older record of human-Pleistocene fauna co-existence for the Patagonian region. The only exception in Pampa corre- sponds to the archaeological site Arroyo Seco 2, which seems to Fig. 2. Biodiversity estimated in Lujanian taxa. Table 3 Temporal and regional faunal distributions. Regions Patagonia Pampa N taxa Intervals Extinct Living N sites Extict Living N sites 13.5e12.5 Ka BP 8 5 8 0 0 0 12.5e11 Ka BP 8 4 11 17 12 2 11e9.5 Ka BP 5 18 15 6 28 12 9.5e7.5 Ka BP 2 12 10 4 23 5 Total N sites 22 38 44 27 63 19 Table 3 Temporal and regional faunal distributions. Table 4 Species with archaeological record. Table 3 T Fig. 2. Biodiversity estimated in Lujanian taxa. L. Miotti et al. / Quaternary International 473 (2018) 173e189 178 Table 4 Species with archaeological record. L. Miotti et al. / Quaternary International 473 (2018) 173e189 179 eet this condition of presenting a high number of mega and large ammal species for 12,600e12,000 RYBP. Nevertheless, the il d i f i b i d f h h i l 6. Characteristics of the species with archaeological recor Th i h id k i hi k Fig. 3. Taxonomic frequency in a) Patagonia, and b) Pampa. y extinct species. yy dessapear in the region. L. Miotti et al. / Quaternary International 473 (2018) 173e189 Fig. 3. Taxonomic frequency in a) Patagonia, and b) Pampa. y extinct species. yy dessapear in the region. Fig. 3. Taxonomic frequency in a) Patagonia, and b) Pampa. y extinct species. yy dessapear in the region. Figs. 2 and 5). 6. Characteristics of the species with archaeological record meet this condition of presenting a high number of mega and large mammal species for 12,600e12,000 RYBP. Nevertheless, the detailed information obtained from the taphonomic analyses indicate that for the inferior levels of the site, humans only hunted/ used few of all the taxa present in the area (Politis, 2014). On the other hand, even though the spatial distribution of faunas with Lujanian elements is greater in Patagonia than in Pampa, the latter presents a greater taxonomic diversity (Tables 1 a-b, 2 and 3 and Figs 2 and 5) meet this condition of presenting a high number of mega and large mammal species for 12,600e12,000 RYBP. Nevertheless, the detailed information obtained from the taphonomic analyses indicate that for the inferior levels of the site, humans only hunted/ used few of all the taxa present in the area (Politis, 2014). On the other hand, even though the spatial distribution of faunas with Lujanian elements is greater in Patagonia than in Pampa, the latter presents a greater taxonomic diversity (Tables 1 a-b, 2 and 3 and Figs 2 and 5) The species that we consider as key in this work correspond to the association of Lujanian mammals corresponding to the Biozone of Equus (A) neogaeus Association, according to Cione et al. (2009), since these are the ones that had better chances of having co- existed a longer time with the ﬁrst humans, and therefore, having being exploited by the hunter-gatherers that colonized both re- gions of South America (Fig. 5). L. Miotti et al. / Quaternary International 473 (2018) 173e189 180 arding the Xenarthra group, several ﬁndings can be ned in Southern Patagonia, though not always its associa- th archaeological sites is related with mylodonts. However, 7, in Fitz Roy locality (#45), Santa Cruz province, during the tions for a sport building, remains of (Panochthus), and others referred to Scelidotherium (A. Carlini, com. pers.) were Both taxa form part of the Lujanian fauna (late Pleistocene) a far, they have not been documented in sites of the Patagoni gion, since only accuracy ascribed those of Bahia Sanguineto and Puerto Deseado (#46) to Lujanian xenarthrans remains Fig. 4. Taxonomic abundance (living and extinct mammals) in a) Patagonia and b) Pampa sites. / Q y ( ) Fig. 4. Taxonomic abundance (living and extinct mammals) in a) Patagonia and b) Pampa sites. 7. Dates of the ﬁrst and last appearance of extinct taxa The results of taxa dates obtained in archaeological sites of Pampa and Patagonia allow supporting the hypothesis that Pleis- tocene large and mega mammals had a period of coexistence with humans of ca. 5000 years (Cione et al., 2009). However, the coex- istence times between humans and mega fauna were different in the different species, in both regions. In Fig. 5 it is shown this sit- uation based on the zooarchaeological information of each region. This analysis together with the zooarchaeological information of the previous paragraph allows suggesting that hairy Xenarthra (sloths) and Cingulates (glyptodonts) had greater possibilities of becoming prey for humans in Pampa region. This difference is based on the fact that, in Pampa region, the Cingulates disappeared by mid-Holocene,ca. 7.5 Ka BP (Politis and Gutierrez, 1998; Gutierrez et al., 2010), while in Patagonia the last records corre- spond to the end of the transition Pleistocene/Holocene, i.e., ca. 10 Ka BP (Borrero and Martin, 2012; Marchionni and Vazquez, 2012; Miotti and Marchionni, 2012; Martin, 2013) (Fig. 5). This may suggest that the potentiality of these animals as resource for human consumption was extended 2500 years more in Pampa than in Patagonia. On the other hand, their most abundant use in the former region is supported by the vast evidence of primary butchering and consumption marks, and the greatest taxonomic richness per taxon (Table 4 and Fig. 3). The presence of notoungulates is very scarce in the analyzed archaeological sites, being absent in the Patagonian record, while, in Pampa region their presence is exclusively in Arroyo Seco 2 site only, with the species Toxodon platensis (Politis et al., 2014; Salemme, 2014). It is observed that this evidence may only support the hypothesis of coexistence between humans and such species since the record corresponds to a single carpal, which is dated 11,750 ± 70 (CAMS16839) 14C BP and does not present evidence of human action. However, this information together with the taxon dates of the site (Politis, 2014) indicate low chances that the species had coexisted during a long time with the ﬁrst settlers of the area, therefore it seems parsimonious to keep the hypothesis of coexis- tence with the humans for a short time, but we do not believe that this species could have form part of the group of resources chosen by humans for consumption. 6. Characteristics of the species with archaeological record Regarding the Xenarthra group, several ﬁndings can be mentioned in Southern Patagonia, though not always its associa- tion with archaeological sites is related with mylodonts. However, in 2007, in Fitz Roy locality (#45), Santa Cruz province, during the excavations for a sport building, remains of (Panochthus), and others referred to Scelidotherium (A. Carlini, com. pers.) were found. Both taxa form part of the Lujanian fauna (late Pleistocene) and, so far, they have not been documented in sites of the Patagonian re- gion, since only accuracy ascribed those of Bahia Sanguineto (#47) and Puerto Deseado (#46) to Lujanian xenarthrans remains (Tonni others referred to Scelidotherium (A. Carlini, com. pers.) were found. Both taxa form part of the Lujanian fauna (late Pleistocene) and, so far, they have not been documented in sites of the Patagonian re- gion, since only accuracy ascribed those of Bahia Sanguineto (#47) and Puerto Deseado (#46) to Lujanian xenarthrans remains (Tonni L. Miotti et al. / Quaternary International 473 (2018) 173e189 181 Fig. 5. Date of ﬁrst and last appearance of species of Lujanian fauna in both regions. a) Patagonia y b) Pampa. g. 5. Date of ﬁrst and last appearance of species of Lujanian fauna in this region the number of archaeofauna elements is over- dimensioned since the most represented parts in almost all the Patagonian sites correspond to dermal bones and in some cases, like in the Milodon Cave and Ba~no Nuevo-1 cave (Fig. 1), to leather fragments with little bones included. This evidence of several dermal bones is a key marker for being able to interpret these ﬁndings in function of the human choice regarding the use of sloths. This recurrent data in different Patagonian caves encouraged the hypothesis of an initial cave occupation by animals, and then, a subsequent use by humans. In this case, there may be a temporal difference between the megafauna occupation and humans. The taphonomic and stratigraphic studies indicate natural death of animals and previous to peoples at caves (Mena and Reyes, 2001; Borrero and Martin, 2012). In others caves, like Huenul 1, Milodon, Los Chingues, the ﬁrst stratigraphic levels contain milodont's dungs. However, in some cases like in El Trebol cave (Fig. 1 #21) signals of use of mylodonts are clearer. 6. Characteristics of the species with archaeological record The excavators inferring that mylodonts were used by humans: “Algunos huesos dermicos pre- sentan una patina negro brillante, indicando que fueron expuestos al fuego cuando aún conservaban restos de tejido blando.” (Hajduk et al., 2009, pp. 959, pp. 959) … “El rasgo mas destacable en estos huesos dermicos (Mylodontinae) es la presencia de claras huellas de corte producidas por el hombre, en algunos casos profundas y reiteradas.” (Hajduk et al., 2009, pp. 960). The hypothesis of the use of dead animal leather and meat should not be discarded by the arguments further discussed. and Carlini, 2008). Such mammals appear stratigraphically in sed- iments of lagoon and swamp type, as well as wind type, and their habitats were those of open savanna and grasslands related to peri- glacial areas and with temperatures lower than today. In both re- gions these basic ecological characteristics for Lujanian mammals were conﬁrmed by independent evidence lines such as palynology, the studies of sea cores, and stable isotopes (Czerwonogora et al., 2011). 7. Dates of the ﬁrst and last appearance of extinct taxa In numerous societies, from hunter-gatherers to agricultural or pastoral, the jaguar image as power symbol and social belonging has been important until today, in ceremonies, artifacts, artistic representations, legends and other narratives as well (see references above). On the other hand, it is worth highlighting that in Patagonia there is only one species recorded, Hippidion saldiasi, while in Pampa the equids were represented by two species, Hippidion sp. (probably H. principale) and Equus (Amerhippus) neogaeus, though these are recorded in an only archaeological site, Arroyo Seco 2 (AS2). Even though in recent works, the archaeologists consider that in Pampa region both species were available, only Equus is the species with evidence of human use (Gutierrez and Johnson, 2014; Salemme, 2014). These marks of cut and fracture impact are still scarce and they were recorded in a bone (radius) of those recovered in the only archaeological site where the species appeared repre- sented (Gutierrez and Johnson, 2014, pp. 124; Salemme, 2014, Table 4.8). In Patagonia Hippidion saldiasi was recorded in fourteen local- ities: Los Toldos 2 and 3, Piedra Museo AEP-1, La María, Túnel cave, Casa del Minero 1, Cave 6 from El Ceibo, Lago Sofía 1 and 4; Fell, Pali Aike, Cueva del Medio, Los Chingues cave, Del Puma cave, and Tres Arroyos rockshelter, being the representation of anatomic units numerous in each site, generally corresponding to individuals of different age groups and presenting clear marks of prey processing. In this region, the time of the last equid appearance reached the early Holocene, around 8.5 Ka BP, recorded with taxon date in cerro Bombero site (Paunero, 2010). This evidence, combined with the gregarious nature of these mammals, an ethology similar to that of camelids, which undoubtedly were the most important preys in both regions, allows stating that the coexistence with equids may have been another factor contributing to adjust this species with the programmed hunting strategies of the ﬁrst inhabitants in the Patagonian region. In such sense, horses are considered a selected resource searched by the ﬁrst settlers of the southern extreme, and in Patagonia, they might have been very interesting in the eco- nomic, social and symbolic spheres. Probably, we should expect new evidence for supporting that a similar use of equids took place among hunter-gatherers in Pampa region. In both regions, the species with more possibilities of having been important prey for humans is the camelids (Figs. 7. Dates of the ﬁrst and last appearance of extinct taxa This reasoning may explain partly the In Patagonia there are no records of glyptodont remains in archaeological sites (Table 4 and Fig. 3) but there have been numerous ﬁndings of giant sloths; though in very few of these ﬁndings there is clear evidence of butchering marks and anthropic burnt (Borrero et al., 1997; Miotti and Cattaneo, 2003; Hajduk et al., 2009, 2012; Lezcano et al., 2010; Marchionni and Vazquez, 2012; Marchionni, 2013; Miotti and Marchionni, 2012, 2014). However, in L. Miotti et al. / Quaternary International 473 (2018) 173e189 182 low archaeological representation of the species at Pampean regional level, since this is indeed frequent in paleontological groups of the end of Pleistocene. evidence in Pampa region, with presence only in site AS 2, their record in Patagonia is much higher in number of sites and in amount of anatomic parts, as well as in amount of cut and chop traces (Nami and Menegaz,1991; Miotti,1998; Miotti and Salemme, 2005; Miotti and Marchionni, 2012; Martin, 2013). In this case, it can also be interpreted that the longest date of the last appearance in Patagonian sites (ca. 9.2 ka BP), together with an ethology similar to that of guanacos, may have contributed to the greatest and longest consumption of these mammals in the most southern region. Horses have a higher numerical representation in Patagonian contexts than in Pampa ones (Fig. 3), being their last appearance date 2000 years earlier in Pampa than in Patagonia (Fig. 5). Their longer coexistence period with humans in Patagonia could have been an important cause for having been more intensely used in this region. However, there are other archaeological markers that allow inferring a more intense use of equids by hunter-gatherers in Patagonia. Among these markers, there are equid images recorded in rock art from Los Toldos, La Maria and Piedra Museo sites (Cardich, 1987; Miotti, 1991, 1993e96; Miotti et al., 2007; Miotti and Carden, 2007; Paunero et al., 2007; Carden, 2009), most of the anatomic units, of different age categories, and abundant and clear cut/blow marks of butchering for the species Hippidion sal- diasi in several Patagonian sites support the hypothesis about economic use of these mammals. The large carnivores, such as the Panthera onça, have a very low archaeological representation in sites of Patagonia (Fig. 5) and null in those of Pampa. 7. Dates of the ﬁrst and last appearance of extinct taxa However, this species that nowadays lives in rain forests as far as the North of Argentina, inhabited both regions towards the end of Pleistocene. In Patagonia, the time of coexis- tence with humans was of 2000 years (Fig. 5), being ca. 10 ka BP the last records of the subspecies Panthera onça mesembrina. In Pampa Panthera onça inhabited until the beginning of the 20th century, but there are no archaeological records of the use given to this species by the ﬁrst settlers in this region. This unequal archaeological ev- idence in both regions brings into questions the different use of these large felines by humans (Table 6). Taking into account that in Pampa the time of coexistence was much longer, the most parsi- monious hypothesis is that the Pampean hunter-gatherers had not used jaguars as economic, social or symbolic resource (Table 6). On the contrary, and although a short period of coexistence was recorded in Patagonia, the archaeological evidence indicates that these animals formed part of, at least, the social and symbolic spheres of the ﬁrst humans (Table 6). The sites where teeth of this species were recorded are Cueva del Medio, La María and El Ceibo. In this last Locality it was also documented a very big painting, assigned to that feline (Cardich, 1987; Miotti and Carden, 2007). This form of appropriation of one of the extinct species corre- sponding to a large carnivore, which at the same time was predator of Pleistocene large and mega mammals, is, in every sense, assignable to the symbolic category. This interpretation considers that the evidence of their remains associated to the ﬁrst archaeo- logical contexts is related with large images, outstanding on painting panels as well as with the appropriation of tusks and paws. This information supports the hypothesis of image appropriation or of parts of these large carnivores as elements that even today are considered mimetic of super natural power in several societies (Gonzalez, 1974; Reichel-Dolmatoff, 1975; Dillehay and Kaulick, 1984-85; Ingold, 1986; Taçon et al., 1996; Politis and Saunders, 2002; Miotti and Carden, 2007), and in that sense, that those items might have been considered as metonymic and/or meta- phoric of prestige and/or power among the ﬁrst Patagonian ex- plorers. However, such evidence is not enough for formulating the incidence in hunting for an economic use. 7. Dates of the ﬁrst and last appearance of extinct taxa 3 and 4), and within them the guanacos, which are considered main resources for hunter-gatherers until late Holocene. The abundance of remains of these mammals in most sites of both regions, along with the fact that their presence continues nowadays, supports such hypothesis. This record that leads to a strategy of hunting specialization during mid Holocene (Miotti and Salemme, 1999; Miotti, 2012, and refer- ences therein) is now outlined as a trend in hunting strategies which may have started at the beginning of human occupation. Table 5 Thus, it is inferred that from the end of the Pleistocene to mid Holocene, biodiversity trends, both in Pampa in Patagonia, were decreasing with some variables in both regions (Fig. 6). The greatest Table 6 Different human uses of mammalian fauna in Pampa and Patagonia regions. Regions Pampa Patagonia Use of Taxa Taxa Economic Social Symbolic Economic Social Symbolic Xenarthrans yes yes yes yes yes yes Equids yes n/d n/d yes yes yes Camelids yes yes n/d yes yes yes Grand Carnivors No Data No yes yes Fig. 6. Faunal diversity in Pampa and Patagonia: temporal trends. 1 to 5 are temporal intervals expressed in ka BP. Fig. 6. Faunal diversity in Pampa and Patagonia: temporal trends. 1 to 5 are temporal intervals expressed in ka BP. Table 6 Different human uses of mammalian fauna in Pampa and Patagonia regions. Regions Pampa Patagonia Use of Taxa Taxa Economic Social Symbolic Economic Social Symbolic Xenarthrans yes yes yes yes yes yes Equids yes n/d n/d yes yes yes Camelids yes yes n/d yes yes yes Grand Carnivors No Data No yes yes 8. Discussion The zooarchaeological, ethological, coeval information between humans and Pleistocenic extinct fauna presented here supports the hypothesis of Broken Zig Zag (see, Cione et al., 2009, 2015, and ci- tations therein) as a varied and complex set of environmental and cultural factors of mammal extinction. In this frame, the amount of taxa of extinct fauna appearing in archaeological sites of ﬁrst colonization in both regions does not exceed the 30% of the total (Fig. 2), therefore they are few as regards those of living species in Holocene which were used, ﬁrstly, as product of human hunting. As As regards other extinct camelids, present in the ﬁrst settlings in both regions, they also have a different representation. While Hemiauchenia paradoxa and Lama gracilis have an ephemeral L. Miotti et al. / Quaternary International 473 (2018) 173e189 183 Table 5 Table 5 Ecological and ethological characteristics of extinct Pleistocene mammals with archaeological record. Lujanian Species behavior Activity feeding habits Potential depredators environment climate Panthera onça lonely crepuscular/ dusk carnivore unknown Forest/moist Savanna tropical/ temperate Smilodon Sp lonely diurnal carnivore without Savanna cool temperate Arctotherium tarijense lonely diurnal/ daytime? omnivore without ? cool temperate Canis (D) avus lonely diurnal/ dusk omnivore unknown Savanna/steppe? cool temperate Scelidotherium Sp ? diurnal/ daytime ? grand carnivores/ human Savanna/grassland cool temperate Lestodon armatus ? diurnal/ daytime ? grand carnivores/ human Savanna/grassland cool temperate Mylodon darwini/listai ? diurnal/ daytime grazer grand carnivores/ human open shrub steppe cool temperate Megatherium Sp lonely? diurnal/ daytime grazer grand carnivores/ human Savanna/grassland cool temperate Glossotherium Sp. lonely? diurnal/ daytime grazer grand carnivores/ human Savanna/grassland cool temperate Eutatus seguini lonely? diurnal/ daytime grazer grand carnivores/ human Savanna/grassland cool temperate Gliptodon Sp lonely? diurnal/ daytime grazer grand carnivores/ human Savanna/grassland cool temperate Panochthus Sp lonely? diurnal/ daytime grazer grand carnivores/ human Savanna/grassland cool temperate Doedicurus Sp lonely? diurnal/ daytime grazer grand carnivores/ human Savanna/grassland cool temperate Sclerocalypthus Sp lonely? diurnal/ daytime grazer grand carnivores/ human Grassland/schrubby cool temperate Toxodon Sp herd? diurnal/ daytime grazer grand carnivores/ human wetland? cool temperate Macrauchenia Sp herd? diurnal/ daytime grazer grand carnivores/ human Grassland/schubby cool temperate Gomphoteriidae herd diurnal/ daytime grazer grand carnivores/ human Grassland/woodland cool temperate Hippidion saldiasi herd diurnal/ daytime grazer grand carnivores/human Plains and mountain cool and wet Equus (A.) neogaeus herd diurnal/ daytime grazer grand carnivores/ human Plains and mountain cool temperate Hemiauchenia paradoxa herd? diurnal/ daytime grazer grand carnivores/ human Grassland/schrubby cool temperate Lama gracilis herd diurnal/ daytime grazer grand carnivores/ human grassland, schubby cool temperate Lama guanicoe herd diurnal/ daytime grazer grand carnivores/ human Grassland/schubby steppe cool temperate Ozotoceros bezoarticus herd diurnal/ daytime grazer grand carnivores/ human Grassland plains tropical/ temperate Table 5 Ecological and ethological characteristics of extinct Pleistocene mammals with archaeological record. a consequence, even though it is observed that the glyptodonts lasted until mid Holocene for Pampa region (ca. 7 ka BP), in Pata- gonia, their disappearance was completed 3000 years before. However, we can suggest that in Patagonia the social and symbolic use of some extinct species may have indeed survived among hunter-gatherers until the end of Mid Holocene. Table 6 Diff Even though the mostly used species was the guanaco in both regions from the beginning of the ﬁrst settlers, the complementary re- sources changed, being the cervids which complemented the eco- nomic resources in Pampa and north Patagonia, while in Patagonia the speciﬁcity about guanacos was more signiﬁcant (Fig. 4). mammal biodiversity recorded archaeologically at the end of Pleistocene or beginning of Holocene was due to the presence of mega and large mammals of Lujanian stock, which decreases in sites of both regions towards the second interval of human occu- pation, ca. 11.5 Ka BP (Fig. 6) because of the extinction process both of mega and large mammals. From that date, the inversion in di- versity trends took place in both regions, between the extinct and present species. The variety of extinct species decreased abruptly and the representation of large living mammals increased. Even though the mostly used species was the guanaco in both regions from the beginning of the ﬁrst settlers, the complementary re- sources changed, being the cervids which complemented the eco- nomic resources in Pampa and north Patagonia, while in Patagonia the speciﬁcity about guanacos was more signiﬁcant (Fig. 4). p y g g ( g ) In Pampa, the ﬁrst records of the Pleistocene fauna use in archaeological sites were later than in Patagonia, which indicates that the interaction (humans-extinct fauna) began roughly 1000 years earlier in Patagonian sites than in Pampa sites. The use of Pleistocenic fauna continued increasing in both regions during the third interval (10.9e9.5 Ka BP) and fourth interval (9.4e8.5 Ka BP), reaching the ﬁfth interval (8.4e7.5 Ka BP) only with present fauna in both regions and the survival of cingulated xenarthrans only in Pampa region until 7 Ka BP (Fig. 6). Xenarthra disappeared 1000 years before in Patagonia ca. 8.5 Ka. BP, when in that region the extinction of all Pleistocene species was completed. Nevertheless, in Pampa, survival of some glyptodonts in environmental shelters of that region reached ca. 7 Ka BP. Particular cases for both regions are those of giant sloths, glyptodonts and large carnivores such as jaguars. In Patagonia these mammal groups lived with humans around 2500 years (Fig. 5), however, the archaeological evidence suggests an opportunist use of giant sloths and jaguars, while the glyptodonts were only recorded in paleontological sites (No 45, 46 and 47 of Tables 1 and 2, and Fig. Table 6 Diff This materiality of the extinguished fauna may also be indicating that these animals had their social importance in the narratives of stories of places and people, possibly in ceremonies, myths and legends. For horses (Equus [A.] and Hippidion) and camelids (Lama gua- nicoe and Lama gracilis), all of them herd mammals of grazing and running habits (Table 5), the archaeological and ethnographic evi- dence indicates a human appropriation through ambush and planned hunting. We can add the pattern of selected places for such practice. Both in Pampa and Patagonia, these places coincide with special sectors and lagoon and/or river banks for speciﬁc activities such as hunting and prey processing. Among the most outstanding sites we can mention Paso Otero 4 and 5, Campo Laborde, Cerro El Sombrero, Cerro Amigo Oeste, Piedra Museo, where the recurrence of these microenvironments working as fauna trappers and as topographic tramps for lurking and capturing preys are present. Moreover, in both regions there are lithic contexts of projectiles such as spears FTP and triangular of straight base are added to these sites (Nami and Menegaz, 1991; Miotti et al., 1999; Mazzanti and Quintana, 2001; Massone, 2004; Messineo et al., 2009; Flegenheimer et al., 2013; Martínez and Gutierrez, 2015; Miotti and Terranova, 2015). This evidence is consistent and reliable and suggests that the equid and camelid appropriation is related with strategies of programmed hunting. These strategies may have been As regards the change of strategies of fauna appropriation, we observe that towards 10 Ka BP the greatest biodiversity increment took place in both regions dominated by present faunas (Figs. 4e6). The contexts of hunting technology and zooarchaeological, until then, indicate that the prevailing strategy may have been the wide generalized hunting, though with a prevalence of large mammals such as guanacos. In both regions, the gregarious animals, such as the camelids and equids in Patagonia, and the camelids, equids and cervids in Pampa, seem to have been predominantly sought by humans as economic resources. However, in all sites, the anatomic richness and the richness of use and consumption evidence was always focused on guanacos. After 8.5 Ka BP, the strategy in both regions changed, turning to a greater speciation on guanaco hunting (Miotti and Salemme, 1999; Miotti, 2012). Table 6 Diff 1), and consequently, the appropriation of all these animals might have inﬂuenced more signiﬁcantly the social and symbolic spheres than those economic. That is, they were used for learning about the new places and their beings but they were not focus of hunting strategies for satisfying their basic needs (food). Another peculiarity, in both regions after 8.5 Ka. BP, is the most diverse use of taxa of both large and small mammals in Pampa region, while in Patagonia the almost exclusive resource was rep- resented in the species Lama guanicoe, which was joined, as a complement, by some cervids in the Andean sector and pinnipedes on the Atlantic coast. The reduction in biodiversity in archaeolog- ical sites may have resulted from the extinction process. An outstanding issue of the fauna variations of both regions is that in Patagonia the taxonomic diversity was always below that recorded in Pampa region (Fig. 6). Despite this difference of a greater mammal variety in Pampa region than in Patagonia, in both regions the guanaco dominated all archaeological contexts before and after the great extinction of Pleistocene species. This supports the hy- pothesis of a greater adaptation between hunters and preys, posi- tioning the guanaco as the main resource from the beginning of colonization. On the other hand, even with a wider biodiversity in Pampa than in Patagonia, the use of fauna was of larger spectrum in the human occupations of both regions until the third interval (Fig. 6), though with main contributions obtained from guanacos. In Pampa, both groups lived longer with humans (Fig. 3), how- ever, the same as in Patagonia, giant sloths acquired an opportunist appropriation, while it is impossible by now to assign any kind of human use of jaguars. This importance was reverted in this Pampean region in the case of glyptodonts (xenarthrans cingu- lates), from which there is clear evidence of economic (Alvarez et al., 2013; Politis et al., 2014; Salemme, 2014; Martínez et al., 2016), but is weakness of social and symbolic use (Table 6). The discussion about anthropological, archaeological and palaeoecologial information allowed us to state that the knowledge about them lasted in the social imaginary, even after their extinc- tion, through rock art, in curated and/or claimed artifacts, such as scutes, paws and teeth, present in some Pampean and Patagonian contexts. Table 6 Diff a consequence, even though it is observed that the glyptodonts lasted until mid Holocene for Pampa region (ca. 7 ka BP), in Pata- gonia, their disappearance was completed 3000 years before. However, we can suggest that in Patagonia the social and symbolic use of some extinct species may have indeed survived among hunter-gatherers until the end of Mid Holocene. Fig. 6. Faunal diversity in Pampa and Patagonia: temporal trends. 1 to 5 are temporal intervals expressed in ka BP. Thus, it is inferred that from the end of the Pleistocene to mid Holocene, biodiversity trends, both in Pampa in Patagonia, were decreasing with some variables in both regions (Fig. 6). The greatest L. Miotti et al. / Quaternary International 473 (2018) 173e189 184 catalyst role in the process of megafauna extinction in South America, is still the most parsimonious (Cione et al., 2009, 2015). Even though, since the earliest peopling humans had used species of extinct and living Pleistocenic mammals, the archaeological ev- idence presented herein for both regions indicates that humans were not the main responsible for the extinction by mass slaughter but rather another factor which destabilized the environments already in crisis about 3000 years before the human arrival in South America. The human settling in a new environment produces changes in the ecosystems from their interaction. The interaction in those ﬁnipleistocenic environments where deep climatic and environmental changes had been taking place (Mc Culloch et al., 2000; Rabassa, 2008) produced the ﬁnal disarticulation of trophic chains with differential extinction structures in different mammal species, and such species did not manage to overcome population decreases which repeatedly occurred before the human arrival in the continent (Cione et al., 2015). mammal biodiversity recorded archaeologically at the end of Pleistocene or beginning of Holocene was due to the presence of mega and large mammals of Lujanian stock, which decreases in sites of both regions towards the second interval of human occu- pation, ca. 11.5 Ka BP (Fig. 6) because of the extinction process both of mega and large mammals. From that date, the inversion in di- versity trends took place in both regions, between the extinct and present species. The variety of extinct species decreased abruptly and the representation of large living mammals increased. Table 6 Diff In this case, the general trend of the ﬁrst times became a hunting strategy of speciﬁcation of resources, centered in the most popular species, the guanaco, with few cervid representatives in northern sites of the region, on the other hand, the only survivors of Pleis- tocenic Lujanian fauna in Patagonia. This form of narrative and transmission, from generation to generation, of the knowledge of environments and beings who were extinct when being materialized in rock art or in ornaments/ offerings, is a symbolic narrative that the ancient settlers may have used for transmitting to their descendants the history of the an- cestors who colonized that place with different characteristics and beings from those they knew (Politis and Saunders, 2002; Rockman, 2003; Miotti and Carden, 2007; Miotti et al., 2015). When the scale of information about extinguished or mythic beings and landscapes lasts in wide regions and can be resigniﬁed by centuries and millenniums in societies, such as the case of the appropriation of many supernatural and/or natural beings (unicorns, dragons, jaguars), it is then when this is considered as social knowledge and culturally learned (Ingold, 1986; Taçon et al., 1996; Saunders, 1998; Meltzer, 2003). The information gathered in this work, resulting from a myriad of sites of Pampa and Patagonia does not allow stating the gener- alization made by Martínez and Gutierrez (2011) about the use of guanacos, since this theory is based on the archaeological evidence of a single site: “Remarkably, in one archaeological site (Paso Otero 5, see, mammal record previous of the extinction showed a large exploitation of extinct mammals and a reduced one of guanacos (Lama guanicoe). After the extinction, hunting was concentrated on guanacos and other relatively small mammals. This could be the representation of an opportunistic behaviour that resulted after the extermination of the spectacular fauna of huge mammals that inhabited South America before the entrance of humans. (Martínez and Gutierrez, 2011, pp. 61). The evidence of few cut marks in anatomic units of mylodonts is present in few sites, AEP1, El Trebol, Paso Otero 5, and AS 2, remaining as a low signal for supporting the hypothesis of a sys- tematic hunting of these animals. Table 6 Diff Therefore, the change in strategy that in previous works (Miotti and Salemme, 1999) we placed between early Holocene and Mid Holocene, with the new regional information we are able to place it around 9.5 Ka BP, at the end of the third interval (Fig. 6). In this sense, the hypothesis, which assigns the ﬁrst humans a L. Miotti et al. / Quaternary International 473 (2018) 173e189 185 where charred/burnt human remains were covered by large Mylodont bones (Hyslop,1988). Third similar case and associated to human burials is that of AS2, in Pampa (Salemme, 2014). In these contexts, though, bones do not present cut or human action traces. Meanwhile in El Trebol cave, osteoderms appear burnt and show bearing anthropic cutmarks (Hajduk et al., 2009, 2012; Lezcano et al., 2010). This evidence reinforces the idea that their associa- tion to the use of leather by human would be a hypothesis to evaluate. by ambush and use of special conditions of some continental aquatic landscapes, found only in some sectors of lagoons and streams, where small elevations present in the surroundings could have allowed concealment of the hunters (see works cited above). This argument has a similar pattern as regards the use of land- scapes, mammal fauna and strategies of collective hunting between hunter-gatherer societies from different parts of America, from the end of Pleistocene to the end of the 20th century (Speth, 1983; Frison and Todd, 1987; Binford, 1991; Miotti, 1993e96; Miotti et al., 1999, 2004; Martínez, 1999, among others). From the paleontological information of these three sites of the central plateau, it can be inferred that Lujanian stock of xenar- thrans, horses and camelids, placed in Bahía Sanguineto (Ameghino, 1880), together with those from Fitz Roy, Puerto Deseado and the only horse individual found in Cerro Bombero, may have been available for the late Pleistocene in the plateau. This information added to that paleo-environmental of the area, mainly palinological (Borromei, 2003; Mancini et al., 2005; Mancini, 2009), supports the hypothesis that this could have been an open grass- land steppe, with shrub patches, similar to Pampean region for the same time span. However, equids of both species have a lower hunting incidence in Pampean region than in Patagonia. Table 6 Diff In this last region Hippidion appears represented in numerous sites, being also more numerous and clearer the evidence of chopping and human consumption than in Pampean sites (Miotti and Cattaneo, 2003; Miotti and Salemme, 2005; Marchionni, 2013). Possibly, as equids are gregarious animals, of open environ- ments and share these with camelids, the hunting strategy of both could have been adjusted well to technology, using the same weapons and lurking forms for their capture. Moreover, in Southern Patagonia it is remarkable that zoomorphic images assigned to horses were represented in several sites such as Cave 6 of Los Toldos, Cave Túnel l of La María and possibly their tracks may have been recorded in the petroglyph of Piedra Museo. In this last case, even though their chronological order is discussed (see Carden, 2009 and reference therein) due to the fact that the petroglyphs lean on the stratigraphic layer corresponding to ca. 7.7e7.4 years BP (Miotti et al., 2003), the social use of equids is brought into question even for times when equids had already disappeared in both re- gions. In this case, the persistence of extinct animals in social memory would be similar to that previously referred to for xenar- thrans. Thus, the symbolic use of that fauna through collective memory of societies could have moved at least 1000 years the idea of Pleistocenic horses that lived in Patagonian steppes. The last appearance in the Patagonian environment was recorded in 8.5 Ka BP, in the paleontological site Cerro Bombero, in Santa Cruz central plateau (Paunero, 2010). As regards the archaeological evidence (taxonomic and anatomic abundance) of guanaco, undoubtedly it is the most important species in the sites since the beginning of the human occupation in both regions. Nevertheless, in this group, the social appropriation goes beyond the economic sphere involve that social and imaginary domains, since the artistic representation of these animals appears in several places of Patagonia until late Holocene (Miotti and Carden, 2007 and reference therein). The zooarchaeological contexts grouped by intervals (Table 3 and Fig. 6) indicate that those of the ﬁrst interval (16 - 10 Ka BP) show a greater biodiversity than those of the second interval (9.9e8.7 Ka BP). For this span only horses and Lama gracilis per- sisted. Besides, during this span, the presence of Lama guanicoe is increased in number of individuals and of represented anatomic parts in all the considered sites. 9. Conclusions The extinction event was certainly spectacular. Some authors have referred to it as a mass extinction (Marshall et al., 1984; La Violette, 2011). However, it was not. Coexistence between ﬁrst human explorers and Pleistocenic megafauna and large mammals was differential in time in each species process. Nonetheless, coexistence remained ca. 5000 years since ﬁrst humans arrived in Pampa and Patagonia. Human/fauna relationships started and ﬁnished ca. 1000 years before in Patagonia (Fig. 6). Even though in Pampa region, the species Panthera onca (jaguar) had a distribution until the beginning of the 20th century, for this region and from the zooarchaeology, we conclude that the extinct subspecies Panthera onca mesembrina was coeval but was not used by the ﬁrst settlers of this region (Table 6). In Pampa the occurrence of few species of Lujanian stock in archaeological contexts (ca. 7.5 Ka BP) allows us to infer that glyptodonts were economically exploited, and probably also so- cially and symbolically used for longer time than in Patagonia. Scouting landscapes (12-11 Ka BP) in Patagonia, and (11e10.5 Ka BP) in Pampa had a lot mega mammals and large mammals avail- able for new hunter-gatherer groups. The greatest biodiversity is recorded in Pampa, in Arroyo Seco 2 site (12 taxa), followed by sites in Patagonia, with the greatest records in Cueva Lago Sofía 4 (5 taxa) (Tables 1 and 2). Teeth and paws of these felines are pieces of high preservation in the archaeological and paleontological records, as a result, if this species played a role in the cosmovision of the ﬁrst settlers of both regions, it could be expected that they had been collected by the ﬁrst settlers, even without ever having had the intention of hunting these carnivores. However, the zooarchaeological evidence of these elements is very fragmented, appearing in three sites so far (Caves Túnel de La María; El Ceibo and Cueva del Medio). Colonization landscapes (11e8.5 Ka BP) in Patagonia, and (10.5e7. ka BP) in Pampa had different economic, social and sym- bolic incidence of the extinct fauna. The only representation of a large feline in rock art of cave 6 of El Ceibo, although it is scarce evidence, supports the hypothesis of a social and symbolic appropriation of these animals, at least for the Patagonian area. However, it must be discarded the idea of eco- nomic use, or programmed hunting of these large felines. Table 6 Diff necessary and long time was spent as it was not frequent to ﬁnd dead jaguars every day. On the other hand, these collars could have been prestigious artifacts of hereditary type, what means that we can trace a biography (Gosden and Marshall, 1999). This allows inferring that their ﬁnding in a deﬁnite historic context is the result of a cultural tradition which could have started hundreds of years ago. opportunistic. If there was really a human intention of consump- tion, this was occasional. Only chopping patterns of these “allegedly preys” were detected in few sites such as Piedra Museo (Marchionni and Vazquez, 2012) and Arroyo Seco 2 (Politis et al., 2014; Gutierrez and Johnson, 2014). Another use given to bones belonging to these animals was as fuel, as seen in Paso Otero 4 (Gutierrez et al., 2010; Martínez and Gutierrez, 2015). There are no artifacts made of these bones what means that this hypothesis of their use as part of technology is even weaker. However, in some cases, the mylodon association of a big deal of dermal little bones as in Cueva Fell, and El Trebol, and large bones used as fuel in Paso Otero 4, may be indicating that these ﬁrst settlers of Patagonia might have used these animals’ leather and bones for different social and symbolic practices. The gomphotherid group belonging to immigrant holartic megafauna is scarcely represented in Pampa and Patagonia regions. It is only recorded in Monte Verde site, in the north of Patagonia, (Fig. 1). In the earliest occupational context, the abundance of Cuveronius sp. genus is of a NISP ¼ 268 specimens, corresponding to a MNI of 1 individual (Dillehay, 1997). The cutmarks and usefulness are mostly represented in task fragments, what allows inferring a human opportunistic proﬁt of any individual found dead, that is why, the hypothesis about programmed hunting of these pro- boscideans is discarded. p The coexistence between the ﬁrst explorers and Pleistocenic megafauna could have been in stages according to different taxa and environmental differences in both regions. In Patagonia (12.5 Ka BP) it would begin 1000 years before and besides it would ﬁnish 1000 years before than in Pampa (ca. 7 ka BP). The large carnivores do not have archaeological record in Pampa, whereas in Patagonia, though with a low record, it is considered that they were used somehow by the ﬁrst settlers (Table 6). Table 6 Diff That is why it is more parsimo- nious to hold the hypothesis of opportunist use (Borrero and Martin, 2012) of any dead animal by non human reasons, and the subsequent attainment of their parts, not for food, but for diverse uses like bones for fuel (Alvarez et al., 2013); or leather. It is sig- niﬁcant that in Ba~no Nuevo-1 cave, dermal bones (osteoderms) could have been associated to the death treatment with the human body wrapping. However, taphonomic and stratigraphic in- vestigations indicate that these bones are not associated to cultural layers (Mena and Reyes, 2001). Similar cases are Fell and Pali Aike, By the evidence discussed at regional scale we understand that megafauna extinction did not interrupt the social and symbolic use that humans made of these giants in Holocene, and that their extinction did not lead to a more opportunistic use of smaller species, but rather, since the beginning of the human settling in both regions, the food subsistence basis was in guanacos, being the extinguished species of complementary and opportunist use. As regards hairy xenarthrans, during the ﬁrst interval it is scarce the representation of individuals and their anatomic parts in the archaeological sites, with which the most feasible hypothesis is that these animals formed part of the colonization landscape of the ﬁrst humans in the region, but their use would be complementary or L. Miotti et al. / Quaternary International 473 (2018) 173e189 186 opportunistic. If there was really a human intention of consump- tion, this was occasional. Only chopping patterns of these “allegedly preys” were detected in few sites such as Piedra Museo (Marchionni and Vazquez, 2012) and Arroyo Seco 2 (Politis et al., 2014; Gutierrez and Johnson, 2014). Another use given to bones belonging to these animals was as fuel, as seen in Paso Otero 4 (Gutierrez et al., 2010; Martínez and Gutierrez, 2015). There are no artifacts made of these bones what means that this hypothesis of their use as part of technology is even weaker. However, in some cases, the mylodon association of a big deal of dermal little bones as in Cueva Fell, and El Trebol, and large bones used as fuel in Paso Otero 4, may be indicating that these ﬁrst settlers of Patagonia might have used these animals’ leather and bones for different social and symbolic practices. Table 6 Diff The images of great felines are present in rock art and in the zooarchaeological record from the presence of paws and tusks. In Magallanes, Arctotherium sp, Smi- lodon sp, and a similar genus of xenarthra are also recorded (Mena and Reyes, 2001; Labarca, 2015), the latter with an archaeological record of a paw in the site Túnel of Santa Cruz central plateau (Skarbun et al., 2015). References Aguerre, A.M., 2003. La Cueva 4 de La Martita y las ocupaciones de 8000 a~nos (Santa Cruz, Argentina). In: Aguerre, A.M. (Ed.), Arqueología y Paleoambiente en la Patagonia Santacruce~na Argentina. Talleres Graﬁcos Nuevo Offset, Buenos Aires, pp. 27e61. Franco, N.V., Martucci, M., Ambrústolo, P., Brook, G., Mancini, M.V., Cirigliano, N., 2010. Ocupaciones humanas correspondientes a la transicion Pleistoceno- Holoceno al sur del Macizo del Deseado: el area de La Gruta (provincia de Santa Cruz, Argentina). Relaciones de la Sociedad Argentina de Antropología XXXV 301e308. Alberdi, M.T., Leone, G., Tonni, E.P., 1995. Evolucion Biologica y Climatica de la Region Pampeana Durante los últimos Cinco Millones de A~nos. Monografías 12, Museo Nacional de Ciencias Naturales, Madrid, p. 423. Frison, G., Todd, L., 1987. The Horner Site. The Type Site of the Cody Cultural Complex. Academic Press, London, U.K. Frontini, R., 2012. El aprovechamiento de animales en valles ﬂuviales y lagunas del sur bonaerense durante el Holoceno. Ph.D, Facultad de Filosofía y Letras. Uni- versidad de Buenos Aires, Buenos Aires. Frontini, R., 2012. El aprovechamiento de animales e b d t l H l Ph D F Alvarez, M.C., Alcaraz, A., Gutierrez, M.A., Martínez, G., 2013. Analisis zooar- queologico del sitio Paso Otero 4 (Partido de Necochea, provincia de Buenos Aires, Argentina). Aportes a la discusion de modelos de subsistencia de la region pampeana. Intersecc. Antropol. 14, 383e398. Gonzalez, A.R., 1974. Arte, Estructura Y Arqueología. Ediciones Nueva Vision, Buenos Aires. Ameghino, F., 1880. La Antigüedad del Hombre en el Plata. Imprenta Coni, Buenos Aires. Gosden, Ch, Marshall, Y., 1999. The cultural biography of objects. World Archaeol. 31 (2), 169e178. Barberena, R., Borrazo, K., Rughini, A., Romero, G., Pompei, M., Llano, C., de Porras, M., Duran, V., Stern, C., Re, A., Estrella, D., Forasiepi, A., Fernandez, F., Chediak, M., Acu~na, L., Gasco, A., Quiroga, M., 2015. Perspectivas arqueologicas para Patagonia Septentrional: sitio Cueva Huenul 1 (Provincia de Neuquen, Argentina). Magallania 43 (1), 1e29. Grayson, D., 1984. Quantitative Zooarchaeology. Topics in the Analysis of Archaeo- logical Faunas. Academic Press. Guilday, J., 1984. Pleistocene extinction and environmental change. In: Martin, P., Klein, R. (Eds.), Quaternary Extinctions a Prehistoric Revolution. The University of Arizona Press, Arizona, pp. 250e258. Binford, L., 1991. A Corporate Caribou Hunt. Documenting the Archaeology of the Past Lifeways. Expedition: the Magazine of the University of Pennsylvania, 33, pp. 33e43, 1. Guthrie, R.D., 1984. References Mosaics, allelochemics, and nutrients: an ecological theory of late pleistocene megafaunal extinctions. In: Martin, P., Klein, R. (Eds.), Quater- nary Extinctions a Prehistoric Revolution. The University of Arizona Press, Arizona, pp. 259e298. Bird, J., 1988. Travels and Archaeology in South of Chile. University Iowa Press, Iowa. Borrero L 1990 Fuego Patagonia bone assemblages and the problem of the gua Bird, J., 1988. Travels and Archaeology in South of Chile. University Iowa Press, Iowa. Borrero, L., 1990. Fuego-Patagonia bone assemblages and the problem of the gua- naco communal hunting. In: Davis, L., Reeves, B. (Eds.), Hunters of the Recent Past. Unwin Hyman, London, pp. 373e399. Borrero, L., 1990. Fuego-Patagonia bone assemblages and the problem of the gua- naco communal hunting. In: Davis, L., Reeves, B. (Eds.), Hunters of the Recent Past. Unwin Hyman, London, pp. 373e399. Gutierrez, M., Johnson, E., 2014. Analisis de los efectos tafonomicos del material oseo faunístico. Interpretaciones sobre los procesos de la formacion del sitio. In: Politis, G., Gutierrez, M., Scabuzzo, C. (Eds.), Estado actual de las Investigaciones en el sitio arqueologico Arroyo Seco 2 (Partido de Tres Arroyos, Provincia de Buenos Aires, Argentina), serie Monograﬁca nº 5. INCUAPA-CONICET-UNICEN, Olavarría, pp. 97e137. y pp Borrero, L.A., Martin, F.M., 2012. Ground sloths and humans in southern Fuego- Patagonia: taphonomy and archaeology. World Archaeol. 44 (1), 102e117. B L A M i F M P i A 1997 L C L S fí 4 Úl i E Borrero, L.A., Martin, F.M., Prieto, A., 1997. La Cueva Lago Sofía 4, Última Esperanza: una madriguera de felino del Pleistoceno tardío. Anales del Instituto de la Patagonia. Serie Ciencias Humanas 25, 103e122. Gutierrez, M.A., Martínez, G., Luchsinger, H., Alvarez, M.C., Barros, M.P., 2010. Investigaciones arqueologicas y geoarqueologicas preliminares en el sitio Paso Otero 4 (Partido de Necochea). In: Beron, M., Luna, L., Bonomo, M., Montalvo, C., Aranda, C., Carrera Aizpitarte, M. (Eds.), Mamul Mapü: pasado y presente desde la arqueología pampeana. Libros del Espinillo, Ayacucho, pp. 69e84. Borromei, A., 2003. Palinology at Piedra Museo locality, Santa Cruz Province, Argentina. In: Miotti, L., Salemme, M., Flegenheimer, N. (Eds.), Where the South Winds Blow: Ancient Evidence of Paleo South Americans. Center for the Studies of the First Americans. A&M University Press, Texas, pp. 113e120. EE.UU. Hajduk, A., Albornoz, A., Lezcano, M., 2009. Nuevas excavaciones en el sitio El Trebol. San Carlos de Bariloche, Provincia de Río Negro. L. Miotti et al. / Quaternary International 473 (2018) 173e189 L. Miotti et al. / Quaternary International 473 (2018) 173e189 187 9.1. In short 9.1. In short Bryan, A., 1986. New Evidences from South American for Early Man Studies. Uni- versity of Alberta, Edmonton. Bryan, A., 1995. Disproof of commonly held assumptions relevant to the peopling of the Americas. Curr. Res. Pleistocene 12, 6e8. What happened when megafauna became extinguished? Palaeo-environmental aspect generated several consequences in restructuration of climates, biota, and water distribution, pro- ducing huge changes for landscapes in both regions. However, from the human point of view, we can say that the changes were economically smaller. In both regions the guanacos were the main faunal resources, however, other species like birds, armadillos, ro- dents and cervids were used as complementary, and promoted a generalist hunter strategy since the ﬁrst human occupations. Carden, N., 2009. Imagenes a traves del tiempo. Arte rupestre y construccion social del paisaje en la Meseta Central de Santa Cruz. Sociedad Argentina de Antro- pología, Buenos Aires. Cardich, A., 1987. Arqueología de Los Toldos y El Ceibo (provincia de Santa Cruz, Argentina). Estud. Atacame~nos 8, 98e117. Cione, A., Tonni, E., Soibelzon, L., 2003. The broken zig-zag: late Cenozoic large mammals and tortoise extinction in South America. Revista del Museo Argen- Cione, A., Tonni, E., Soibelzon, L., 2003. The broken zig-zag: late Cenozoic large mammals and tortoise extinction in South America. Revista del Museo Argen- tino de Ciencias Naturales 5 (1), 1e19. tino de Ciencias Naturales 5 (1), 1e19. Cione, A., Tonni, E., Soibelzon, L., 2009. Did humans cause the late pleistocene-early Holocene mammalian extinctions in South America in a context of shrinking open areas? In: Haynes, G. (Ed.), American Megafaunal Extinctions at the End of the Pleistocene. Springer Netherlands, pp. 125e144. However, from the social and symbolic appropriation the Pleistocenic giants probably were important beings which after their extinction became myths and legends, remaining among Pampean and Patagonian people long time. Cione, A., Gasparini, G., Soibelzon, E., Soibelzon, L., Tonni, E., 2015. The Great American Biotic Interchange a South American Perspective. Springer, Netherlands. Cordero, J.A., 2011. Arqueofauna de las ocupaciones tempranas de cueva Traful I, provincia del Neuquen, Argentina. Arqueología 17, 161e194. In scientiﬁc thought we are in agreement “If those giant Plesi- tocenic bugs encourage our curiosity, maybe, despite being extinct, they will continue somehow alive just like some hundreds of centuries ago”. (Fari~na and Vizcaíno, 1995, pp. 97). Appendix A. Supplementary data Flegenheimer, N., Miotti, L., Mazzia, N., 2013. In: Graf, K., Ketron, C., Waters, M. (Eds.), Rethinking Early Objects and Landscapes in the Southern Cone. Palae- oamerican Odyssey, Santa Fe, pp. 359e376. Supplementary data related to this article can be found at https://doi.org/10.1016/j.quaint.2018.01.004. Franco, N., Borrero, L., 2003. Chorrillo Malo 2: initial peopling of the upper Santa Cruz basin, Argentina. In: Miotti, L., Salemme, M., Flegenheimer, N. (Eds.), Ancient Evidence for Paleo South Americans: From where the South Winds Blow. Center for the Study of First Americans. Texas A&M University Press, pp. 149e152. Aknowledgments Dillehay, T., 1997. Monte Verde: A Late Pleistocene Settlement in Chile. The Archaeological Context and Interpretation, Vol. 2. Smithsonian Press, Washington. We thank colleagues for help provided along the way, Alberto Cione for discussions concerning the Pleistocene mammals at issue in this paper, Darío Hermo, Rafael Suarez and the anonymous ref- erees for valuable comments on the manuscript, and Diego Gobbo for creating the map. This work was done within the framework of the projects PICT-ANCyT-176 and PI-665 UNLP. ́ Dillehay, T., Kaulick, P., 1984-85. Aproximacion Metodologica: el Comportamiento del Jaguar y la Organizacion Socio-Espacial Humana. Relaciones de la Sociedad Argentina de Antropología XVI, pp. 27e35. Duran, V., 1986/1987. Estudio tecno-tipologico de los raspadores del sitio El Verano, Cueva 1. Patagonia Centro Meridional, Santa Cruz, Argentina. Anales de Arqueología y Etnología de la Universidad Nacional de Cuyo 41e42, 129e163. F i~ R Vi í S 1995 H l di il ~ D d d l Fari~na, R., Vizcaíno, S., 1995. Hace solo diez mil a~nos. Donde se trata de como era la fauna que habito America del Sur antes de los indios. Editorial Fin de Siglo, Coleccion Prometeo, Buenos Aires. L. Miotti et al. / Quaternary International 473 (2018) 173e189 Czerwonogora, A., Fari~na, R., Tonni, E., 2011. Diet and isotopes of late pleistocene ground sloths: ﬁrst results for lestodon and glossotherium (Xenarthra, Tardi- grada). Neues Jahrbuch für Geologie und Pal€aontologie 262, 257e266. De Nigris, M.E., 2004. El Consumo en Grupos Cazadores Recolectores: Un Ejemplo Zooarqueologico de Patagonia Meridional. Sociedad Argentina de Antropología, Buenos Aires. 9. Conclusions Both pieces of evidence account for a social knowledge and symbolic use of these animals (Rockman, 2003), which could have remained for many generations in the collective imaginary, even after their extinction. This collective memory may indicate that these animals certainly entered the cosmologic sphere of the ﬁrst settlers, that is why, their appropriation expressed in images and in tooth and paw recollection would evoke their presence in the social collective of the ﬁrst hunter-gatherers. In Patagonia region the main resource hunted was Lama gua- nicoe, meanwhile Hippidion and L. gracilis were complementary, and Ground giant sloths were occasional. Only some parts of Pan- thera onca and other great carnivores were used, and these parts may have been obtained from ﬁnding dead animals but no from hunting. However, interaction human/animals should have been a high impact on social and symbolic spheres (Table 6). Archaeological record in rock art and daily contexts reinforces this hypothesis. In Pampa L. guanicoe was the main species exploited, L. gracilis, and ground sloths were occasional and/or complementary resources. It is worth highlighting that in several societies, from current hunter-gatherers, such as Nukak groups from Amazonia, to Andean state societies (Dillehay and Kaulick, 1984-85; Saunders, 1998), the jaguar ﬁgure is conceived as one of a supernatural being with great power. This power could be transferred or granted to people through the possession of paws and tusks or the leather of these animals. Possessing these pieces, in general, did not result from the chasing and systematic hunting of these carnivores, but rather from collecting them after ﬁnding dead individuals. In this sense, it is worth thinking about the social importance that a tooth collar ac- quired, since for manufacturing it, dozens of elements were As regards camelids, even though Lama gracilis and Hemi- auchenia paradoxa were available in both regions until ca. 9 Ka BP, the evidence of use of both is stronger in the Patagonian region than in that Pampean, where it can be inferred only the coexistence but not the human use. Thus, those of greater chances of a taxa-human coexistence span were xenarthrans cingulates in Pampa and equids in Patagonia. Therefore, equids are outstanding in Patagonia as economic, social and symbolic resource, whereas the Pampean evidence is even weaker to be able to infer relevance in any of these three aspects in this region. References Early human remains from Ba~no Nuevo-1 cave, central patagonian andes, Chile. Quat. Int. 109e110, 113e121. La Violette, P., 2011. Evidence for a solar ﬂare cause of the pleistocene mass extinction. 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https://openalex.org/W4387603507	https://www.mdpi.com/1660-3397/21/10/534/pdf?version=1697167738	English	null	Multicopy Chromosome Integration and Deletion of Negative Global Regulators Significantly Increased the Heterologous Production of Aborycin in Streptomyces coelicolor	Marine drugs	2,023	cc-by	11,394	Citation: Li, J.-Y.; Liang, J.-Y.; Liu, Z.-Y.; Yi, Y.-Z.; Zhao, J.; Huang, Z.-Y.; Chen, J. Multicopy Chromosome Integration and Deletion of Negative Global Regulators Signiﬁcantly Increased the Heterologous Production of Aborycin in Streptomyces coelicolor. Mar. Drugs 2023, 21, 534. https://doi.org/ 10.3390/md21100534 Keywords: RiPP; marine Streptomyces; phoU (SCO4228); wblA (SCO3579); SCO1712; orrA (SCO3008); gntR (SCO1678) Academic Editor: Um Soohyun Received: 13 September 2023 Revised: 10 October 2023 Accepted: 11 October 2023 Published: 13 October 2023 Jia-Yi Li 1, Jun-Yu Liang 1, Zhao-Yuan Liu 1, Yue-Zhao Yi 1, Jing Zhao 1,2,3 , Zhi-Yong Huang 4,5,* and Jun Chen 1,2,3,* Jia-Yi Li 1, Jun-Yu Liang 1, Zhao-Yuan Liu 1, Yue-Zhao Yi 1, Jing Zhao 1,2,3 , Zhi-Yong Huang 4,5,* and Jun Chen 1,2,3,* 1 Department of Marine Biological Science & Technology, College of Ocean and Earth Sciences, Xiamen University, Xiamen 361102, China; 22320191151003@stu.xmu.edu.cn (J.-Y.L.); y__vette@sina.com (J.-Y.L.); liuzhaoyuan2919@163.com (Z.-Y.L.); 22320182201398@stu.xmu.edu.cn (Y.-Z.Y.); sunnyzhaoj@xmu.edu.cn (J.Z.) y ( ); ( ); y j (J ) 2 State-Province Joint Engineering Laboratory of Marine Bioproducts and Technology, Xiamen University, Xiamen 361102, China 3 Xiamen City Key Laboratory of Urban Sea Ecological Conservation and Restoration, Xiamen University, Xiamen 361102, China 4 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China 5 National Technology Innovation Center of Synthetic Biology, Tianjin 300308, China * C d h @ ib (Z YH ) h j @ d (J C ) 5 National Technology Innovation Center of Synthetic Biology, Tianjin 300308, China gy y gy, j , * Correspondence: huang_zy@tib.cas.cn (Z.-Y.H.); chenjun@xmu.edu.cn (J.C.) Abstract: Aborycin is a type I lasso peptide with a stable interlocked structure, offering a favorable framework for drug development. The aborycin biosynthetic gene cluster gul from marine sponge- associated Streptomyces sp. HNS054 was cloned and integrated into the chromosome of S. coelicolor hosts with different copies. The three-copy gul-integration strain S. coelicolor M1346::3gul showed superior production compared to the one-copy or two-copy gul-integration strains, and the total titer reached approximately 10.4 mg/L, i.e., 2.1 times that of the native strain. Then, ﬁve regulatory genes, phoU (SCO4228), wblA (SCO3579), SCO1712, orrA (SCO3008) and gntR (SCO1678), which reportedly have negative effects on secondary metabolism, were further knocked out from the M1346::3gul genome by CRISPR/Cas9 technology. While the ∆SCO1712 mutant showed a signiﬁcant decrease (4.6 mg/L) and the ∆phoU mutant showed no signiﬁcant improvement (12.1 mg/L) in aborycin production, the ∆wblA, ∆orrA and ∆gntR mutations signiﬁcantly improved the aborycin titers to approximately 23.6 mg/L, 56.3 mg/L and 48.2 mg/L, respectively, which were among the highest heterologous yields for lasso peptides in both Escherichia coli systems and Streptomyces systems. Thus, this study provides important clues for future studies on enhancing antibiotic production in Streptomyces systems. marine drugs marine drugs marine drugs 1. Introduction Aborycin is a class I lasso peptide and was ﬁrst isolated from Streptomyces sp. SP9440 as a novel anti-HIV metabolite with the origin name RP 71,955 [1,2]. It was independently rediscovered from soil S. griseoﬂavus Tü 4072 as an antibiotic [3], then from deep-sea Streptomyces sp. SCSIO ZS0098 as an anti-infective natural product [4] and from marine sponge-associated Streptomyces sp. MG010 as an antibacterial marker for screening gain- of-function mutants [5]. It was also predicted to be expressed in the mangrove soil strain Streptomyces sp. EMB24 [6]. Aborycin has a typical lasso topology in which the N-terminal 9 amino acids form a macrocyclic ring, and the C-terminal 12 amino acids form a tail that folds back and threads through the ring [2]. Two disulﬁde bonds between the ring and the tail further increase the structural stability and distinguish class I lasso peptides from others [7,8]. Citation: Li, J.-Y.; Liang, J.-Y.; Liu, Z.-Y.; Yi, Y.-Z.; Zhao, J.; Huang, Z.-Y.; Chen, J. Multicopy Chromosome Integration and Deletion of Negative Global Regulators Signiﬁcantly Increased the Heterologous Production of Aborycin in Streptomyces coelicolor. Mar. Drugs 2023, 21, 534. https://doi.org/ 10.3390/md21100534 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/marinedrugs Mar. Drugs 2023, 21, 534. https://doi.org/10.3390/md21100534 Mar. Drugs 2023, 21, 534 2 of 13 Lasso peptides are a growing class of bioactive bacterial peptides with unique lasso topology, which differentiates them from other members within the much larger riboso- mally synthesized and post-translationally modiﬁed peptide (RiPP) superfamily [9,10]. The compact and constrained topology endows most lasso peptides with remarkable ther- mal and proteolytic stability and favors peptide–protein interactions, accounting for the diverse biological activities of lasso peptides, mainly as enzyme inhibitors and receptor antagonists [7,11]. Robust scaffolds of lasso peptides have attracted attention towards drug development, such as epitope grafting [12], the incorporation of noncanonical amino acids [13] and protein fusion [14]. While these modiﬁcations provide the opportunity to develop novel biological activities with therapeutic potential, they always lead to lower production levels [15]. Moreover, genome mining approaches have greatly accelerated lasso peptide discovery in recent years. Since the ﬁrst lasso peptide was isolated by genome min- ing in 2008 [16], the number of lasso peptides discovered by such approaches has steadily increased [11]. By applying the RODEO algorithm, >1400 lasso peptide biosynthetic gene clusters (BGCs) were identiﬁed from DNA sequence databases, a great increase over the pre- viously known numbers [17]. Although lasso peptide BGCs are widely distributed among bacteria, only approximately 80 lasso peptides were previously characterized [18]. There- fore, both drug development and functional characterization require effective production systems to explore this rich source of lasso peptides and their modiﬁcations. The heterologous production of lasso peptides in E. coli often provides higher yields than the use of native producers. However, this production advantage in E. coli seems to be conﬁned to lasso peptides from Proteobacteria and is less viable for clusters from other phyla [11,15]. Specially, inefﬁciency in expressing genes with high GC content, the lack of metabolic precursors and the lack of detoxiﬁcation mechanisms are the main weakness of the E. coli system in terms of expressing natural products from actinomycetes [19,20]. Recently, Streptomyces hosts, especially S. coelicolor, S. lividans and S. albus, have shown signiﬁcant potential for producing lasso peptides from actinobacte- ria [17,21,22]. Several technological advances have also been achieved for Streptomyces systems, such as CRISPR/Cas9 genome editing technology [23,24] and the multiplexed site-speciﬁc genome engineering (MSGE) method [25]. Genome editing offers a rapid way to modify regulatory elements involving secondary metabolism and therefore impact heterologous production. The MSGE method led to the successful development of a panel of S. coelicolor heterologous hosts, in which up to ﬁve copies of BGCs could be integrated into the speciﬁc sites of the host chromosome in a single step, leading to signiﬁcant yield improvements [25]. In our previous study, an aborycin BGC was identiﬁed from a marine sponge-associated Streptomyces sp. HNS054 [5]. This provided an opportunity to produce this lasso peptide heterologously in Streptomyces systems. Thus, in this study, a Strepto- myces system for aborycin production that is compatible with up-to-date technologies was established. By increasing the integrated copy numbers of the aborycin BGC in the host chromosome and by deleting the negative global regulatory genes involved in secondary metabolism by genome editing, the production of aborycin was signiﬁcantly improved. This study provides a useful reference to improve Streptomyces systems for the produc- tion of lasso peptides and their modiﬁcations and thus beneﬁts drug development and functional characterization. . General Information on the Aborycin Gene Cluster from Streptomyces sp. HNS054 Cloning of the Gul BGC and Construction of Strains for Heterologous Expression of Aborycin 2.2. Cloning of the Gul BGC and Construction of Strains for Heterologous Expression of Aborycin The cloning procedure of the gul BGC is shown in Figure S1. The 14 kb fragment containing the gul BGC was amplified from Streptomyces sp. HNS054 by high-fidelity PCR and subsequently cloned and inserted into the integrative vector pSAT209, yielding pSAT-GUL (Figure S2). The plasmid was fully sequenced, and the results confirmed that no mutation occurred within the plasmid frame nor the gul ORFs. Then, the plasmid pSAT-GUL was transferred to S. coelicolor strains M1146, M1246, M1346, M1446 and M1546 by conjugation. As a blank control, the empty plasmid pSAT209 was also trans- ferred to S. coelicolor M1146. The numbers of successful attP-attB recombinations in excon- jugants were determined by PCR. The results showed that strains M1146::gul (Figure S3A), M1246::2gul (Figure S3B) and M1346::3gul (Figure S3C) were successfully constructed. However, four-copy and five-copy integrations were unsuccessful after four rounds of conjugation and screening. The cloning procedure of the gul BGC is shown in Figure S1. The 14 kb fragment containing the gul BGC was ampliﬁed from Streptomyces sp. HNS054 by high-ﬁdelity PCR and subsequently cloned and inserted into the integrative vector pSAT209, yielding pSAT-GUL (Figure S2). The plasmid was fully sequenced, and the results conﬁrmed that no mutation occurred within the plasmid frame nor the gul ORFs. Then, the plasmid pSAT-GUL was transferred to S. coelicolor strains M1146, M1246, M1346, M1446 and M1546 by conjugation. As a blank control, the empty plasmid pSAT209 was also transferred to S. coelicolor M1146. The numbers of successful attP-attB recombinations in exconjugants were determined by PCR. The results showed that strains M1146::gul (Figure S3A), M1246::2gul (Figure S3B) and M1346::3gul (Figure S3C) were successfully constructed. However, four-copy and ﬁve-copy integrations were unsuccessful after four rounds of conjugation and screening. . General Information on the Aborycin Gene Cluster from Streptomyces sp. HNS054 2.1. General Information on the Aborycin Gene Cluster from Streptomyces sp. HNS054 As previously reported [5], the aborycin gene cluster gul (named after Gulei Town, where the host marine sponge was collected from its coastal sea) from marine sponge- associated Streptomyces sp. HNS054 consists of 14 ORFs spanning an approximately 12 kb region from gulR1 to gulR2 (Figure 1). Sequence alignment showed that the DNA sequence of this region shares 98% identity to the aborycin gene cluster abo from Streptomyces sp. SCSIO ZS0098 [4]. The gul cluster also shows high identity to the siamycin-I gene cluster msl from Streptomyces sp. M-271 [26] in gene sequence and gene organization (Figure 1). GulA Mar. Drugs 2023, 21, 534 3 of 13 uence es sp 3 of 13 uence es sp encodes a 42-residue peptide with a leader peptide at its N-terminus and a 21-residue core peptide (CLGIGSCNDFAGCGYAVVCFW) at its C-terminus. The primary and secondary structures of aborycin and siamycin-I are almost the same, except that the residues at the 4th and 17th positions are switched (Figure 1). Due to the high similarity between aborycin and siamycin-I, commercial reagents of siamycin-I could be used as a reference to locate the high-performance liquid chromatography (HPLC) signal of aborycin. msl from Streptomyces sp. M-271 [26] in gene sequence and gene organization (Figure 1). GulA encodes a 42-residue peptide with a leader peptide at its N-terminus and a 21-resi- due core peptide (CLGIGSCNDFAGCGYAVVCFW) at its C-terminus. The primary and secondary structures of aborycin and siamycin-I are almost the same, except that the res- idues at the 4th and 17th positions are switched (Figure 1). Due to the high similarity be- tween aborycin and siamycin-I, commercial reagents of siamycin-I could be used as a ref- erence to locate the high-performance liquid chromatography (HPLC) signal of aborycin. Figure 1. General information on aborycin and siamycin-I and their BGCs, sequences and secondary structures. Sequence differences between aborycin and siamycin-I are marked in red. Figure 1. General information on aborycin and siamycin-I and their BGCs, sequences and secondary structures. Sequence differences between aborycin and siamycin-I are marked in red. Figure 1. General information on aborycin and siamycin-I and their BGCs, sequences and secondary structures. Sequence differences between aborycin and siamycin-I are marked in red. Figure 1. General information on aborycin and siamycin-I and their BGCs, sequences and secondary structures. Sequence differences between aborycin and siamycin-I are marked in red. 2.2. 2.3. Extraction and Detection of Aborycin 2.3. Extraction and Detection of Aborycin A peak at m/z = 1082.43 (z = 2) accounted for approximately 90% of the total mass. (D). The standard curve of concentrations of the HPLC peak areas of aborycin. f resins. Siamycin I was used as a control. The loading quantities of each test were adjusted to represent the same amount of the supernatant. (C). Mass spectrum analysis of the matter abundance in the m/z range [150–2000] of the 23.2 min collection from the sample enriched by the AB-8 resin. A peak at m/z = 1082.43 (z = 2) accounted for approximately 90% of the total mass. (D). The standard curve of concentrations of the HPLC peak areas of aborycin. 2.3. Extraction and Detection of Aborycin 2.3. Extraction and Detection of Aborycin The 23.2 min HPLC peaks of extractives obtained from the supernatant of M1346::3gul by butanone or by macroporous adsorption resins. Siamycin-I was used as a control. The loading quantities of each test were adjusted to represent the same amount of the supernatant. (C). Mass spectrum analysis of the matter abundance in the m/z range [150–2000] of the 23.2 min collection from the sample enriched by the AB-8 resin. A peak at m/z = 1082.43 (z = 2) accounted for approximately 90% of the total mass. (D). The standard curve of concentrations of the HPLC peak areas of aborycin. Figure 2. Extraction of aborycin. (A). The extraction procedure. (B). The 23.2 min HPLC peaks o extractives obtained from the supernatant of M1346::3gul by butanone or by macroporous adsorp Figure 2. Extraction of aborycin. (A). The extraction procedure. (B). The 23.2 min HPLC peaks of Figure 2. Extraction of aborycin. (A). The extraction procedure. (B). The 23.2 min HPLC peaks o extractives obtained from the supernatant of M1346::3gul by butanone or by macroporous adsorp tion resins. Siamycin-I was used as a control. The loading quantities of each test were adjusted to represent the same amount of the supernatant. (C). Mass spectrum analysis of the matter abundance in the m/z range [150–2000] of the 23.2 min collection from the sample enriched by the AB-8 resin. A peak at m/z = 1082.43 (z = 2) accounted for approximately 90% of the total mass. (D). The standard curve of concentrations of the HPLC peak areas of aborycin. f Figure 2. Extraction of aborycin. (A). The extraction procedure. (B). The 23.2 min HPLC peaks of extractives obtained from the supernatant of M1346::3gul by butanone or by macroporous adsorption resins. Siamycin-I was used as a control. The loading quantities of each test were adjusted to represent the same amount of the supernatant. (C). Mass spectrum analysis of the matter abundance in the m/z range [150–2000] of the 23.2 min collection from the sample enriched by the AB-8 resin. A peak at m/z = 1082.43 (z = 2) accounted for approximately 90% of the total mass. (D). The standard curve of concentrations of the HPLC peak areas of aborycin. represent the same amount of the supernatant. (C). Mass spectrum analysis of the matter abundanc in the m/z range [150–2000] of the 23.2 min collection from the sample enriched by the AB-8 resin. 2.3. Extraction and Detection of Aborycin 2.3. Extraction and Detection of Aborycin With siamycin-I as the control, the M1346::3gul strain was used to test whether abo- rycin was successfully produced. Aborycin was extracted, as shown in Figure 2A. The butanone extract had an HPLC signal at 23.2 min, which was the same as the signal of siamycin-I (Figure 2B). In the following test of the macroporous adsorption resin method, the AB-8 resin showed high enrichment efficiency for aborycin that was equivalent to that With siamycin-I as the control, the M1346::3gul strain was used to test whether aborycin was successfully produced. Aborycin was extracted, as shown in Figure 2A. The butanone extract had an HPLC signal at 23.2 min, which was the same as the signal of siamycin-I (Figure 2B). In the following test of the macroporous adsorption resin method, the AB-8 resin showed high enrichment efﬁciency for aborycin that was equivalent to that of the butanone method. Mass spectrum analysis conﬁrmed that a substance with a relative molecular mass of m/z = 1082.43 (z = 2) accounted for the majority of the composition at the 23.2 min peak of the AB-8-enriched samples (Figure 2C). The MS data of this substance were perfectly matched to the aborycin data from a previous report [5]. Thus, two facts were conﬁrmed by these tests: First, the M1346::3gul strain can express aborycin. Second, Mar. Drugs 2023, 21, 534 4 of 13 position his sub compared with the butanone method, the AB-8 resin method offered a safer, easier and cheaper method to enrich aborycin from the fermentation supernatant. This method was thus applied in the following quantiﬁcation studies: After pure aborycin was isolated from 5.6 L of culture broth containing the M1346::3gul strain, a precise standard curve of concentrations to the HPLC peak areas of aborycin was obtained (Figure 2D). By applying this curve, aborycin production from different strains or conditions could be quickly quantitated by HPLC. The 1H NMR spectral data of aborycin were also obtained to conﬁrm the structure (Figure S4).i y , g p y ond, compared with the butanone method, the AB-8 resin method offered a safer, easie and cheaper method to enrich aborycin from the fermentation supernatant. This method was thus applied in the following quantification studies: After pure aborycin was isolated from 5.6 L of culture broth containing the M1346::3gul strain, a precise standard curve o concentrations to the HPLC peak areas of aborycin was obtained (Figure 2D). 2.3. Extraction and Detection of Aborycin 2.3. Extraction and Detection of Aborycin By applying this curve, aborycin production from different strains or conditions could be quickly quantitated by HPLC. The 1H NMR spectral data of aborycin were also obtained to con firm the structure (Figure S4). Figure 2. Extraction of aborycin. (A). The extraction procedure. (B). The 23.2 min HPLC peaks o extractives obtained from the supernatant of M1346::3gul by butanone or by macroporous adsorp tion resins. Siamycin-I was used as a control. The loading quantities of each test were adjusted to represent the same amount of the supernatant. (C). Mass spectrum analysis of the matter abundance in the m/z range [150–2000] of the 23.2 min collection from the sample enriched by the AB-8 resin. A peak at m/z = 1082.43 (z = 2) accounted for approximately 90% of the total mass. (D). The standard curve of concentrations of the HPLC peak areas of aborycin. 2 4 Yield Comparison among Different Strains Figure 2. Extraction of aborycin. (A). The extraction procedure. (B). The 23.2 min HPLC peaks of extractives obtained from the supernatant of M1346::3gul by butanone or by macroporous adsorption resins. Siamycin-I was used as a control. The loading quantities of each test were adjusted to represent the same amount of the supernatant. (C). Mass spectrum analysis of the matter abundance in the m/z range [150–2000] of the 23.2 min collection from the sample enriched by the AB-8 resin. A peak at m/z = 1082.43 (z = 2) accounted for approximately 90% of the total mass. (D). The standard curve of concentrations of the HPLC peak areas of aborycin. Fi 2 E i f b i (A) Th i d (B) Th 23 2 i HPLC k Figure 2. Extraction of aborycin. (A). The extraction procedure. (B). The 23.2 min HPLC peaks of extractives obtained from the supernatant of M1346::3gul by butanone or by macroporous adsorp- tion resins. Siamycin-I was used as a control. The loading quantities of each test were adjusted to represent the same amount of the supernatant. (C). Mass spectrum analysis of the matter abundance in the m/z range [150–2000] of the 23.2 min collection from the sample enriched by the AB-8 resin. A peak at m/z = 1082.43 (z = 2) accounted for approximately 90% of the total mass. (D). The standard curve of concentrations of the HPLC peak areas of aborycin. Y ld ff Figure 2. Extraction of aborycin. (A). The extraction procedure. (B). 2.4. Yield Comparison among Different Strains Titers of aborycin from different strain 2.4. Yield Comparison among Different Strains Discussion In this study, efforts were made to use the Streptomyces system for the heterologous production of aborycin. First, the cloning procedure was simplified. The 14 kb genomic fragment containing the gul gene cluster was amplified by high-fidelity PCR, and sequenc- ing confirmed its accuracy. High-fidelity PCR techniques have been developed to amplify long DNA fragments with lengths as long as 15–20 kb. The cloning of RiPP gene clusters Table 1. Information on the genes to be knocked out. Trial Gene Code Symbol Relevant Features References J01 SCO4228 phoU ∆phoU mutant showed 6-fold increase in ACT production when phosphate starvation [27] J04 SCO3579 wblA ∆wblA mutant showed 1.5-fold increase in doxorubicin production [28] J05 SCO1712 SCO1712 ∆SCO1712 mutant showed 1.62-fold or 1.22-fold increase in ACT or RED production, respectively [29] J07 SCO3008 orrA ∆orrA mutant showed great increase in ACT and RED production [30] J08 SCO1678 gntR ∆gntR mutant altered the secondary metabolite proﬁle of S. coelicolor [31] 3. Discussion In this study, efforts were made to use the Streptomyces system for the heterologous production of aborycin. First, the cloning procedure was simpliﬁed. The 14 kb genomic fragment containing the gul gene cluster was ampliﬁed by high-ﬁdelity PCR, and sequenc- ing conﬁrmed its accuracy. High-ﬁdelity PCR techniques have been developed to amplify long DNA fragments with lengths as long as 15–20 kb. The cloning of RiPP gene clusters could beneﬁt from these developments because a large portion of RiPP gene clusters have lengths below 15 kb (Table S3). In a recent review on newly discovered RiPPs, out of the 33 listed gene clusters, 25 (76%) had lengths below 15 kb ([32], Table S4). Second, to facili- tate foreign DNA integration following genome editing, the integrative vector pSET152 Trial Gene Code Symbol Relevant Features References J01 SCO4228 phoU ΔphoU mutant showed 6-fold increase in ACT production when phosphate starvation [27] J04 SCO3579 wblA ΔwblA mutant showed 1.5-fold increase in doxorubicin production [28] J05 SCO1712 SCO1712 ΔSCO1712 mutant showed 1.62-fold or 1.22-fold increase in ACT or RED production, respectively [29] J07 SCO3008 orrA ΔorrA mutant showed great increase in ACT and RED production [30] J08 SCO1678 gntR ΔgntR mutant altered the secondary metabolite profile of S. 2.4. Yield Comparison among Different Strains Titers of aborycin from different strain 2.4. Yield Comparison among Different Strains The ΔwblA strain was significantly (p < 0.01) higher than the M1346::3gul strain, with a titer of 23.6 ± 10.2 mg/L, i.e., 4.8 times that of the native strain or 2.3 times that of the strain before wblA knockout. Greater improvements were displayed in the ΔorrA and ΔgntR mutants, which had titers of 56.3 ± 18.0 and 48.2 ± 15.1 mg/L, respectively (Figure 3). Figure 3. Aborycin titers from different strains. HNS054, the wild-type strain Streptomyces sp. HNS054. ΔphoU—ΔgntR, gene knockout strains from S. coelicolor M1346::3gul. Orange block, aver- age production from supernatant extracted by the AB-8 method. Blue block, average production from mycelia extracted by the methanol method. Error bar, standard deviation of total titers (super- natant + mycelia). Data were counted from triplicate experiments. *, p < 0.001. n.s., p > 0.05. Table 1. Information on the genes to be knocked out. Figure 3. Aborycin titers from different strains. HNS054, the wild-type strain Streptomyces sp. HNS054. ∆phoU—∆gntR, gene knockout strains from S. coelicolor M1346::3gul. Orange block, average production from supernatant extracted by the AB-8 method. Blue block, average production from mycelia extracted by the methanol method. Error bar, standard deviation of total titers (supernatant + mycelia). Data were counted from triplicate experiments. , p < 0.001. n.s., p > 0.05. Table 1. Information on the genes to be knocked out. f Figure 3. Aborycin titers from different strains. HNS054, the wild-type strain Streptomyces sp. HNS054 ΔphoU ΔgntR gene knockout strains from S coelicolor M1346::3gul Orange block aver Figure 3. Aborycin titers from different strains. HNS054, the wild-type strain Streptomyces sp. Figure 3. Aborycin titers from different strains. HNS054, the wild-type strain Streptomyces sp. HNS054. ΔphoU—ΔgntR, gene knockout strains from S. coelicolor M1346::3gul. Orange block, aver- age production from supernatant extracted by the AB-8 method. Blue block, average production from mycelia extracted by the methanol method. Error bar, standard deviation of total titers (super- natant + mycelia). Data were counted from triplicate experiments. , p < 0.001. n.s., p > 0.05. Table 1. Information on the genes to be knocked out Figure 3. Aborycin titers from different strains. HNS054, the wild-type strain Streptomyces sp. HNS054. ∆phoU—∆gntR, gene knockout strains from S. coelicolor M1346::3gul. Orange block, average production from supernatant extracted by the AB-8 method. Blue block, average production from mycelia extracted by the methanol method. Error bar, standard deviation of total titers (supernatant + mycelia). 2.4. Yield Comparison among Different Strains Titers of aborycin from different strain 2.4. Yield Comparison among Different Strains coelicolor [31] Trial Gene Code Symbol Relevant Features References J01 SCO4228 phoU ∆phoU mutant showed 6-fold increase in ACT production when phosphate starvation [27] J04 SCO3579 wblA ∆wblA mutant showed 1.5-fold increase in doxorubicin production [28] J05 SCO1712 SCO1712 ∆SCO1712 mutant showed 1.62-fold or 1.22-fold increase in ACT or RED production, respectively [29] J07 SCO3008 orrA ∆orrA mutant showed great increase in ACT and RED production [30] J08 SCO1678 gntR ∆gntR mutant altered the secondary metabolite proﬁle of S. coelicolor [31] 2.4. Yield Comparison among Different Strains Titers of aborycin from different strain 2.4. Yield Comparison among Different Strains Drugs 2023, 21, 534 5 of 13 ts were cin pro- 5 of 13 ts were cin pro- ∆wblA strain was signiﬁcantly (p < 0.01) higher than the M1346::3gul strain, with a titer of 23.6 ± 10.2 mg/L, i.e., 4.8 times that of the native strain or 2.3 times that of the strain before wblA knockout. Greater improvements were displayed in the ∆orrA and ∆gntR mutants, which had titers of 56.3 ± 18.0 and 48.2 ± 15.1 mg/L, respectively (Figure 3). than that of the M1346::3gul strain but not significantly different (p > 0.05). The ΔwblA strain was significantly (p < 0.01) higher than the M1346::3gul strain, with a titer of 23.6 ± 10.2 mg/L, i.e., 4.8 times that of the native strain or 2.3 times that of the strain before wblA knockout. Greater improvements were displayed in the ΔorrA and ΔgntR mutants, which had titers of 56.3 ± 18.0 and 48.2 ± 15.1 mg/L, respectively (Figure 3). ∆wblA strain was signiﬁcantly (p < 0.01) higher than the M1346::3gul strain, with a titer of 23.6 ± 10.2 mg/L, i.e., 4.8 times that of the native strain or 2.3 times that of the strain before wblA knockout. Greater improvements were displayed in the ∆orrA and ∆gntR mutants, which had titers of 56.3 ± 18.0 and 48.2 ± 15.1 mg/L, respectively (Figure 3). than that of the M1346::3gul strain but not significantly different (p > 0.05). The ΔwblA strain was significantly (p < 0.01) higher than the M1346::3gul strain, with a titer of 23.6 ± 10.2 mg/L, i.e., 4.8 times that of the native strain or 2.3 times that of the strain before wblA knockout. Greater improvements were displayed in the ΔorrA and ΔgntR mutants, which had titers of 56.3 ± 18.0 and 48.2 ± 15.1 mg/L, respectively (Figure 3). ∆wblA strain was signiﬁcantly (p < 0.01) higher than the M1346::3gul strain, with a titer of 23.6 ± 10.2 mg/L, i.e., 4.8 times that of the native strain or 2.3 times that of the strain before wblA knockout. Greater improvements were displayed in the ∆orrA and ∆gntR mutants, which had titers of 56.3 ± 18.0 and 48.2 ± 15.1 mg/L, respectively (Figure 3). than that of the M1346::3gul strain but not significantly different (p > 0.05). 2.4. Yield Comparison among Different Strains Titers of aborycin from different strain 2.4. Yield Comparison among Different Strains Titers of aborycin from different strains are shown in Figure 3. Adding the superna tant and mycelial products, the native HNS054 strain average yielded 4.9 ± 2.6 mg/L abo- rycin. No aborycin signal was detected from the blank control M1146::pSAT209 and the one-copy strain M1146::gul. Signals became obvious for the M1246::2gul and M1346::3gu strains, which had average titers of 1.8 ± 1.0 mg/L and 10.4 ± 1.6 mg/L, i.e., 0.4 times and 2.1 times that of the native strain, respectively. Both supernatant and mycelia contributed to the production, accounting for approximately 38 percent and 62 percent, respectively. Then the M1346::3gul strain was selected for genetic modification to verify whether Titers of aborycin from different strains are shown in Figure 3. Adding the supernatant and mycelial products, the native HNS054 strain average yielded 4.9 ± 2.6 mg/L aborycin. No aborycin signal was detected from the blank control M1146::pSAT209 and the one-copy strain M1146::gul. Signals became obvious for the M1246::2gul and M1346::3gul strains, which had average titers of 1.8 ± 1.0 mg/L and 10.4 ± 1.6 mg/L, i.e., 0.4 times and 2.1 times that of the native strain, respectively. Both supernatant and mycelia contributed to the production, accounting for approximately 38 percent and 62 percent, respectively. Then, the M1346::3gul strain was selected for genetic modification to verify whether aborycin production could be further improved by deleting negative regulatory genes (Table 1), which were reported to act at higher levels of the gene-regulatory networks to Then, the M1346::3gul strain was selected for genetic modiﬁcation to verify whether aborycin production could be further improved by deleting negative regulatory genes (Table 1), which were reported to act at higher levels of the gene-regulatory networks to control secondary metabolism, whose knockout mutants always resulted in an increase in secondary metabolism. The ∆phoU, ∆wblA, ∆SCO1712, ∆orrA and ∆gntR mutants were successfully constructed (Figures S5 and S6). A titer study showed that the aborycin production of the M1346::3gul ∆phoU strain was 12.1 ± 0.7 mg/L, which was slightly higher than that of the M1346::3gul strain but not signiﬁcantly different (p > 0.05). The Mar. 2.4. Yield Comparison among Different Strains Titers of aborycin from different strain 2.4. Yield Comparison among Different Strains Data were counted from triplicate experiments. , p < 0.001. n.s., p > 0.05. Table 1 Information on the genes to be knocked out Figure 3. Aborycin titers from different strains. HNS054, the wild-type strain Streptomyces sp. HNS054. ΔphoU—ΔgntR, gene knockout strains from S. coelicolor M1346::3gul. Orange block, aver- age production from supernatant extracted by the AB-8 method. Blue block, average production from mycelia extracted by the methanol method. Error bar, standard deviation of total titers (super- natant + mycelia). Data were counted from triplicate experiments. , p < 0.001. n.s., p > 0.05. Table 1. Information on the genes to be knocked out Figure 3. Aborycin titers from different strains. HNS054, the wild-type strain Streptomyces sp. HNS054. ∆phoU—∆gntR, gene knockout strains from S. coelicolor M1346::3gul. Orange block, average production from supernatant extracted by the AB-8 method. Blue block, average production from mycelia extracted by the methanol method. Error bar, standard deviation of total titers (supernatant + mycelia). Data were counted from triplicate experiments. , p < 0.001. n.s., p > 0.05. Table 1 Information on the genes to be knocked out age production from supernatant extracted by the AB-8 method. Blue block, average production from mycelia extracted by the methanol method. Error bar, standard deviation of total titers (super- natant + mycelia). Data were counted from triplicate experiments. , p < 0.001. n.s., p > 0.05. T bl 1 I f ti th t b k k d t HNS054. ∆phoU—∆gntR, gene knockout strains from S. coelicolor M1346::3gul. Orange block, average production from supernatant extracted by the AB-8 method. Blue block, average production from mycelia extracted by the methanol method. Error bar, standard deviation of total titers (supernatant + mycelia). Data were counted from triplicate experiments. *, p < 0.001. n.s., p > 0.05. Table 1. Information on the genes to be knocked out. Trial Gene Code Symbol Relevant Features References J01 SCO4228 phoU ΔphoU mutant showed 6-fold increase in ACT production when phosphate starvation [27] J04 SCO3579 wblA ΔwblA mutant showed 1.5-fold increase in doxorubicin production [28] J05 SCO1712 SCO1712 ΔSCO1712 mutant showed 1.62-fold or 1.22-fold increase in ACT or RED production, respectively [29] J07 SCO3008 orrA ΔorrA mutant showed great increase in ACT and RED production [30] J08 SCO1678 gntR ΔgntR mutant altered the secondary metabolite profile of S. coelicolor [31] 3. In this stu 3. Discussion In this study, efforts were made to use the Streptomyces system for the heterologous production of aborycin. First, the cloning procedure was simplified. The 14 kb genomic fragment containing the gul gene cluster was amplified by high-fidelity PCR, and sequenc- ing confirmed its accuracy. High-fidelity PCR techniques have been developed to amplify long DNA fragments with lengths as long as 15–20 kb. The cloning of RiPP gene clusters In this study, efforts were made to use the Streptomyces system for the heterologous production of aborycin. First, the cloning procedure was simpliﬁed. The 14 kb genomic fragment containing the gul gene cluster was ampliﬁed by high-ﬁdelity PCR, and sequenc- ing conﬁrmed its accuracy. High-ﬁdelity PCR techniques have been developed to amplify long DNA fragments with lengths as long as 15–20 kb. The cloning of RiPP gene clusters could beneﬁt from these developments because a large portion of RiPP gene clusters have lengths below 15 kb (Table S3). In a recent review on newly discovered RiPPs, out of the 33 listed gene clusters, 25 (76%) had lengths below 15 kb ([32], Table S4). Second, to facili- tate foreign DNA integration following genome editing, the integrative vector pSET152 In this study, efforts were made to use the Streptomyces system for the heterologous production of aborycin. First, the cloning procedure was simplified. The 14 kb genomic fragment containing the gul gene cluster was amplified by high-fidelity PCR, and sequenc- ing confirmed its accuracy. High-fidelity PCR techniques have been developed to amplify long DNA fragments with lengths as long as 15–20 kb. The cloning of RiPP gene clusters In this study, efforts were made to use the Streptomyces system for the heterologous production of aborycin. First, the cloning procedure was simpliﬁed. The 14 kb genomic fragment containing the gul gene cluster was ampliﬁed by high-ﬁdelity PCR, and sequenc- ing conﬁrmed its accuracy. High-ﬁdelity PCR techniques have been developed to amplify long DNA fragments with lengths as long as 15–20 kb. The cloning of RiPP gene clusters could beneﬁt from these developments because a large portion of RiPP gene clusters have lengths below 15 kb (Table S3). In a recent review on newly discovered RiPPs, out of the 33 listed gene clusters, 25 (76%) had lengths below 15 kb ([32], Table S4). Second, to facili- tate foreign DNA integration following genome editing, the integrative vector pSET152 Mar. In this stu 3. Discussion Drugs 2023, 21, 534 6 of 13 was modiﬁed to pSAT209 to prevent challenges related to antibiotic resistance. Third, the extraction of aborycin from the supernatant was optimized. Aborycin is a peptide with am- phiphilic characteristics and thus has reversible afﬁnity to certain macroporous adsorption resins. In this case, AB-8 resin was found to be such a resin, adsorbing aborycin in a water solution and desorbing it in methanol. The optimal AB-8 resin method condition is that the supernatant and AB-8 resin are 10:1 (v/v) mixed for 4 h to overnight, and then the resin is washed with a 5 × resin volume of 10% methanol following the desorption of the aborycin from the resin by a 2 × resin volume of methanol for 1 h. Compared with the butanone method [4,5], the AB-8 resin method recovered a majority of the aborycin from the super- natant with minimal organic solvent and minimal labor. The desorbed extract is also much cleaner than the butanone extract. By simply adsorbing and desorbing, approximately 40% of the total production was recovered from the culture supernatant. A similar situation occurred with the heterologous expression of sviceucin, where approximately 1/3 of the product was released in the culture supernatant [21]. Large-scale production would discard the supernatant because of the high cost of solvent extraction. With this optimization, the product in the supernatant could be recovered at a low cost. We speculated that this theory and operation could be applied to other RiPPs due to their peptide nature. Finally, the multiple chromosomal integration of foreign gene clusters following CRISPR/Cas9 genome editing was successfully achieved, and the best case resulted in a 25-fold increase in aborycin production compared to the native strain. Multiple chromosomal integration of foreign BGC was proven to be a mature technique to increase heterologous expression. The approach was successfully demonstrated in S. coelicolor [25], S. albus J1074 [33] and S. lividans [34]. In this study, one-copy integration showed no signal, while three-copy integration showed a 2.1-fold increase compared to the native strain. However, more advanced techniques were needed to obtain four- or ﬁve-copy integration in one single step. In our attempts to transfer other BGCs into these M1146- M1546 hosts, the number of exconjugants decreased rapidly with increasing integrated copy number. It was previously speculated that a high copy number of chromosomal integration caused the accumulation of target products, endangering bacterial growth [25]. In this stu 3. Discussion However, the titer of aborycin was further improved 5.4-fold by orrA gene knockout, implying that the Streptomycete actually tolerates higher concentrations of aborycin. CRISPR/Cas-based genome editing tools provide swift, accurate and traceless ways to modify the genomes of Streptomyces [35]. A straightforward way to exploit the CRISPR/Cas9 tools was to delete negative regulator genes. This study proposed deleting genes that were reported to have a negative impact on secondary metabolism at a high level of the regulation networks. Five mutants, ∆phoU, ∆wblA, ∆SCO1712, ∆orrA and ∆gntR, were successfully constructed from the M1346::3gul strain. g Although the detailed function of the phoU gene was unclear, it was speculated to be involved in the pho regulon, which responds to phosphate starvation. PhoR senses such conditions, and then PhoP is phosphorylated following PhoP-P binding to speciﬁc sequences named PHO boxes, thus activating or repressing a set of genes [36]. Under phosphate starvation conditions, the ∆phoU mutants showed an approximately six-fold increase in the production of actinorhodin [27]. Our study showed that aborycin production in the ∆phoU mutants was not signiﬁcantly improved from that in the strain before mutation when strains were cultured in R5 medium. Moreover, the growth of the ∆phoU mutants on MS-agar was retarded in a 9-day morphology observation (Figure 4). This type of the mutant requires more optimization before it can be applied in antibiotic production. Mar. Drugs 2023, 21, 534 Mar. Drugs 2023, 21, x F 7 of 13 7 of 13 Figure 4. The growth of strains on MS-agar plates from 3 days to 9 days. ΔphoU—ΔgntR, g knockout strains from S. coelicolor M1346::3gul. The wblA gene was reported as a pleiotropic downregulator of antibiotic biosynthe Figure 4. The growth of strains on MS-agar plates from 3 days to 9 days. ∆phoU—∆gntR, gen knockout strains from S. coelicolor M1346::3gul. Figure 4. The growth of strains on MS-agar plates from 3 days to 9 days. ΔphoU—ΔgntR, gen knockout strains from S. coelicolor M1346::3gul. Figure 4. The growth of strains on MS-agar plates from 3 days to 9 days. ∆phoU—∆gntR, gene knockout strains from S. coelicolor M1346::3gul. The wblA gene was reported as a pleiotropic downregulator of antibiotic biosynthesis in S. coelicolor. ΔwblA mutants exhibited a defect in sporulation, achieved higher biomass than the wild type, and overproduced secondary metabolites [37]. Overproduction of an- tibiotics by disruption of the wblA orthologs was also observed in other Streptomyces bac- teria [38]. In this stu 3. Discussion This study confirmed that the production of aborycin in the ΔwblA mutant was significantly improved by two-fold. Higher growth and defects in sporulation were also observed (Figure 4). The wblA gene was reported as a pleiotropic downregulator of antibiotic biosynthesis in S. coelicolor. ∆wblA mutants exhibited a defect in sporulation, achieved higher biomass than the wild type, and overproduced secondary metabolites [37]. Overproduction of antibiotics by disruption of the wblA orthologs was also observed in other Streptomyces bacteria [38]. This study conﬁrmed that the production of aborycin in the ∆wblA mutant was signiﬁcantly improved by two-fold. Higher growth and defects in sporulation were also observed (Figure 4). SCO1712 is a member of the TetR family. TetR family transcriptional regulators are among the most common prokaryotic transcriptional regulators. When SCO1712 was overexpressed or disrupted, ACT production decreased or increased compared with that i S li l M145 i l i h SCO1712 i l i i d l SCO1712 is a member of the TetR family. TetR family transcriptional regulators are among the most common prokaryotic transcriptional regulators. When SCO1712 was overexpressed or disrupted, ACT production decreased or increased compared with that in Mar. Drugs 2023, 21, 534 8 of 13 S. coelicolor M145, respectively, suggesting that SCO1712 is a pleiotropic downregulator of antibiotic biosynthesis in S. coelicolor [29]. It was further speculated that there is a syner- gistic effect between SCO1712 and precursor ﬂux pathways in antibiotic production [39]. Unfortunately, in this study, after SCO1712 knockout, the production of aborycin decreased signiﬁcantly. This provides ideas for future optimizations of precursor ﬂux pathways or mediums for this mutant. OhkA (SCO1596)—OrrA (SCO3008) is a group of prokaryotic two-component reg- ulatory systems with highly similar transcriptomic features. ∆orrA mutants lead to the signiﬁcant overproduction of antibiotics and the downregulation of the bld, chp, rdl, and wbl genes associated with morphological development [30]. Correspondingly, we found that ∆orrA mutants overproduced approximately 4.4-fold more aborycin than the strains before mutation (Figure 3). Their morphological development was also similar to that of the ∆wblA mutants (Figure 4). These results implied that the OrrA regulatory system likely covered the wbl regulatory system and controlled a wider range of resources for antibiotic production. The bacterial GntR family is one of the most abundant groups of helix-turn-helix transcription factors that respond appropriately to metabolite microenvironments [40]. In this stu 3. Discussion It was reported that deletion of a GntR-like gene allowed for platensimycin and platencin overproduction in S. platensis [41]. GntR (SCO1678) of S. coelicolor encodes a repressor protein to control the gluconate operon, which enables Streptomyces to utilize gluconate in the media [31]. No obvious evidence has linked GntR (SCO1678) to antibiotic overproduc- tion to date. Interestingly, both the overproduction of aborycin (Figure 3) and overgrowth (Figure 4) were observed from the ∆GntR mutants in this study. No additional gluconate was added to the R5 or MS-agar media to obtain these results. These observations pro- vide a signiﬁcant gene that is worthy of further study to discover underlying metabolic regulatory mechanisms. To further improve the production of aborycin, we attempted to double knockout the genes orrA and gntR. It was difﬁcult to obtain enough spores from the ∆orrA strains. Thus, we tried to knock out the orrA gene from the M1346::3gul ∆gntR strain. The cloning process was difﬁcult, and positive clones showed contradictory yields. We reason it in part to genetic instability, which might result from unknown genetic changes by one round of multi-sites of attP/attB recombination and two rounds of Cas9-based CRISPR. Thus, the cloning strategy should be carefully planned to achieve double knockout or triple knockout. It could be feasible that before the integration of the heterogeneous BGCs, stable S. coelicolor M1346-based double-knockout or triple-knockout hosts should be constructed by one round of multiplex genome editing [23,25] to reduce genetic instability risks, which might be brought from two or more rounds of CRISPR. g g Although great production was achieved by these genetic modiﬁcations, we believe the Streptomyces systems could be further improved. With more global regulators that govern secondary metabolism being characterized [42], it is worth manipulating them one by one to obtain further knowledge. 4. Materials and Methods 4.1. Strains, Plasmids and Primers 4.1. Strains, Plasmids and Primers The strains and plasmids used in this study are listed in Table S1, and the primers used in this study are listed in Table S2. The construction of the related vectors is shown in Figures S1 and S2. 4.3. Gene Knockout in S. coelicolor M1346::3gul by the CRISPR/Cas9 Method g y The phoU (SCO4228), wblA (SCO3579), SCO1712, orrA (SCO3008) and gntR (SCO1678) genes (Table 1) in S. coelicolor M1346::3gul were independently knocked out by the CRISPR/Cas9 genome editing method [24]. To generate the ∆phoU mutant, the sgRNA expression cassette (f1 fragment) was PCR ampliﬁed with the plasmid pKCcas9dO as the template and f1J01- fwd/f1gRNA-R as primers. The upstream (f2 fragment) and downstream (f3 fragment) regions of the phoU gene were PCR ampliﬁed with the primer pairs f2J01-fwd/rev and f3J01-fwd/rev, respectively. Then, three DNA fragments were assembled by overlapping PCR using primers f1J01-fwd and f3J01-rev. The resulting DNA fragment was cloned and inserted into pKCCas9 using a one-step cloning kit (Cat # C113, Vazyme, Nanjing, China) to yield pKCCas9-dJ01. The obtained plasmid was introduced into the M1346::3gul strain by conjugal transfer. The correct knockouts were veriﬁed by PCR using primers ID-dJ01- fwd/rev. By using the J04, J05, J07 and J08 sets of primers and the same procedure, pKCCas9- dJ04, -dJ05, -dJ07 and -dJ08 were generated and yielded the ∆wblA, ∆SCO1712, ∆orrA and ∆gntR mutants after successful conjugation into the M1346::3gul strain, respectively. The positions of the spacers, primers and target genes are shown in Figure S5. 4.2. Construction of Heterologous Expression Strains to Produce Aborycin 4.2. Construction of Heterologous Expression Strains to Produce Aborycin Foreign DNA integration could be fulﬁlled by the integrative plasmid pSET152 [43], and genome editing could be executed by the suicide plasmid pKCcas9 [24]. However, both vectors contain the apramycin resistance gene aac(3)IV. To achieve BGC integration following genome editing, the antibiotic-resistance markers between the two genetic ma- nipulations should be different. Thus, the aac(3)IV gene in pSET152 was replaced by the Mar. Drugs 2023, 21, 534 9 of 13 9 of 13 ampicillin resistance gene bla from the pUC57 plasmid and the thiostrepton resistance gene tsr from the pGM1190 plasmid. The resulting plasmid was named pSAT209 (Figure S1). The Genomic DNA of Streptomyces sp. HNS054 was extracted from the later logarithmic phase cells using a bacterial genomic DNA extraction kit (Cat # DP2001, Biotake, Beijing, China). Using primers 054LasF and 054LasR, a 14 kb DNA fragment containing the gul BGC (located at 15932–29864 nt of GenBank: AC003_RS35325) was ampliﬁed from the Strep- tomyces sp. HNS054 genome (Assembly: GCF_001044185.1). After gel puriﬁcation, the gul fragment was assembled with EcoRV-linearized pSAT209 using a one-step cloning kit (Cat # C113, Vazyme, Nanjing, China). The resulting vector pSAT-GUL (Figure S1) was sequenced to conﬁrm the accuracy. Then, it and the control plasmid pSAT209 were transferred into E. coli ET12567/pUZ8002 competent cells and conjugated into S. coelicolor M1146-M1546 hosts by standard protocols [44]. Exconjugants were randomly picked to check attP-attB recombination by PCR with the following primers: The forward primer ID-oriT-fw targets a position approximately 400 bp upstream of the attP locus in the vector pSAT-GUL. The reverse primers, ID-native-attB-Rev, ID-CPK-attB-Rev, ID-RED-attB-Rev, ID-CDA-attB-Rev and ID-ACT-attB-Rev, each target a position downstream of an individual attB locus in the S. coelicolor M1546 genome. The integrated copy number of the gul gene cluster in the host genome was determined by the number of positive attP-attB recombinations [25]. 4.4. Metabolite Analysis The wild-type Streptomyces sp. HNS054 and the S. coelicolor strains were grown on MS solid medium to achieve sporulation. Approximately 108 spores (or 40 µL mycelia store) were transferred to 40 mL R5 medium with appropriate antibiotics and cultured at 200 rpm and 28 ◦C for 3 days as the seed solution. Ten milliliters of the seed was transferred to 500 mL of R5 medium and cultured at 200 rpm and 28 ◦C for 9 days to complete the fermentation. Aborycin was extracted, as shown in Figure 2A. The fermented broth was centrifuged to separate the supernatant from the mycelia and then independently extracted. The supernatant was extracted by two methods. The ﬁrst method was butanone extraction three times, and then the butanone phase was concentrated by rotary evaporation. Then, four macroporous adsorption resins, AB-8, DC201-C, NK-9 and D101 (Hecheng New Material, Zhengzhou, China), were tested for their aborycin recovery efﬁciencies. The AB-8 resin had a high enrichment ratio for aborycin (Figure 2B); therefore, AB-8 was used as the second method to extract aborycin. Because the AB-8 resin method was safer, easier and cheaper, all aborycin titers of the supernatants in this study were obtained by this method. The mycelium was extracted three times with 300 mL methanol and then concentrated by rotary evaporation. Extracts from both the supernatant and mycelia were adjusted to a ﬁnal concentration of approximately 5 mg/mL in methanol and ﬁltered with 0.22 µm ﬁlter Mar. Drugs 2023, 21, 534 10 of 13 10 of 13 membranes before HPLC or LC—MS analysis. Analytical HPLC was performed using a Shimadzu Prominence LC-20A (Shimadzu, Tokyo, Japan) with a 2.6 × 250 mm Ultimate XB-C18 column (Welch, Shanghai, China). The elution conditions were 1% solvent B for 0–7 min and then a linear gradient to 95% solvent B (solvent B: 0.1% formic acid in CH3CN; solvent A: 0.1% formic acid in H2O) for 7–30 min at a ﬂow rate of 1 mL/min and monitored at 277 nm. To locate the aborycin signal, the commercial siamycin-I reagent (Adipogen Life Sciences, San Diego, CA, USA) was used as the control. Because the amino acid sequences between aborycin and siamycin-I were the same except the switched residues at the 4th and 17th positions (Figure 1), it is reasonable to expect the HPLC signal position of aborycin to be slightly different from that of siamycin-I. 4.5. Puriﬁcation of Aborycin, the Calibration Curve and 1H-NMR Spectra 4.5. Puriﬁcation of Aborycin, the Calibration Curve and 1H-NMR Spectra A 5.6 L culture broth of the M1346::3gul strain was prepared as described in the previous section. The crude extract was enriched by the AB-8 resin method from the supernatant and then puriﬁed by semipreparative HPLC with a YMC-PACK ODS-A column (10 × 250 mm, ϕ 5 µm, YMC, Kyoto, Japan). F1–F4 fractions near the target position were collected, and the F2 fraction was determined by analytical HPLC to be the major fraction containing aborycin. Then, approximately 22 mg of the F2 fraction was obtained after rotary evaporation. Ten milligrams of the F2 fraction was dissolved in 2 mL of methanol, and analytical HPLC was run as described in the previous section. The 23.2 min peaks were collected and concentrated to approximately 4 mg of pure aborycin in the form of a white powder. A methanol solution of aborycin was prepared with a precise concentration of 1.00 mg/mL and then diluted to a series of concentrations. These dilutions were subjected to analytical HPLC, and the 23.2 min peak areas were recorded. A standard curve of concentrations to HPLC peak areas of aborycin was then constructed (Figure 2D), and the following formula was deduced. y = 4.970 x + 0.046 (R2 = 0.999) (1) (1) where y (106·mAU·min) is the peak area of the HPLC signal of aborycin, and x (mg/mL) is the concentration. Approximately 3.5 mg of aborycin was dissolved in 500 µL DMSO, and the 1H NMR (600 MHz) spectra of aborycin were obtained using a Bruker Avance III HD 600 MHz spectrometer (Bruker, Billerica, MA, USA). 4.4. Metabolite Analysis LC—MS analysis was performed using a Q Exactive spectrometer (Thermo Scientiﬁc, Waltham, MA, USA) with the same elution program as HPLC. The software MZmine 2 [45] was used to assess the chromatograms. References 1. Helynck, G.; Dubertret, C.; Mayaux, J.F.; Leboul, J. Isolation of RP 71955, a new anti-HIV-1 peptide secondary metabolite. J. Antibiot. 1993, 46, 1756–1757. [CrossRef] [PubMed] 2. Fréchet, D.; Guitton, J.D.; Herman, F.; Faucher, D.; Helynck, G.; Monegier du Sorbier, B.; Ridoux, J.P.; James-Surcouf, E.; Vuilhorgne, M. Solution structure of RP 71955, a new 21 amino acid tricyclic peptide active against HIV-1 virus. Biochemistry 1994, 33, 42–50. [CrossRef] [PubMed] 3. Potterat, O.; Stephan, H.; Metzger, J.W.; Gnau, V.; Zähner, H.; Jung, G. Aborycin—A tricyclic 21-peptide antibiotic isolated from Streptomyces griseoﬂavus. Liebigs Ann. Chem. 1994, 1994, 741–743. [CrossRef] p y g ﬂ g 4. Shao, M.; Ma, J.; Li, Q.; Ju, J. Identiﬁcation of the anti-infective aborycin biosynthetic gene cluste 4. Shao, M.; Ma, J.; Li, Q.; Ju, J. Identiﬁcation of the anti-infective aborycin biosynthetic gene cluster from deep-sea-derived Streptomyces sp. SCSIO ZS0098 enables production in a heterologous host. Mar. Drugs 2019, 17, 127. [CrossRef] [PubMed] 4. Shao, M.; Ma, J.; Li, Q.; Ju, J. Identiﬁcation of the anti-infective aborycin biosynthetic gene cluster from deep-sea-derived Streptomyces sp. SCSIO ZS0098 enables production in a heterologous host. Mar. Drugs 2019, 17, 127. [CrossRef] [PubMed] 5. Liu, T.; Huang, Z.; Gui, X.; Xiang, W.; Jin, Y.; Chen, J.; Zhao, J. Multi-omics comparative analysis of Streptomyces mutants obtained byiterative atmosphere and room-temperature plasma mutagenesis. Front. Microbiol. 2020, 11, 630309. [CrossRef] 6. Goel, N.; Singh, R.; Sood, S.; Khare, S.K. Investigation of Streptomyces sp. strain EMB24 secondary metabolite proﬁle has unraveled , ; g, ; , ; g, ; J , ; , J ; , J p y p y obtained byiterative atmosphere and room-temperature plasma mutagenesis. Front. Microbiol. 2020, 11, 630309. [CrossRef] 6. Goel, N.; Singh, R.; Sood, S.; Khare, S.K. Investigation of Streptomyces sp. strain EMB24 secondary metabolite proﬁle has unraveled its extraordinary antibacterial potency against drug resistant bacteria Mar Biotechnol 2022 24 1168 1175 [CrossRef] y p p p g , , [ ] 6. Goel, N.; Singh, R.; Sood, S.; Khare, S.K. Investigation of Streptomyces sp. strain EMB24 secondary metabolite proﬁle has unraveled its extraordinary antibacterial potency against drug-resistant bacteria. Mar. Biotechnol. 2022, 24, 1168–1175. [CrossRef] its extraordinary antibacterial potency against drug-resistant bacteria. Mar. Biotechnol. 2022, 24, 1168–1175. [CrossRef] 7. Maksimov, M.O.; Pan, S.J.; James Link, A. Lasso peptides: Structure, function, biosynthesis, and engineering. Nat. Prod. Rep. 2012, 29, 996–1006. [CrossRef] 8. 5. Conclusions Acknowledgments: We gratefully acknowledge Yinhua Lu (College of Life Sciences, Shanghai Normal University) and Xiuhua Pang (School of Life Sciences, Shandong University) for their valuable support regarding host strains, plasmids and research suggestions. Acknowledgments: We gratefully acknowledge Yinhua Lu (College of Life Sciences, Shanghai Normal University) and Xiuhua Pang (School of Life Sciences, Shandong University) for their valuable support regarding host strains, plasmids and research suggestions. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions This study demonstrated improvements in the S. coelicolor system for aborycin over- production. The maximum shake ﬂask titer was over 50 mg/L, which is comparable to the best examples of E. coli systems or Streptomyces systems for lasso peptide heterologous expression [15]. We conﬁrmed that increasing the copy number of chromosome integra- tion and disrupting the negative global regulators governing secondary metabolism were effective ways to improve antibiotic production in S. coelicolor systems. GntR (SCO1678) was proposed to be a signiﬁcant target that is worthy of further studies on metabolic regulatory mechanisms. The optimized extraction method, which could efﬁciently recover aborycin from the supernatant, also contributed to the total titer. Due to the similar chemi- cal properties of RiPPs to aborycin, this tactic is also suitable for the development of other RiPP relatives. By applying these modiﬁcations to Streptomyces systems, the drug develop- ment and functional characterization of new lasso peptides from genome mining will be greatly improved. Mar. Drugs 2023, 21, 534 11 of 13 11 of 13 Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/md21100534/s1, Figure S1: Construction of the integrative vector for aborycin expression; Figure S2: Cloning of the gul gene cluster; Figure S3: PCR validation of gul cluster integration in the attB loci. Figure S4: 1H NMR (600 MHz) spectrum of aborycin in DMSO. Figure S5: CRISPR/Cas9 constructions of the ﬁve gene-knockout mutants from M1346::3gul; Figure S6: PCR screening for positive mutants; Table S1: Information of plasmids and strains; Table S2: Primer sequence used in this study; Table S3: The length of RiPP biosynthetic gene clusters; Table S4: RiPP examples reported by Montalbán-López et al [46–56]. Author Contributions: Conceptualization, J.-Y.L. (Jia-Yi Li) and J.C.; methodology, J.-Y.L. (Jia-Yi Li), J.-Y.L. (Jun-Yu Liang) and Z.-Y.L.; validation, Y.-Z.Y.; investigation, J.C. and Z.-Y.H.; resources, J.Z. and J.C.; writing—original draft preparation, J.-Y.L. (Jia-Yi Li) and J.C.; writing—review and editing, J.-Y.L. (Jia-Yi Li) and J.C.; supervision, J.C. and Z.-Y.H.; project administration, J.C.; funding acquisition, J.C. and Z.-Y.H. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Key R&D Projects of Hainan Province Science and Technology Cooperation Project, grant number ZDYF2019204; and the General Program from the National Natural Science Foundation of China, grant number 41977200. Institutional Review Board Statement: Not applicable. Data Availability Statement: Not applicable. 13. Piscotta, F.J.; Tharp, J.M.; Liu, W.R.; Link, A.J. Expanding the chemical diversity of lasso peptide MccJ25 with genetically encoded noncanonical amino acids. Chem. Commun. 2015, 51, 409–412. [CrossRef] [PubMed] 14. 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https://openalex.org/W2808286624	http://pdf.blucher.com.br/designproceedings/cidi2017/050.pdf	Portuguese	null	O design e a emoção: Uma análise da abordagem do Design Emocional nos artigos publicados em periódicos brasileiros	Blucher Design Proceedings	2,018	cc-by	3,956	Emotional design, Brazilian periodicals, scientific articles. This work aims to present a brief overview of the researches related to the theme Emotional Design, published in the magazines Estudos em Design and InfoDesign. Based on an analysis that aims to delimit and relate characteristics of construction and elaboration of the structure of the article, as well as observation of the theoretical foundation, main authors used and the knowledge generated by each one of the works, it was possible, although with a small sample, List characteristics of thinking and construction of texts that can point out tendencies and characteristics present in the works related to the area of design and emotion. 1 Introdução Grande parte dos estudos em design e emoção no Brasil e no mundo tem se focado no desenvolvimento de modelos de descrição da interação emocional entre os indivíduos e seus produtos, o que contribui tanto para o desenvolvimento de projetos com base nas emoções, como para o desenvolvimento de ferramentas de auxílio (identificação, medição e etc.) ao processo de Design. É crescente a produção acadêmica no país que se utilizada dos fundamentos do design emocional para observar os critérios mais subjetivos no processo de desenvolvimento, uso e descarte dos artefatos produzidos pelos designers. Assim, este trabalho busca observar de que maneira o tema Design Emocional vem sendo trabalhado nos principais periódicos de design do Brasil, verificando quais os autores mais fortemente trabalhados, qual o processo de construção e avaliação dos trabalhos desenvolvidos acerca do tema. Para tanto, foram escolhidas para análise as revistas Estudos em Design e InfoDesign, sob o critério de melhor avaliação no Qualis/CAPES, política de acesso livre e integração com associações e congressos de design do país. A partir disso, foram escolhidos os artigos para o estudo, selecionados por suas palavras-chaves. Estes foram análise em duas fases: a primeira por meio de uma ficha de análise do artigo (FAA), desenvolvida anteriormente, onde foram observados critérios de fluidez do texto, clareza na metodologia, condições de falseamento e validação da pesquisa. Na segunda fase da pesquisa, foi desenvolvida uma tabela de comparação dos artigos observando fundamentação teórica, autores utilizados, linhas de raciocínio construída, bem como objetivos de pesquisa e resultados alcançados, buscando assim apresentar um breve panorama das pesquisas desenvolvidas sobre o tema nos últimos anos. Design and emotion: An analysis of the Emotional Design approach in articles published in Brazilian periodicals. Laís Helena Gouveia Rodrigues; Fabio Ferreira da Costa Campos; Lucas Moreno Cavalcanti Araújo. Design emocional, periódicos brasileiros, artigos científicos. Este trabalho visa apresentar um breve panorama das pesquisas relacionada ao tema Design emocional, publicadas nas revistas Estudos em Design e a InfoDesign. Com base em uma análise que visa delimitar e relacionar características de construção e elaboração da estrutura do artigo, bem como observação da fundamentação teórica, principais autores utilizados e o conhecimento gerado por cada um dos trabalhos, foi possível, ainda que com uma pequena amostra, listar características de pensamento e construção de textos que podem apontar tendências e características presentes nos trabalhos ligados à área de design e emoção. Emotional design, Brazilian periodicals, scientific articles. O design e a emoção: Uma análise da abordagem do Design Emocional nos artigos publicados em periódicos brasileiros. Design and emotion: An analysis of the Emotional Design approach in articles published in Brazilian periodicals. Anais do 8º CIDI e 8º CONGIC Guilherme Santa Rosa; Cristina Portugal (orgs.) Sociedade Brasileira de Design da Informação – SBDI Natal \| Brasil \| 2017 ISBN 978-85-212-1305-5 O design e a emoção: Uma análise da abordagem do Design Emocional nos artigos publicados em periódicos brasileiros. 8th CIDI 8th Information Design International Conference 8th CONGIC 8th Information Design Student Conference Blucher Design Proceedings Junho 2018, num. 1, vol. 4 proceedings.blucher.com.br O design e a emoção: Uma análise da abordagem do Design Emocional nos artigos publicados em periódicos brasileiros. 8th CIDI 8th Information Design International Conference 8th CONGIC 8th Information Design Student Conference Blucher Design Proceedings Junho 2018, num. 1, vol. 4 proceedings.blucher.com.br Blucher Design Proceedings Junho 2018, num. 1, vol. 4 proceedings.blucher.com.br 2. Os níveis de design emocional de Norman É possível encontrar diversos autores que tratam do tema atualmente, discutindo sua fundamentação teórica e sua a relação com a neurociência, além da elaboração de modelos de Anais do 8º CIDI e 8º CONGIC Guilherme Santa Rosa; Cristina Portugal (orgs.) Sociedade Brasileira de Design da Informação – SBDI Natal \| Brasil \| 2017 ISBN 978-85-212-1305-5 Anais do 8º CIDI e 8º CONGIC Guilherme Santa Rosa; Cristina Portugal (orgs.) Sociedade Brasileira de Design da Informação – SBDI Natal \| Brasil \| 2017 ISBN 978-85-212-1305-5 Proceedings of the 8th CIDI and 8th CONGIC Guilherme Santa Rosa; Cristina Portugal (orgs.) Sociedade Brasileira de Design da Informação – SBDI Natal\| Brazil \| 2017 ISBN 978-85-212-1305-5 Proceedings of the 8th CIDI and 8th CONGIC Guilherme Santa Rosa; Cristina Portugal (orgs.) Sociedade Brasileira de Design da Informação – SBDI Natal\| Brazil \| 2017 ISBN 978-85-212-1305-5 535 análise e esquemas para avaliação e aferição dos sentimentos dos consumidores em relação aos objetos. Entretanto, devido a extensão do trabalho desenvolvido, será apresentado apenas um breve resumo do que defende o autor mais citado nos artigos analisados, a ser observado no tópico posterior. 8th CIDI 8th Information Design International Conference 8th CONGIC 8th Information Design Student Conference Blucher Design Proceedings Junho 2018, num. 1, vol. 4 proceedings.blucher.com.br 535 análise e esquemas para avaliação e aferição dos sentimentos dos consumidores em relação aos objetos. Entretanto, devido a extensão do trabalho desenvolvido, será apresentado apenas um breve resumo do que defende o autor mais citado nos artigos analisados, a ser observado no tópico posterior. Norman (2008) defende também que nós somos resultados de três níveis diferentes de estrutura cerebral: o nível visceral - automático e pré-programado responsável por julgamentos rápidos; o nível comportamental – que se refere aos processos cerebrais que controlam a maior parte de nossas ações e o nível reflexivo – que é a parte contemplativa do cérebro, ligado a interpretação, compreensão e raciocínio. Esses três níveis operam sempre em conjunto na nossa relação com os objetos presentes em nosso entorno, sendo o design visceral responsável pelo primeiro impacto com o produto, o design comportamental relacionado aos aspectos funcionais do artefato e o design reflexivo ligado a interpretações mais subjetivas e pessoais com relação ao produto. O design visceral pode ser definido como a nossa relação com o mundo que nos cerca. 2. Os níveis de design emocional de Norman São os estímulos básicos que, apesar da nossa evolução natural, ainda podemos captar em determinado ambiente ou produto. Segundo Norman (2008), o amor que nós, seres humanos, possuímos por sabores e cheiros doces, cores claras e altamente saturadas vêm exatamente desta co-evolução, da ligação entre pessoas e as características básicas da natureza, despertadas para perpetuação das espécies. O autor afirma ainda que os seres humanos selecionam os objetos de seu entorno por tamanho, cor e aparência. Tudo o que estamos biologicamente predispostos a considerar esteticamente agradável advém desta característica da evolução das espécies, sendo a cultura em que o ser humano está inserido, o maior balizador na hora de considerar qual destes elementos podem ser racionalmente decisivos para sua escolha. O design comportamental reflete-se totalmente no uso do produto, a eficácia e eficiência de sua usabilidade é a base para a aprovação neste nível, extremamente enfatizado por Norman em seu livro The design of everyday things. Neste livro o autor afirma que do ponto de vista comportamental, quatro componentes são cruciais para um bom design: função, compreensibilidade, usabilidade e sensação física. Assim, este nível está basicamente ligado as satisfações das necessidades físicas dos usuários, seja aperfeiçoando um produto já existente que não atenda de forma eficiente a função para o qual foi projetado ou criando-o, buscando perceber as necessidades do consumidor e sua percepção em relação ao artefato. De acordo com Norman (2008), as emoções negativas se manifestam exatamente quando há falta de compreensão ao sentirem frustradas e sem controle na situação, o que pode gerar irritação, falta de controle e até mesmo raiva. O nível reflexivo é mais racional dos três caracterizados por Norman (2008), nele tudo tem a ver com a cultura, com a compreensão que o usuário tem de si mesmo e da sociedade em que vive. Segundo Norman (2008), esta visão aborda as lembranças e significados dos objetos para cada pessoa, além de relaciona-se também com a autoimagem e o significado que um objeto pode ter perante a sociedade. “Quer desejemos admitir ou não, todos nós nos preocupamos com a imagem que apresentamos aos outros” (NORMAN, 2008, p. 107). O valor reflexivo pode até superar algumas dificuldades comportamentais. Quando, muitas vezes, escolhemos um produto não pela praticidade ou facilidade de uso, mas sim pelo orgulho de exibir um artefato socialmente diferenciado ou inovador. 3. As revistas brasileiras de design Há atualmente 33 periódicos registrados no portal da Capes com o título contendo a palavra “design”, abrangendo todas as áreas do conhecimento. Destes, apenas 3 são brasileiros. Para este trabalho, escolhemos apenas os periódicos mais bem avaliados, com política de acesso livre aos conteúdos e relacionados a associações e congresso de design realizados no país. Assim, foram selecionadas para análise as revistas: Estudos em design e Infodesign, ambas com avaliação Qualis B1, que oferece a estes periódicos um fator de impacto de entre 2,499 e 1,300, sendo este medido pelo Institute for Scientific Information(ISI). 2. Os níveis de design emocional de Norman O autor afirma ainda que um produto pode representar muito mais do que simplesmente a função que desempenha ou para que foi desenvolvido. Anais do 8º Congresso Internacional de Design da Informação \| CIDI 2017 Proceedings of the 8th Information Design International Conference \| CIDI 2017 536 Portal Capes. Optou-se por não restringir a área do conhecimento, pois muitos periódicos de design estão inclusos em áreas relacionadas, como Arquitetura, por exemplo. Nessa busca foram encontrados 33 periódicos em que o título contivesse a palavra “design”, sendo apenas 3 periódicos brasileiros. Para este trabalho, escolhemos apenas os periódicos mais bem avaliados, com política de acesso livre aos conteúdos e relacionados a associações e congresso de design realizados no país. Portal Capes. Optou-se por não restringir a área do conhecimento, pois muitos periódicos de design estão inclusos em áreas relacionadas, como Arquitetura, por exemplo. Nessa busca foram encontrados 33 periódicos em que o título contivesse a palavra “design”, sendo apenas 3 periódicos brasileiros. Para este trabalho, escolhemos apenas os periódicos mais bem avaliados, com política de acesso livre aos conteúdos e relacionados a associações e congresso de design realizados no país. Assim, foram selecionadas para análise as revistas: Estudos em design e InfoDesign, ambas com avaliação Qualis B1. O Qualis, por sua vez, afere a qualidade dos artigos e de outros tipos de produção, a partir da análise da qualidade dos veículos de divulgação, ou seja, periódicos científicos. A classificação do Qualis como B1 para estas publicações oferecem a estes periódicos um fator de impacto de entre 2,499 e 1,300, sendo este medido pelo Institute for Scientific Information (ISI). Após selecionados os periódicos a serem analisados, foi realizado uma busca em ambos os sites para verificação dos artigos relacionados ao tema trabalhado no estudo. Foram utilizadas palavras- chaves que facilitassem a procurar dos artigos. Não foi delimitado um período de tempo para a busca das publicações, ficando este elemento condicionado a disponibilidade de arquivos em cada um dos periódicos: Revista Estudos em Design de 2008 a 2016 e Revista InfoDesign de 2009 a 2015. Assim, foram encontrados 5 artigos na revista Estudos em Design e 4 artigos na revista Infodesign, publicados entre os anos de 2009 a 2016. Deste total, foram retirados os trabalhos que tratavam diretamente de design emocional, restando apenas 6 artigos para uma análise mais criteriosa. Estes artigos foram avaliados com base em uma ficha anteriormente elaborada intitulada Ficha de análise do artigo (FAA), que analisa critérios importantes para uma boa organização dos elementos estruturais de um artigo, fluidez do texto, clareza na metodologia, condições de falseamento e validação da pesquisa. Esses elementos foram organizados na tabela de avaliação do artigo como um Check-list da seguinte maneira: Informações do periódico; Identificação do artigo; Análise da estrutura do artigo (título, resumo, introdução, corpo do texto, metodologia, conclusão e referencias); Considerações gerais sobre o artigo e Avaliação geral do artigo, tendo cada um desses elementos subtópicos que se relacionam com os critérios listados acima listados. Nesse momento, a intenção é a compressão estrutural de cada um dos elementos, observando os pontos de tensão e características positivas apresentadas, o que facilitará a compreensão do panorama geral dos artigos analisados. Com base nas informações encontradas a partir da Ficha de análise do artigo (FAA), foi possível desenvolver uma segunda análise dos trabalhos, observando aspectos importantes acerca da fundamentação teórica, autores utilizados, linhas de raciocínio construída, bem como objetivos de pesquisa e resultados alcançados, buscando assim apresentar um breve panorama das pesquisas desenvolvidas pelos estudantes, professores e profissionais acerca do tema Design emocional nos últimos anos, como será observado no tópico a seguir. Anais do 8º Congresso Internacional de Design da Informação \| CIDI 2017 Proceedings of the 8th Information Design International Conference \| CIDI 2017 4. Metodologia A pesquisa exploratória teve início com a busca dos periódicos mais representativos no site do 536 Portal Capes. Optou-se por não restringir a área do conhecimento, pois muitos periódicos de design estão inclusos em áreas relacionadas, como Arquitetura, por exemplo. Nessa busca foram encontrados 33 periódicos em que o título contivesse a palavra “design”, sendo apenas 3 periódicos brasileiros. Para este trabalho, escolhemos apenas os periódicos mais bem avaliados, com política de acesso livre aos conteúdos e relacionados a associações e congresso de design realizados no país. 8th CIDI 8th Information Design International Conference 8th CONGIC 8th Information Design Student Conference Blucher Design Proceedings Junho 2018, num. 1, vol. 4 proceedings.blucher.com.br Anais do 8 Congresso Internacional de Design da Informação \| CIDI 2017 Proceedings of the 8th Information Design International Conference \| CIDI 2017 Anais do 8º Congresso Internacional de Design da Informação \| CIDI 2017 5. Resultados A partir da análise individual de cada artigo foi possível mapear características que podem indicar tendências para as pesquisas em relação ao Design emocional nas revistas analisadas. É importante ressaltar inicialmente que o número de artigos analisados é muito pequeno para apontar características que possam ser levadas como tendências no país, entretanto, pode oferecer um direcionamento para pesquisas futuras que abarquem um maior número periódicos e publicações. Nesta pesquisa foram selecionados trabalhos publicado entre os anos de 2009 a 2016, foi percebido uma crescente evolução na qualidade geral dos artigos, que pode advir não só de um aprimoramento no processo de submissão, bem como um maior interesse pelo tema e aumento da bibliografia em língua brasileira. Nos resumos, apenas 2 dos 6 artigos analisados conseguiram oferecer um panorama geral das informações relatadas no artigo, apresentando claramente a estrutura completa do trabalho, com informações sobre metodologia e apontando os resultados. Na introdução temos um avanço em relação a qualidade geral do tópico, entretanto, não é incomum observarmos uma introdução extensa, onde 40% do texto é uma descrição do estado da arte dos conteúdos. No corpo do texto tem-se duas questões importantes a serem observadas: a organização estrutural e o encadeamento das ideias. Nesse momento, foram observadas apenas as informações 537 estruturais. Foi possível perceber uma decrescente evolução no uso de citações diretas na revista Estudo em Design, e o não uso da revista InfoDesign, o que pode apontar uma preocupação maior dos autores com a fluidez do texto. 8th CIDI 8th Information Design International Conference 8th CONGIC 8th Information Design Student Conference Blucher Design Proceedings Junho 2018, num. 1, vol. 4 proceedings.blucher.com.br 537 estruturais. Foi possível perceber uma decrescente evolução no uso de citações diretas na revista Estudo em Design, e o não uso da revista InfoDesign, o que pode apontar uma preocupação maior dos autores com a fluidez do texto. estruturais. Foi possível perceber uma decrescente evolução no uso de citações diretas na revista Estudo em Design, e o não uso da revista InfoDesign, o que pode apontar uma preocupação maior dos autores com a fluidez do texto. Ainda em relação ao corpo de texto é possível notar um uso claro das referências para construção dos artigos e um bom encadeamento das ideias, além de bom esclarecimento de termos importantes para um correto entendimento dos argumentos apresentados para o raciocínio. Anais do 8º Congresso Internacional de Design da Informação \| CIDI 2017 Proceedings of the 8th Information Design International Conference \| CIDI 2017 5. Resultados Há ainda alguns textos referenciados entre as informações relatadas dos experimentos para complementar as informações apresentadas. A maioria usa publicações recentes: entre 1999 a 2012. Na metodologia foram encontrados os maiores entraves dos artigos analisados: 2 dos 6 artigos analisados, objetiva desenvolver uma revisão de literatura sobre o conteúdo do Design Emocional, entretanto, não oferece informações sobre quais os procedimentos utilizados para busca das fontes, e/ou como foram analisadas as obras. Os demais artigos apresentando métodos de procedimentos para utilização de técnicas e equipamentos no campo do design emocional. A metodologia fica clara do ponto de vista das atividades realizadas, entretanto não apresenta as condições de falseamento da pesquisa, apesar de listar características de inclusão e exclusão de elementos. Na conclusão 4 dos 6 artigos analisados não apresentam respostas em relação aos objetivos, bem como limitações de pesquisa e contribuição acadêmica do estudo. Todos os trabalhos deixaram a desejar relação as respostas às condições de falseamento da pesquisa, já que, por não terem sido apresentadas na metodologia, não poderiam ser respondidas na conclusão. A segunda parte da análise dos resultados foi uma pesquisa mais aprofundada na fundamentação teórica dos artigos analisados. Nesta fase, foi percebido uma forte tendência para a construção de revisões de literatura, o que pode sugerir a incipiência no país de estudos na área, devido à recente tradução dos trabalhos internacionais. Além do próprio tempo de evolução das pesquisas em design emocional, área relativamente recente nos estudos da interação humano-objeto. Confrontando os artigos publicados entre os anos de 2009 a 2010 com os artigos elaborados entre os anos 2015 e 2016 é possível notar claramente uma evolução na percepção das abordagens dos principais autores e uma melhor discussão entre os fundamentos selecionados para o corpo teórico e a proposta de metodologia de pesquisa sugerida pelo artigo, o que sugere uma expansão dos estudos relacionados ao design emocional na comunidade científica brasileira. Nota-se ainda uma tendência forte no uso de referência específicas para construção do corpo do texto, os autores mais fortemente citados nos trabalhos analisados são: Donald Norman, Patrick Jordan, Beatriz Russo & Paul Hekkert, Pieter Desmet e Paul Ekman. Por fim, é possível notar ainda que todos os artigos analisados tratam da relação dos estudos do design emocional com outras áreas do conhecimento humano, o que denota a interdisciplinaridade do tema. analisado. Como desdobramento deste trabalho, podem ser realizados análises mais aprofundadas com um maior número de artigos, incluindo os trabalhos apresentados em eventos científicos de design que também são publicados e organizados pelos periódicos analisados. A inclusão de trabalhos internacionais também pode oferecer uma outra perspectiva de observação, por meio da comparação dos trabalhos desenvolvidos no Brasil e fora do país. A depender da quantidade de trabalhos analisados e da relevância destes, há, ainda, a possibilidade de elaboração de recomendações que possam vir a contribuir com o processo de construção de artigos científicos, além de clarificar o raciocínio proposto pela área de conhecimento analisada neste trabalho. 538 6. Conclusões Ao termino do trabalho, foi possível observar que os artigos analisados possuem em sua maioria uma abordagem significativa para o campo do design emocional por possibilitar a integração entre várias áreas do conhecimento humano. Esta é uma característica inerente as pesquisas no campo das emoções, a interdisciplinaridade ajuda a maturar os estudos na área, que ainda é bem recente se comparada a outras do design e projetação de artefatos. É nítido, porém, que os trabalhos ainda precisam de uma observação criteriosa em relação a elaboração, descrição e verificação dos experimentos científicos realizados, para que estes possam ser reproduzidos de forma correta e, assim, contribuam para propagação do conhecimento científico na área do design emocional. Em relação as limitações de pesquisa se faz importante ressaltar que os números de periódicos e artigos analisados neste trabalho são bem pequenos para serem tomados como verdade em relação ao conhecimento científico construído no país a respeito do tema Design emocional. Entretanto, as características encontradas podem apresentar uma tendência de construção e pensamento em relação ao tema. Para construção de pesquisas futuras, se faz importante, ainda, uma observação aprofundada dos instrumentos de pesquisa, observando critérios de inclusão e exclusão de elementos analisado e sua viabilidade para outros trabalhos científicos que não possuam o formato 538 analisado 8th CIDI 8th Information Design International Conference 8th CONGIC 8th Information Design Student Conference Blucher Design Proceedings Junho 2018, num. 1, vol. 4 proceedings.blucher.com.br 538 analisado. Como desdobramento deste trabalho, podem ser realizados análises mais aprofundadas com um maior número de artigos, incluindo os trabalhos apresentados em eventos científicos de design que também são publicados e organizados pelos periódicos analisados. A inclusão de trabalhos internacionais também pode oferecer uma outra perspectiva de observação, por meio da comparação dos trabalhos desenvolvidos no Brasil e fora do país. A depender da quantidade de trabalhos analisados e da relevância destes, há, ainda, a possibilidade de elaboração de recomendações que possam vir a contribuir com o processo de construção de artigos científicos, além de clarificar o raciocínio proposto pela área de conhecimento analisada neste trabalho. 8th CIDI 8th Information Design International Conference 8th CONGIC 8th Information Design Student Conference Blucher Design Proceedings Junho 2018, num. 1, vol. 4 proceedings.blucher.com.br 8th CIDI 8th Information Design International Conference 8th CIDI 8th Information Design International Conference 8th CIDI 8th Information Design International Conference 7. Referências ALMEIDA, C. S. A técnica de análise de micro expressões faciais (METT II-Short) na avaliação da qualidade de jogos digitais: um estudo preliminar de viabilidade. InfoDesign. Revista Brasileira de Design da Informação: São Paulo, v. 6, n. 3 (2009), p. 50 – 57. ALMEIDA, C. S. A técnica de análise de micro expressões faciais (METT II-Short) na avaliação da qualidade de jogos digitais: um estudo preliminar de viabilidade. InfoDesign. Revista Brasileira de Design da Informação: São Paulo, v. 6, n. 3 (2009), p. 50 – 57. ALMEIDA, C. S. Análise emocional de produtos de design baseada em expressão facial. InfoDesign. Revista Brasileira de Design da Informação: São Paulo, v. 7. n.3 (2011), p. 19-27. BRAGA, M. C.; KUREBAYASHI, T. Objetos de expressão e questionamento: consumo afetivo e formulação de diretrizes pessoais. Estudos em Design: Rio de Janeiro, v. 22, n. 2 (2014), p. 115 – 134. CAPES/MEC. Portal de periódicos. Disponível em: http://www.periodicos.capes.gov.br/ Acessado em: 20 de novembro de 2016. FREITAS, R. F.; CARVALHO, C. O.; MENESCAL, R. E. Design Emocional e o designer como interpretador de desejos e necessidades: Revisão de Literatura. Estudo em Design: Rio de Janeiro, V.18, n.1 (2010). NORMAN, D. A. Design emocional: porque adoramos (ou detestamos os objetos do dia-a-dia). Tradução de Ana Deiró. Rio de Janeiro: Rocco, 2008. QUEIROZ, S. G.; CARDOSO, C. L.; GONTIJO, L. A. Design Emocional e Semiótica: caminhos para obter respostas emocionais dos usuários. Estudo em Design: Rio de Janeiro, V.17, n.1 (2009). SANTOS, J. R.; TONETTO, L. M. Design e emoção na qualificação das experiências de educação: redução da ansiedade na busca de cursos de mestrado. Estudos em Design: Rio de Janeiro, v. 24, n. 1 (2016), p. 104 – 123. 8. Autores Laís Rodrigues – UFPE; Brasil; lais_hgr@hotmail.com Fábio Campos – UFPE; Brasil; fc2005@gmail.com Lucas Cavalcanti – UFPE; Brasil; lucascavalcanti@gmail.com Anais do 8º Congresso Internacional de Design da Informação \| CIDI 2017 Proceedings of the 8th Information Design International Conference \| CIDI 2017
https://openalex.org/W2799969944	https://www.frontiersin.org/articles/10.3389/fendo.2018.00201/pdf	English	null	Association Between Progesterone Elevation on the Day of Human Chronic Gonadotropin Trigger and Pregnancy Outcomes After Fresh Embryo Transfer in In Vitro Fertilization/Intracytoplasmic Sperm Injection Cycles	Frontiers in endocrinology	2,018	cc-by	11,923	Edited by: Carlo Alviggi, University of Naples Federico II, Italy 1 ANDROFERT, Andrology and Human Reproduction Clinic, Campinas, Brazil, 2 Division of Urology, Department of Surgery, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, Brazil, 3 Faculty of Health, Aarhus University, Aarhus, Denmark, 4 BIRTH, Kolkata, India, 5 Mumbai Fertility Center – Kamala Polyclinic & Nursing Home, Mumbai, India, 6 Indira IVF Group, Udaipur, India, 7 Sir Ganga Ram Kolmet Hospital, New Delhi, India, 8 Oasis Centre for Reproductive Medicine, Hyderabad, India, 9 Srishti Assisted Fertility & Advanced Laparoscopy Center, Srishti Hospital, Moolakulam, India, 10 Nirmiti Clinic, Centre for Assisted Reproduction, Chinchwad, India, 11 Shobha Nursing Home, Solapur, India, 12 ARMC IVF Fertility Centre, Kozhikode, India, 13 Merck Limited, Mumbai, India Reviewed by: Roberta Venturella, Università degli studi Magna Graecia di Catanzaro, Italy Alessandro Conforti, University of Naples Federico II, Italy Progesterone elevation (PE) during the late follicular phase of controlled ovarian stimu- lation in fresh embryo transfer in vitro fertilization (IVF)/intracytoplasmic sperm injection cycles has been claimed to be associated with decreased pregnancy rates. However, the evidence is not unequivocal, and clinicians still have questions about the clinical validity of measuring P levels during the follicular phase of stimulated cycles. We reviewed the existing literature aimed at answering four relevant clinical questions, namely (i) Is gonadotropin type associated with PE during the follicular phase of stimulated cycles? (ii) Is PE on the day of human chorionic gonadotropin (hCG) associated with negative fresh embryo transfer IVF/intracytoplasmic sperm injection (ICSI) cycles outcomes in all patient subgroups? (iii) Which P thresholds are best to identify patients at risk of implan- tation failure due to PE in a fresh embryo transfer? and (iv) Should a freeze all policy be adopted in all the cycles with PE on the day of hCG? The existing evidence indicates that late follicular phase progesterone rise in gonadotropin releasing analog cycles is mainly caused by the supraphysiological stimulation of granulosa cells with exogenous follicle-stimulating hormone. Yet, the type of gonadotropin used for stimulation seems to play no significant role on progesterone levels at the end of stimulation. Furthermore, PE is not a universal phenomenon with evidence indicating that its detrimental conse- quences on pregnancy outcomes do not affect all patient populations equally. Patients with high ovarian response to control ovarian stimulation are more prone to exhibit PE at the late follicular phase. Review Review published: 26 April 2018 doi: 10.3389/fendo.2018.00201 published: 26 April 2018 doi: 10.3389/fendo.2018.00201 Association Between Progesterone Elevation on the Day of Human Chronic Gonadotropin Trigger and Pregnancy Outcomes After Fresh Embryo Transfer in In Vitro Fertilization/Intracytoplasmic Sperm Injection Cycles Sandro C. Esteves1,2,3, Gautam Khastgir 4, Jatin Shah5, Kshitiz Murdia6, Shweta Mittal Gupta7, Durga G. Rao8, Soumyaroop Dash9, Kundan Ingale10, Milind Patil11, Kunji Moideen12, Priti Thakor13 and Pavitra Dewda13* Sandro C. Esteves1,2,3, Gautam Khastgir 4, Jatin Shah5, Kshitiz Murdia6, Shweta Mittal Gupta7, Durga G. Rao8, Soumyaroop Dash9, Kundan Ingale10, Milind Patil11, Kunji Moideen12, Priti Thakor13 and Pavitra Dewda13* Correspondence: Pavitra Dewda pavitra.dewda@merckgroup.com Specialty section: This article was submitted to Reproduction, a section of the journal Frontiers in Endocrinology Specialty section: This article was submitted to Reproduction, a section of the journal Frontiers in Endocrinology Received: 17 January 2018 Accepted: 10 April 2018 Published: 26 April 2018 However, in studies showing an overall detrimental effect of Correspondence: Pavitra Dewda pavitra.dewda@merckgroup.com INTRODUCTION Currently, many clinicians have questions about the clinical validity of measuring P levels during the follicular phase of stimu- lated cycles. Among several concerns, it is not clear which patients might benefit from P monitoring and what would be the practical implications of PE to pregnancy success in an IVF/ICSI program. In this review, we summarize the recent evidence concerning the clinical implications of PE on Assisted Reproductive Technology (ART) cycles outcomes and identify gaps of knowledge as well as opportunities for future research. Progesterone (P) is essential before and during pregnancy as it plays a critical role in supporting the endometrium and hence survival of the conceptus (1). In the natural cycle, preovulatory P secretion facilitates the action of estrogen on the pituitary; the latter is the key factor to induce the mid-cycle luteinizing hormone (LH) peak. Progesterone also stimulates the mid-cycle follicle-stimulating hormone (FSH) surge, which is important to support the expression of LH receptors in the granulosa layer (2, 3). Notably, most circulating P (~95%) is produced in the intrafollicular compartment by the granulosa cells (GCs) via the action of 3β-HSD that catalyzes the conversion of pregnenolone (delta-4 pathway) under LH influence (4, 5). After ovulation, the corpus luteum is formed and P is produced in both luteinized theca and GCs under the effect of endogenous LH activity (6). In early pregnancy, human chorionic gonadotropin (hCG) secreted by syncytio trophoblast cells rescues the corpus luteum and main- tains luteal function until placental steroidogenesis is established (7). As such, progesterone elevation (PE) and its sustained levels have been considered essential to elicit the endocrine signals responsible for initiating the period of endometrial receptivity to embryo implantation (8, 9).i Citation: Esteves SC, Khastgir G, Shah J, Murdia K, Gupta SM, Rao DG, Dash S, Ingale K, Patil M, Moideen K, Thakor P and Dewda P (2018) Association Between Progesterone Elevation on the Day of Human Chronic Gonadotropin Trigger and Pregnancy Outcomes After Fresh Embryo Transfer in In Vitro Fertilization/Intracytoplasmic Sperm Injection Cycles. Front. Endocrinol. 9:201. doi: 10.3389/fendo.2018.00201 April 2018 \| Volume 9 \| Article 201 Frontiers in Endocrinology \| www.frontiersin.org 1 Esteves et al. PE and Pregnancy Outcomes PE on pregnancy rates, the adverse effect of PE on endometrial receptivity seems to be offset, at least in part, by the availability of good quality embryo for transfer in women with a high ovarian response. Given the limitations of the currently available assays to measure progesterone at low ranges, caution should be applied to adopt specific cutoff values above which the effect of progesterone rise could be considered detrimental and to recommend “freeze-all” based solely on pre-defined cutoff points. PE on pregnancy rates, the adverse effect of PE on endometrial receptivity seems to be offset, at least in part, by the availability of good quality embryo for transfer in women with a high ovarian response. Given the limitations of the currently available assays to measure progesterone at low ranges, caution should be applied to adopt specific cutoff values above which the effect of progesterone rise could be considered detrimental and to recommend “freeze-all” based solely on pre-defined cutoff points. Keywords: assisted reproductive technology, controlled ovarian stimulation, human chorionic gonadotropin trigger, intracytoplasmic sperm injection, in vitro fertilization, late follicular phase, pregnancy outcomes, progesterone elevation Keywords: assisted reproductive technology, controlled ovarian stimulation, human chorionic gonadotropin trigger, intracytoplasmic sperm injection, in vitro fertilization, late follicular phase, pregnancy outcomes, progesterone elevation Eligibility Criteriai Specifically, our study was designed to answer clinical questions raised by the group of authors with clinical experience in IVF/ ICSI (SE, GK, JS, KM, SG, DR, SD, KI, and MP), namely (i) Is gonadotropin type associated with PE during the follicular phase of stimulated cycles? (ii) Is PE on the day of hCG associated with negative fresh embryo transfer IVF/ICSI outcomes in all patient subgroups? (iii) Which P thresholds are best to identify patients at risk of implantation failure due to PE in a fresh embryo transfer? and (iv) Should a freeze all policy be adopted in all the cycles with PE on the day of hCG? Therefore, eligible studies were those that provided evidence to answer one or more questions above. Late follicular phase PE, commonly defined as P levels of 1.5 ng/ml (4.77 nmol/l) or greater at the day of hCG trigger, has been reported in 6–30% of controlled ovarian stimulation (COS) cycles (10–14). The observation of worse pregnancy outcomes in fresh embryo transfer in vitro fertilization (IVF)/ intracytoplasmic sperm injection (ICSI) cycles among patients with PE compared to non-PE has prompted clinicians to monitor progesterone levels during the late follicular phase or at the day of hCG trigger. A policy of freezing all embryos from a fresh IVF/ ICSI cycle and replacing the embryos in a subsequent cycle has been advocated as a solution to avoid the potential negative effect of PE on pregnancy (15, 16). Search Criteria An extensive search of studies published in the past 10 years by examining the relationship between P levels on the day of hCG trigger and pregnancy outcomes in fresh embryo transfer IVF/ICSI cycles was performed using PubMed and MEDLINE. The start and end dates for the searches were January 2006 and February 2017, respectively. Frontiers in Endocrinology \| www.frontiersin.org Is Gonadotropin Type Associated With PE During the Follicular Phase of Stimulated Cycles?h The menotropin versus recombinant FSH IVF trial (MERIT) compared to ongoing pregnancy rates (OPR) in 731 young, nor- mogonadotrophic women undergoing IVF after stimulation with highly purified human menopausal gonadotrophin (HP-hMG; n = 363) or recombinant FSH (rFSH; n = 368) (25). The threshold value for defining serum PE in this study was 4 nmol/l (1.25 ng/ml), measured on the last day of stimulation. The serum P levels were higher in rFSH-treated patients than in HP-hMG-treated patients, with the former showing a higher incidence of PE (23%, 3.4 nmol/l versus 11%, 2.6 nmol/l; p < 0.001). In the study men- tioned above, patients showing higher P values had lower fresh embryo implantation rate (HP-hMG group: 24 versus 19%, not statistically significant; rFSH group: 23 versus 11%, p = 0.025). The authors concluded that late follicular phase PE affected fresh embryo transfer IVF/ICSI outcomes, with PE being more com- mon with the use of rFSH than HP-hMG, however, it is important to note that values of PE are below 4.77 nmol/l (1.5 ng/l) in both groups implying statistically significant values could be clinically irrelevant (25). Collectively, these findings suggest that gonadotropin type does not play a major role to PE in COS. During follicular phase, the GCs are supraphysiologically stimulated by gonadotropins, which may result in increased serum P levels (36). As the pituitary is normally suppressed by GnRH analogs during COS, the serum P levels in stimulated cycles represent the total follicle output of GCs (17, 33). The findings that LH activity provided by rLH of hCG content in hMG preparations is unable to influence P levels is explained by the fact that cytochrome 17a-hydroxylase-C17, 20 lyase (P450-17α), the key enzyme driving the conversion of intrafollicular P to estradiol, is virtually absent in the intrafol- licular compartment (36–38), which makes the conversion of P to estradiol negligible in humans (39). PE is rather dependent on the overall GC output and is, therefore, associated with the number of follicles, oocytes, and E2 levels. Notwithstanding, some evidence suggests that the use of rFSH may be associated with higher P output than urinary gonadotropin normal and high responders, due to the higher potency of rFSH than urinary products (35, 40). Contrary results were reported by Andersen et al. RESULTS AND DISCUSSION The search retrieved a total of 31 articles that specifically addressed at least one of the formulated clinical questions. The Table S1 in Supplementary Material lists the eligible studies included to address the key questions. Study Selectionh In non-gonadotropin releasing hormone (GnRH) analog cycles, premature PE can be explained by an early preovulatory LH elevation (17, 18), which results in endometrial asynchrony that ultimately affects implantation and pregnancy (19). In con- trast, follicular phase PE cannot be attributed to premature LH surge in GnRH analog cycles, since the pituitary is suppressed (20–24). Furthermore, whereas some investigators have reported an inverse association between pregnancy rates in fresh embryo transfer IVF/ICSI cycles and PE, the evidence is not unequivocal thus making the universal application of the freeze-all policy debatable (11). The overall strategy for study identification and data extraction was based on the following key words: “assisted reproductive technol- ogy,” “controlled ovarian stimulation,” “intracytoplasmic sperm injection,” “in vitro fertilization,” “progesterone levels,” “hCG trigger,” “pregnancy outcomes,” with the filters “humans,” and “English language.” Data only published in conference or meeting proceedings, websites, or books were not included. Articles were only included if hCG trigger alone followed by conventional luteal phase support, which specifically means luteal supplementation with progesterone administration, and fresh embryo transfers were carried out. Oocyte donation and frozen-thawed embryo April 2018 \| Volume 9 \| Article 201 Frontiers in Endocrinology \| www.frontiersin.org 2 PE and Pregnancy Outcomes Esteves et al. rFSH alone, rFSH combined with rLH, HP-hMG alone, and rFSH combined with HP-hMG on PE. The authors found that P levels in the late follicular phase were associated with the number of oocytes retrieved and serum estradiol levels, irrespective of type of GnRH analog, but not with the type of gonadotropin administered. Furthermore, there was no associa- tion between PE and duration of stimulation or FSH requirement. Subsequently, Requena et al. corroborated these aforementioned findings by showing that the mean serum P levels did not differ significantly with respect to the type of gonadotropin used for COS: rFSH + rLH (n = 377, P: 1.01 ng/ml), rFSH alone (n = 728, P: 1.06 ng/ml), rFSH + HP-hMG (n = 1,375; P: 1.30 ng/ml), and HP-hMG alone (n = 370; P: 1.10 ng/ml) (33). Last, a randomized, open-label, assessor-blind study comparing the efficacy and safety of HP-hMG and rFSH for COS reported that at the end of stimulation, the mean P levels were was not significantly different between HP-hMG (3.1 ± 3.4 nmol/l) and rFSH (3.1 ± 3.3 nmol/l) treatment groups (34). The characteristics and main findings of the studies mentioned above are summarized in Table 1. Study Selectionh transfer cycles were excluded. Case reports and letters were not considered. Articles published before the start search date and book-chapter citations provided conceptual content only. Is Gonadotropin Type Associated With PE During the Follicular Phase of Stimulated Cycles?h who per- formed a post hoc analysis of data from a randomized controlled trial (RCT) that included 475 women younger than 40 years subjected to IVF/ICSI (26). The primary aim of the study was to investigate whether the addition of recombinant LH (rLH) or FSH from day 6 of stimulation onward impacted pregnancy rates in GnRH agonist cycles. Moreover, the authors evaluated whether the addition of rLH in the second half of the follicular phase influenced serum P levels measured on the day of hCG trigger. P levels were determined on day 1, i.e., before exogenous FSH administration, and on the day of hCG trigger. Although patients receiving rLH had higher LH levels at the end of stimula- tion than those receiving rFSH alone, there were no differences in pregnancy rates and late follicular phase P levels between groups. In this study, P levels were associated with both the number of developing follicles and retrieved oocytes and partly to the late follicular phase LH concentration. Interestingly, higher pregnancy rates were reported in women with P concentration >7 nmol/l (>2.20 ng/ml) in the late follicular phase, which coincided with those developing high number of follicles (26). Frontiers in Endocrinology \| www.frontiersin.org Of patients with CFA, only stimulation 5.4% (13/239 patients) showed a PE above 1.5 ng/ml on day of hCG trigger, whereas patients with rFSH stimulation had a significant higher incidence of PE (18.3%; 62/339 patients) (p < 0.001) The threshold value for defining serum PE was 1.5 ng/ml. PE was analyzed on day 8 of stimulation Is PE on the Day of hCG Associated With Negative Fresh Embryo Transfer IVF-ICSI Outcomes in All Patient Subgroups? In both trials, daily rFSH was continued until three follicles reached >17 mm in size For ENGAGE study: stimulation protocol included either a single injection of 150 mg CFA or daily injections of 200 IU rFSH in the first week of stimulation, using a standard GnRH antagonist protocol For PURSUE study: stimulation protocol included either a single injection of 150 mg of CFA or daily 300 IU of rFSH for the first week, again using a standard GnRH antagonist protocol. In both trials, daily rFSH was continued until three follicles reached >17 mm in size retrieved oocytes, late-follicular phase progesterone concentra- tions was not associated with clinical pregnancy rates (CPRs). COS with hMG and/or FSH in a GnRH agonist protocol did not affect oocyte quality and pregnancy rate (41). The findings of Miller et al. were corroborated by Hamdine et al. who conducted a prospective intervention study including 158 IVF-ICSI patients (42). The authors showed that the incidence of PE (>1.5 ng/ml) was 13.3%, but OPRs were not significantly different between patients with normal P levels and PE (27.0 versus 19.0%). Furthermore, no differential impact of early or late GnRH antagonist initiation on the effect of elevated or normal P on OPR was observed. Likewise, the study of Andersen et al. mentioned above (26) reported that despite being strongly associated with the number of follicles and retrieved oocytes, late-follicular phase progesterone concentra- tions was not associated with clinical pregnancy rates (CPRs). Recently, it has been suggested that the impact of PE on IVF/ ICSI may vary according to the affected population. In one study, Griesinger and colleagues analyzed the data from six IVF/ICSI clinical trials to investigate the impact of P measured on the day of hCG trigger on fresh embryo transfer pregnancy outcomes, using the cutoff point of 1.5 ng/ml. Patients were stratified according to the number of oocytes retrieved after COS: low (1–5 oocytes), normal (6–18 oocytes), and high (>18 oocytes). The incidence of PE was 4.5 and 19.0% in low responders and high responders, Recently, it has been suggested that the impact of PE on IVF/ ICSI may vary according to the affected population. In one study, Griesinger and colleagues analyzed the data from six IVF/ICSI clinical trials to investigate the impact of P measured on the day of hCG trigger on fresh embryo transfer pregnancy outcomes, using the cutoff point of 1.5 ng/ml. For ENGAGE study: stimulation protocol included either a single injection of 150 mg CFA or daily injections of 200 IU rFSH in the first week of stimulation, using a standard GnRH antagonist protocol For PURSUE study: stimulation protocol included either a single injection of 150 mg of CFA or daily 300 IU of rFSH for the first week, again using a standard GnRH antagonist protocol. In both trials, daily rFSH was continued until three follicles reached >17 mm in size Controlled ovarian stimulation was performed with HP-hMG or rFSH in a GnRH antagonist cycle with compulsory single-blastocyst transfer on day 5 in one fresh or subsequent frozen blastocyst replacement in natural cycles The threshold value for defining serum PE was 3.18 nmol/l (1.0 ng/ml) Is PE on the Day of hCG Associated With Negative Fresh Embryo Transfer IVF-ICSI Outcomes in All Patient Subgroups? (26) Study included a total of 475 women age with 40 years, undergoing IVF or ICSI with a regular (21–35 days) menstrual cycle and basal serum FSH concentration of <10 IU/l on menstrual cycle day 2–5 Stimulation was performed with GnRH agonist and FSH in one group (n = 247), in another group (n = 228) patients were administered GnRH agonist, rFSH with rLH from day 6 of stimulation. P concentration was measured on day 1, prior to exogenous FSH administration, and on the day of hCG administration The threshold value for defining serum PE was 4.77 nmol/l (1.5 ng/ml) The average progesterone concentration on the day of ovulat induction (day of HCG administrat did not differ between those who received embryo transfer and those who did not [mean ± SD, 4.38 ± 3.90 nmol/l, (n = 419), versus 3.99 ± 2.16 nmol/l, (n = 56 respectively] The threshold value for defining serum PE was 4 nmol/l (1.25 ng/ml) Stimulation was performed with highly purified human menopausal gonadotropin (HP-hMG; n = 363) or rFSH (n = 368). P concentration was measured on the last day of stimulation The serum P levels were higher in rFSH-treated patients than in the HP-HMG-treated patients, with the former showing a higher incidence of PE (23 versus 11%; p < 0.001) Stimulation was performed with GnRH agonist and FSH in one group (n = 247), in another group (n = 228) patients were administered GnRH agonist, rFSH with rLH from day 6 of stimulation. Requena et al. (33) A total of 2,850 infertile women who were classified as high responders (high response was defined as women who had ≥20 oocytes retrieved or whose estradiol levels were ≥3,000 pg/ml) and were undergoing assisted reproduction techniques for the last 2 years were included in this retrospective study Is PE on the Day of hCG Associated With Negative Fresh Embryo Transfer IVF-ICSI Outcomes in All Patient Subgroups? P concentration was measured on day 1, prior to exogenous FSH administration, and on the day of hCG administration The average progesterone concentration on the day of ovulation induction (day of HCG administration) did not differ between those who received embryo transfer and those who did not [mean ± SD, 4.38 ± 3.90 nmol/l, (n = 419), versus 3.99 ± 2.16 nmol/l, (n = 56), respectively] The threshold value for defining serum PE was following in different groups: <0.5 ng/ml (<p10), 0.50–0.70 ng/ml (p10–p25), 0.71–1.00 ng/ml (p25–p50), 1.01–1.40 ng/ml (p50–p75), 1.41–1.80 ng/ml (p75–p90), and >1.81 ng/ml (>p90) Ovarian stimulation was performed by one of four possible methods: recombinant follicle stimulating hormone (rFSH) alone; rFSH combined with recombinant luteinizing hormone (rLH); highly purified human-menopausal gonadotropin (HP-hMG) alone; or rFSH combined with HP-hMG No significant differences in the mean progesterone concentration with respect to the type of gonadotropin that was used for ovarian stimulation: rFSH alone (n = 728, progesterone 1.06 ng/ml), rFSH + rLH (n = 377, progesterone 1.01 ng/ml), HP-hMG alone (n = 370; progesterone 1.10 ng/ml), and rFSH + HP-hMG (n = 1,375; progesterone 1.30 ng/ml) Devroey et al. (34) Study included women aged 21–34 years with a body mass index (BMI) of 18–25 kg/m2; primarydiagnosis of infertility being unexplained infertility or mild male factor; eligible for ICSI, infertile for12 months before randomization; with regular menstrual cycles of 24–35 days Lawrenz et al. (35) ENGAGE study: a total of 1,506 women aged 18–36 years undergoing IVF stimulation cycles PURSUE study: a total of 1,390 women aged 35–42 years undergoing IVF stimulation cycles In both studies, women had a body weight of between 50 and 90 kg, regular menstrual cycles The average serum P level and the proportion of patients with serum P concentrations above 4 nmol/l at the end of stimulation (16% in the HP-hMG group and 14% in the rFSH group) were similar between the treatment groups For ENGAGE study: stimulation protocol included either a single injection of 150 mg CFA or daily injections of 200 IU rFSH in the first week of stimulation, using a standard GnRH antagonist protocol For PURSUE study: stimulation protocol included either a single injection of 150 mg of CFA or daily 300 IU of rFSH for the first week, again using a standard GnRH antagonist protocol. Lawrenz et al. (35) ENGAGE study: a total of 1,506 women aged 18–36 years undergoing IVF stimulation cycles PURSUE study: a total of 1,390 women aged 35–42 years undergoing IVF stimulation cycles In both studies, women had a body weight of between 50 and 90 kg, regular menstrual cycles The threshold value for defining serum PE was 4.77 nmol/l (1.5 ng/ml) Requena et al. (33) A total of 2,850 infertile women who were classified as high responders (high response was defined as women who had ≥20 oocytes retrieved or whose estradiol levels were ≥3,000 pg/ml) and were undergoing assisted reproduction techniques for the last 2 years were included in this retrospective study The threshold value for defining serum PE was following in different groups: <0.5 ng/ml (<p10), 0.50–0.70 ng/ml (p10–p25), 0.71–1.00 ng/ml (p25–p50), 1.01–1.40 ng/ml (p50–p75), 1.41–1.80 ng/ml (p75–p90), and >1.81 ng/ml (>p90) Is PE on the Day of hCG Associated With Negative Fresh Embryo Transfer IVF-ICSI Outcomes in All Patient Subgroups? g p In a systematic review and meta-analysis including 63 studies and over 60,000 IVF/ICSI cycles, Venetis et al. reported that late follicular phase PE was detrimental to fresh embryo transfer pregnancy rates. PE affected pregnancy success with levels as low as 0.8–1.1 ng/ml (OR: 0.79, 95% CI: 0.67–0.95), which increased as the level reaches 1.2 ng/ml (OR: 0.67, 95% CI: 0.53–0.84), and becomes stable thereafter (12). Along the same lines, Bosh et al. showed an association between P levels >1.5 ng/ml at the day of hCG administration and fresh embryo transfer IVF/ICSI pregnancy outcomes. The authors examined their database of 4,032 patients subjected to IVF/ICSI and found that OPRs were higher in patients with serum P levels ≤1.5 ng/ml than those with P levels >1.5 ng/ml (31.0 versus 19.1%; P = 0.00006) (10). In another study, Kolibianakis et al. (27) pooled data from five COS-IVF trials (28–32) using either GnRH antagonists or agonists that evaluated the impact of the type of gonadotropin, However, the evidence is not unequivocal as Miller et al. showed that the elevation of P level in 293 patients subjected to April 2018 \| Volume 9 \| Article 201 3 PE and Pregnancy Outcomes Esteves et al. Table 1 \| Characteristics of included studies to discuss if gonadotropin type is associated with progesterone elevation (PE) during the follicular phase of stimulated cycles. Study and year (reference) Patient characteristics Ovarian stimulation regimen Progesterone threshold PE incidence Menotrophin versus recombinant FSH (rFSH) IVF trial (MERIT) (25) Study included a total of 731 young, normogonadotropic women undergoing IVF, patients were divided in two groups Stimulation was performed with highly purified human menopausal gonadotropin (HP-hMG; n = 363) or rFSH (n = 368). P concentration was measured on the last day of stimulation The threshold value for defining serum PE was 4 nmol/l (1.25 ng/ml) The serum P levels were higher i rFSH-treated patients than in the HP-HMG-treated patients, with t former showing a higher incidenc of PE (23 versus 11%; p < 0.001 Andersen CY et al. Which P Thresholds Are Best to Identify Patients at Risk of Implantation Failure due to PE in a Fresh Embryo Transfer?f Some studies have examined the impact of different P thresholds on fresh embryo transfer IVF/ICSI pregnancy outcomes. In one study, Xu et al. assessed the effect of PE on the day of hCG on pregnancy following fresh and frozen embryo transfer (FET) as a function of the ovarian response to COS: high (≥20 oocytes; n = 2,023), poor (≤4 oocytes; n = 27), or intermediate (n = 8,205) (14). The P cutoff points associated with decreased pregnancy outcomes in fresh embryo transfer cycles were 1.5 ng/ml for poor responders, 1.75 ng/ml for intermediate responders, and 2.75 ng/ml for high responders. The authors also observed that P mean values differed according to ovarian response (1.89 ± 0.66 ng/ml in high responders, 1.47 ± 0.47 ng/ml in intermediate responders, and 1.18 ± 0.48 ng/ml in poor responders, p < 0.001), with PE being more common in the high ovarian response group than intermediate and poor ovarian response groups.f Similar results were reported by Requena et al. retrospectively analyzing the effect of PE on fresh embryo transfer IVF/ICSI pregnancy outcomes of 2,850 women classified as high respond- ers (33). The high ovarian response was defined as having ≥20 oocytes or estradiol ≥3,000 pg/ml. The patients were subgrouped into six categories based on the level of serum P on day of hCG, as <0.5, 0.50–0.70, 0.71–1.00, 1.01–1.40, 1.41–1.80, and >1.81 ng/ml. The authors observed that P levels neither had a negative impact on the oocyte quality and endometrial receptivity nor did it affect pregnancy success. Only in the group of P level >1.80 ng/ml there was a marginally significant negative impact on pregnancy rates (OR: 0.73, 95% CI: 0.61 to 0.99) (33). Last, a retrospective analysis of 1,800 IVF/ICSI cycles performed by Cruz et al., who stratified patients by high (P > 1.5 ng/ml) or low (P < 1.5 ng/ ml) concluded that PE in high ovarian responders did not impact fresh embryo transfer IVF/ICSI outcomes. The authors showed no significant differences in the analyzed parameters, namely, number of retrieved oocytes (17.2 ± 0.8 versus 17.3 ± 0.4), number of transferred embryos (1.81 ± 0.08 versus 1.85 ± 0.02), pregnancy rate (59.9 versus 54.6%), implantation rate (41.2 versus 39.7%), and miscarriage rate (22.6 versus 28.6%) for high and low progesterone levels respectively in case of high ovarian response (43). Which P Thresholds Are Best to Identify Patients at Risk of Implantation Failure due to PE in a Fresh Embryo Transfer?f Furthermore, whereas OPRs were negatively affected by PE in fresh embryo transfer IVF/ICSI cycles, no detrimental effect was reported in the FET cycles. A multivariate logistic regression analysis showed that the rise in P level was associated with the number of oocytes retrieved, FSH dose, and estradiol values on the day of HCG administration. Last, the authors found that the detrimental effect of PE on the implantation rate and OPR were independent of oocyte quality (14). It has been suggested that the likely reason for the negligible effect of PE on cycle outcome of high responders relates to the availability of high-quality embryos for transfer, including blas- tocysts, which could circumvent a possible adverse endometrial environment for implantation (42, 44). This hypothesis has been confirmed by the data of an early study by Papanikolaou et al., in which the impact of PE on pregnancy outcome was compared between day 3 and blastocyst single embryo transfers. The authors’ analyzed data from 482 patients undergoing single ET after COS with GnRH antagonist associated with rFSH. The incidence of PE (P above 1.5 ng/ml on the day of hCG administration) was 18.2%. The authors reported that PE did not affect pregnancy outcome in fresh blastocyst transfer cycles. In contrast, even a modest rise in P affected the pregnancy outcome in patients with day 3 embryo transfers (29).l In another study, Venetis et al. retrospectively analyzed 3,296 fresh IVF/ICSI cycles to assess the effect of PE on live birth rates (LBR) (13). A bivariate analysis reported no statistical difference in the LBRs when patients with normal P values (<1.5 ng/ml) were compared to patients with PE (≥1.5 ng/ml; OR: 0.78, 95% confidence interval (CI): 0.56–1.09). However, a multivariable regression analysis showed an overall decrease in LBR in the elevated P level group (OR: 0.68, 95% CI: 0.48–0.97). The study also assessed whether the effect of PE on LBR was associated with the ovarian response. There was no statistically significant impact of PE on LBRs in both low (<6 oocytes, n = 796 cycles) and high (>18 oocytes, n = 730 cycles) ovarian response groups, whereas negative effect of PE was reported in normal responders (6–18 oocytes, n = 1,770 cycles) with threshold levels as low as 0.9 ng/ml being significant. The authors reported that the number of oocytes and female age were the most common confounding factors for PE (13). Is PE on the Day of hCG Associated With Negative Fresh Embryo Transfer IVF-ICSI Outcomes in All Patient Subgroups? Patients were stratified according to the number of oocytes retrieved after COS: low (1–5 oocytes), normal (6–18 oocytes), and high (>18 oocytes). The incidence of PE was 4.5 and 19.0% in low responders and high responders, April 2018 \| Volume 9 \| Article 201 Frontiers in Endocrinology \| www.frontiersin.org 4 PE and Pregnancy Outcomes Esteves et al. respectively. Overall, OPRs per started cycle were significantly lower in women with PE [odds ratio (25): 0.55; 95% CI: 0.37–0.81]. However, a subgroup analysis showed that P level >1.5 ng/ml was associated with decreased pregnancy rates in low to normal responders, but not in high responders (11). In their study, OPRs in high responders with PE were higher than normal responder counterparts. In women without P elevation, the observed OPR increased from 29.9% (1–5 oocytes) to 39.2% (>18 oocytes). By contrast, women with elevated P showed an increase in OPR from 18.2% (1–5 oocytes) to 43.2% (>18 oocytes). Compared with the subjects without P elevation, the observed OPR were numerically lower in all subsets for the women with elevated P except for the high responders (>18 oocytes). showing an overall detrimental effect of PE on pregnancy rates, the adverse effect of PE on endometrial receptivity seems to be offset, at least in part, by the availability of good quality embryo for transfer in women with a high ovarian response. In contrast, elevated P level results in reduced pregnancy in patients with a low ovarian response, who tends to have poorer quality embryos that counterpart with high ovarian response. Table 2 summarizes the studies discussed above. Frontiers in Endocrinology \| www.frontiersin.org There was no significant differences in the analyzed parameters, namely, number of retrieved oocytes (17.2 ± 0.8 versus 17.3 ± 0.4), number of transferred embryos (1.81 ± 0.08 versus 1.85 ± 0.02), pregnancy rate (59.9 versus 54.6%), implantation rate (41.2 versus 39.7%), and miscarriage rate (22.6 versus 28.6%) for high and low progesterone levels, respectively in case of high ovarian response Which P Thresholds Are Best to Identify Patients at Risk of Implantation Failure due to PE in a Fresh Embryo Transfer?f In conclusion, there is conflicting data on the impact of late follicular phase PE on fresh embryo transfer IVF/ICSI outcomes. Although PE has been associated with decreased pregnancy rates in several studies, PE does not seem to affect all patient popula- tions equally with high responders with PE achieving similar pregnancy success than counterparts without PE. In studies In a recent cohort study performed by Shufaro et al., the impact of late follicular phase PE was also assessed as a func- tion of ovarian response to COS. The authors included 8,649 IVF/ICSI cycles performed in women aged 33.9 ± 5.8 years in April 2018 \| Volume 9 \| Article 201 5 PE and Pregnancy Outcomes Esteves et al. Table 2 \| Characteristics of included studies to discuss if progesterone elevation (PE) on the day of hCG was associated with negative fresh embryo transfer IVF-ICS outcomes in all patient subgroups. Reference and place of study conducted Design Patient population Intervention/method Results Venetis et al. (13) country: Greece Retrospective analysis A total of 3,296 women undergoing fresh IVF/ICSI Simple bivariate analyses and multivariate analyses was done to compare PE and LBR according to serum P levels ≤1.5 versus >1.5 ng/ml on the day of HCG administration and compared among low (<6 oocytes), normal (6–18 oocytes), and high (>18 oocytes) responders PE negatively impacted pregnancy success with levels as low as 0.8–1.1 ng/ml (OR: 0.79, 95% CI: 0.67–0.95). The magnitude of effect size increased as P level reached 1.2 ng/ml (OR 0.67, 95% CI: 0.53–0.84), and became stable thereafter Andersen et al. (26) country: Europe (22 centers): 10 in Denmark, 2 in Finland, 4 in Norway, and 6 in Sweden Retrospective analysis A total of 475 patients undergoing IVF/ICSI following ovarian stimulation with GnRH agonist and rFSH with or without rLH administration from day 6 of stimulation were included The study was aimed to explore the association between the number of eggs and live birth outcomes, a likelihood logistic model was used to compare progesterone concentrations in two groups Progesterone concentration was strongly associated with the number of follicles and retrieved oocytes. There was no significant association between the late-follicular phase progesterone concentration and clinical pregnancy rate Griesinger et al. Which P Thresholds Are Best to Identify Patients at Risk of Implantation Failure due to PE in a Fresh Embryo Transfer?f (11) country: meta analysis data collected from different study IVF centers in USA and Europe Retrospective combined analysis 1,866 women undergoing IVF/ICSI with available serum P levels on the day of hCG Univariate and multivariate analyses was done to assess association between elevated P level on the day of hCG, according to serum P levels ≤1.5 versus >1.5 ng/ml and compared among low (1–5 oocytes retrieved), normal (6–18 oocytes), and high (>18 oocytes) responders The incidence of PE was 4.5 and 19.0% in low responders and high responders, respectively. Overall, OPRs per started cycle were significant lower in women with PE (OR: 0.55; 95% CI: 0.37–0.81). However, a subgroup analysis showed that P level >1.5 ng/ml was associated with decreased pregnancy rates in low to norm responders, but not in high responders Requena et al. (33) country: Spain Retrospective cohort study A total of 2,850 women were classified on basis of basis of P level into following groups: <0.5 ng/ml (<p10), 0.50–0.70 ng/ml, (p10–p25), 0.71–1.00 ng/ml (p25–p50), 1.01–1.40 ng/ml (p50–p75), 1.41–1.80 ng/ml (p75–p90), and >1.81 ng/ml (>p90) CPR and implantation rate was assessed on the basis of five distinct serum P levels P levels neither had a negative impact on the oocyte quality and endometrial receptivity nor d it affect pregnancy success. Only in the group of P level >1.80 ng/ml there was a marginally significant negative impact on pregnancy rates (OR: 0.73, 95% CI: 0.61–0.99) Cruz et al. The study aimed to determine the influence of high progesterone levels on clinical outcomes in high ovarian response Progesterone concentration was strongly associated with the number of follicles and retrieved oocytes. There was no significant association between the late-follicular phase progesterone concentration and clinical pregnancy rate Which P Thresholds Are Best to Identify Patients at Risk of Implantation Failure due to PE in a Fresh Embryo Transfer?f (33) country: Spain Retrospective study A retrospective analysis of 1,800 cycles comparing high (>1.5 ng/ml) or low (<1.5 ng/ml) progesterone levels in patients undergoing controlled stimulation classified as high responders (E2 > 3,000 pg/ml) was done The study aimed to determine the influence of high progesterone levels on clinical outcomes in high ovarian response There was no significant differences in the analyzed parameters, namely, number of retrieved oocytes (17.2 ± 0.8 versus 17.3 ± 0.4 number of transferred embryos (1.81 ± 0.08 versus 1.85 ± 0.02), pregnancy rate (59.9 versu 54.6%), implantation rate (41.2 versus 39.7%), and miscarriage rate (22.6 versus 28.6%) for hig and low progesterone levels, respectively in cas of high ovarian response IVF, in vitro fertilization; LBR, live birth rates; RCT, randomized controlled trials; hMG, human menopausal gonadotropin; rhFSH, recombinant human follicle stimulating hormone; CPR, clinical pregnancy rates; FET, frozen-embryo transfer; GnRha, gonadotropin releasing hormone agonist; P, progesterone; OPR, ongoing pregnancy rate; ET, embryo transfer; hCG, human chorionic gonadotropin. Results PE negatively impacted pregnancy success with levels as low as 0.8–1.1 ng/ml (OR: 0.79, 95% CI: 0.67–0.95). The magnitude of effect size increased as P level reached 1.2 ng/ml (OR: 0.67, 95% CI: 0.53–0.84), and became stable thereafter Simple bivariate analyses and multivariate analyses was done to compare PE and LBR according to serum P levels ≤1.5 versus >1.5 ng/ml on the day of HCG administration and compared among low (<6 oocytes), normal (6–18 oocytes), and high (>18 oocytes) responders The study was aimed to explore the association between the number of eggs and live birth outcomes, a likelihood logistic model was used to compare progesterone concentrations in two groups The incidence of PE was 4.5 and 19.0% in low responders and high responders, respectively. Overall, OPRs per started cycle were significantly lower in women with PE (OR: 0.55; 95% CI: 0.37–0.81). However, a subgroup analysis showed that P level >1.5 ng/ml was associated with decreased pregnancy rates in low to normal responders, but not in high responders Univariate and multivariate analyses was done to assess association between elevated P level on the day of hCG, according to serum P levels ≤1.5 versus >1.5 ng/ml and compared among low (1–5 oocytes retrieved), normal (6–18 oocytes), and high (>18 oocytes) responders P levels neither had a negative impact on the oocyte quality and endometrial receptivity nor did it affect pregnancy success. Which P Thresholds Are Best to Identify Patients at Risk of Implantation Failure due to PE in a Fresh Embryo Transfer?f Only in the group of P level >1.80 ng/ml there was a marginally significant negative impact on pregnancy rates (OR: 0.73, 95% CI: 0.61–0.99) CPR and implantation rate was assessed on the basis of five distinct serum P levels There was no significant differences in the analyzed parameters, namely, number of retrieved oocytes (17.2 ± 0.8 versus 17.3 ± 0.4), number of transferred embryos (1.81 ± 0.08 versus 1.85 ± 0.02), pregnancy rate (59.9 versus 54.6%), implantation rate (41.2 versus 39.7%), and miscarriage rate (22.6 versus 28.6%) for high and low progesterone levels, respectively in case of high ovarian response IVF, in vitro fertilization; LBR, live birth rates; RCT, randomized controlled trials; hMG, human menopausal gonadotropin; rhFSH, recombinant human follicle stimulating hormone; CPR, clinical pregnancy rates; FET, frozen-embryo transfer; GnRha, gonadotropin releasing hormone agonist; P, progesterone; OPR, ongoing pregnancy rate; ET, embryo transfer; hCG, human chorionic gonadotropin. whom pituitary suppression was used (GnRH agonist: 49.8% of cycles; GnRH antagonist: 50.2% of cycles). The mean number of follicles >14 mm in diameter recorded before hCG admin- istration was 8.07 ± 3.31 (range: 3–15 follicles). In addition to measure late follicular phase P levels, the authors determined the progesterone-to-follicle index (PFI), which was considered to be more representative of the intrinsic follicular properties that are related to cycle outcome than the total blood P. The PFI was calculated by dividing the blood P by the number of follicles ≥14 mm. The CPR was calculated against the range of PFI values and serum P levels. Overall, the mean late follicular phase P level was 2.22 ± 1.33 nmol/l, and the mean PFI was 0.32 ± 0.25 nmol/l follicle on the day of hCG. The (reverse) ORs for pregnancy after a fresh embryo transfer were 1.11 [95% confidence interval (CI): 1.07–1.16] for serum P and 4.10 (95% CI: 3.18–5.28) for the PFI. These authors reported that elevated P levels were associated with a lower pregnancy rate only when they reached the >93rd percentile. In contrast, the PFI was inversely and linearly related to the pregnancy rate for the whole range of values. The authors concluded that a late increase in P level was detrimental if it resulted from increased P production per follicle (high PFI), but not if it caused by additional follicular recruitment (45). Last, recent reports have examined the issue from a dif- ferent perspective. In one study, Lee et al. PE negatively impacted pregnancy success with levels as low as 0.8–1.1 ng/ml (OR: 0.79, 95% CI: 0.67–0.95). The magnitude of effect size increased as P level reached 1.2 ng/ml (OR: 0.67, 95% CI: 0.53–0.84), and became stable thereafter The incidence of PE was 4.5 and 19.0% in low responders and high responders, respectively. Overall, OPRs per started cycle were significantly lower in women with PE (OR: 0.55; 95% CI: 0.37–0.81). However, a subgroup analysis showed that P level >1.5 ng/ml was associated with decreased pregnancy rates in low to normal responders, but not in high responders CPR and implantation rate was assessed on the basis of five distinct serum P levels Which P Thresholds Are Best to Identify Patients at Risk of Implantation Failure due to PE in a Fresh Embryo Transfer?f The serum P con- centration on the LH surge day in those women without PE was 3.2 ± 1.0 nmol/l (0.9–4.9) [mean ± SD (range)], whereas in those with PE it was 6.4 ± 1.7 nmol/l (5.0–15.3); the serum E2 concentration was 1,082 ± 329 pmol/l (376–2,222) and 1,045 ± 428 pmol/l (440–2,649), respectively. There were no significant differences in both clinical and ongoing PR (39.0 versus 37.3% and 32.5 versus 31.7%) between those with and without PE on the day of LH surge. The incidence of PE in frozen-thawed embryo transfers in subsequent natural cycles (FET-NC) was not significantly different than that in stimulated cycles (15). In another study, Lai et al. evaluated the relationship between serum levels of P and estradiol (P:E ratio) measured on the day of hCG administration on ART clinical outcomes of 139 infertile women with normal ovarian reserve treated with a long GnRH-a protocol. The authors showed that P:E ratio was significantly higher among patients with premature luteinization (PL, n = 41), defined by a P:E ≥ 1.2 using receiver operator characteristic analysis, than in non-PL (n = 98) group (2.4 ± 1.5 and 0.6 ± 0.3, respectively), but P:E ratio did not correlate with pregnancy outcomes after a fresh embryo transfer (46). Should a “Freeze-All” Policy be Adopted in All the Cycles With PE on the Day of hCG? Based on the existing literature, it is clear that the association between PE and IVF/ICSI success does not follow a binomial distribution, i.e., PE not always halt implantation regardless of the threshold level adopted. Therefore, the next critical question for clinicians would be to know the absolute pregnancy reduction rate in the face of PE. According to the largest systematic review and meta-analysis published to date, the OR for pregnancy reduction associated with PE was 0.64 (95% CI: 0.54–0.76). Transformation of the OR mentioned above into absolute pregnancy rate reduction (APRR) translated in 10.1% APRR (95% CI: 6–14%) (12). Using these assumptions, it is possible to estimate the net effect of performing fresh embryo transfer in cases of PE for a given IVF Program. In Table 3, we estimated the net effect of PE on pregnancy for an IVF center performing 1,000 cycles per year with an overall baseline pregnancy rate of 40% per fresh embryo transfer, considering three different scenarios of PE incidence as commonly reported in the literature. Which P Thresholds Are Best to Identify Patients at Risk of Implantation Failure due to PE in a Fresh Embryo Transfer?f According to these estimations, the net effect on pregnancy reduction for the overall population subjected to fresh embryo transfer IVF/ICSI cycles in a given IVF center would range from 0.5 to 3.0% in the best and worst case scenarios, respectively. It means clinicians in that hypothetical center would need to monitor P levels in 1,000 cycles and intervene in 50–300 cycles with PE to potentially avoid 2–12 implantation failures by applying the freeze-all strategy. Notably, despite improvements in cryopreservation techniques and an overall favorable outcome with the transfer of frozen-thawed vitrified embryos (42, 50, 51), pregnancy rates reported by individual centers still vary, with an success rate of approximately 50% (52). As a result, the final net effect of PE on pregnancy rates may be offset even further.h A critical aspect to discuss about P thresholds concerns the shortcomings of the currently rapid immunoassays for steroid determination. These assays have been associated with poorly agreeable results due to low assay specificity, poor optimization of methods over the large concentration ranges observed clinically, and inadequate standardization (47, 48). Although direct immu- noassay platforms are fully automated, easy-to-use, inexpensive, and allow rapid detection with a high throughput, there is limited data regarding the performance and precision of these immu- noassay systems, particularly in the lower range of detectable P concentrations (<2.5 ng/ml) (48). For automated immunoassay platforms, intra-assay variability is generally expressed as an aver- aged variability across discrete analyte standards spanning low, middle, and high ranges of P levels. It has been argued that these systems should not be used clinically for low detection limits such as measuring P with values corresponding to ~1 ng/ml. Due to the non-uniform sensitivity of P measurement assays, especially in the low range of P, thus, caution should be applied to adopt any P threshold level for managing purposes, such as the recom- mendation of freezing all embryos in cycles with P elevation on day of hCG administration based on a single measurement and using specific low range P cutoff points (49). ff Thus, fresh embryo transfers in IVF/ICSI cycles with PE should not be disregarded, particularly when supranumerary embryos will make cryopreservation inevitable. Which P Thresholds Are Best to Identify Patients at Risk of Implantation Failure due to PE in a Fresh Embryo Transfer?f assessed the effect Frontiers in Endocrinology \| www.frontiersin.org April 2018 \| Volume 9 \| Article 201 6 PE and Pregnancy Outcomes Esteves et al. of the duration of preovulatory PE on pregnancy rates (15). Persistence of PE for 2 days or more was significantly associated with a reduction in the CPR (39.4 versus 20.7%; p < 0.001). The overall incidence of PE was 28.4% (n = 173 of 610). Among them, 83.2% (n = 144) had 1 day of PE, 12.7% (n = 22) had 2 days, and 4.1% (n = 7) had 3 days of PE. The serum P con- centration on the LH surge day in those women without PE was 3.2 ± 1.0 nmol/l (0.9–4.9) [mean ± SD (range)], whereas in those with PE it was 6.4 ± 1.7 nmol/l (5.0–15.3); the serum E2 concentration was 1,082 ± 329 pmol/l (376–2,222) and 1,045 ± 428 pmol/l (440–2,649), respectively. There were no significant differences in both clinical and ongoing PR (39.0 versus 37.3% and 32.5 versus 31.7%) between those with and without PE on the day of LH surge. The incidence of PE in frozen-thawed embryo transfers in subsequent natural cycles (FET-NC) was not significantly different than that in stimulated cycles (15). In another study, Lai et al. evaluated the relationship between serum levels of P and estradiol (P:E ratio) measured on the day of hCG administration on ART clinical outcomes of 139 infertile women with normal ovarian reserve treated with a long GnRH-a protocol. The authors showed that P:E ratio was significantly higher among patients with premature luteinization (PL, n = 41), defined by a P:E ≥ 1.2 using receiver operator characteristic analysis, than in non-PL (n = 98) group (2.4 ± 1.5 and 0.6 ± 0.3, respectively), but P:E ratio did not correlate with pregnancy outcomes after a fresh embryo transfer (46). of the duration of preovulatory PE on pregnancy rates (15). Persistence of PE for 2 days or more was significantly associated with a reduction in the CPR (39.4 versus 20.7%; p < 0.001). The overall incidence of PE was 28.4% (n = 173 of 610). Among them, 83.2% (n = 144) had 1 day of PE, 12.7% (n = 22) had 2 days, and 4.1% (n = 7) had 3 days of PE. Frontiers in Endocrinology \| www.frontiersin.org April 2018 \| Volume 9 \| Article 201 Which P Thresholds Are Best to Identify Patients at Risk of Implantation Failure due to PE in a Fresh Embryo Transfer?f Furthermore, recent evi- dence indicates that individualization of the luteal phase support by addition of LH activity to progesterone supplementation for fresh ET can overcome the luteal phase deficiency in infertile patients submitted to IVF/ICSI cycles with GnRH antagonist cotreatment who underwent fresh embryo transfer. There are still concerns that the adoption of a universal segmentation policy Table 3 \| Different scenarios to evaluate net effect of PE on pregnancy for an IVF center performing 1,000 cycles per year. Cycles Scenario 1 Scenario 2 Scenario 3 Cycles with PE (%) 5 15 30 Cycles with PEa (N) 50 150 300 Expected pregnancies in the subgroup of PEb (N) 20 60 120 Achieved pregnancies corrected by APRRc (N) 18 54 108 Overall pregnancy reduction per 1,000 cycles; N (%) 2 (0.5%)d 6 (1.5%)d 12 (3.0%)d aPer 1,000 cycles. bConsidering 40% as the overall PR per fresh embryo transfer. cConsidering 10% absolute pregnancy rate reduction (APRR). d400 pregnancies would be expected overall per 1,000 cycles. Table 3 \| Different scenarios to evaluate net effect of PE on pregnancy for an IVF center performing 1,000 cycles per year. if Collectively, these observations indicate that there is still no clarity on the cutoff value of P above which it negatively affects pregnancy success in fresh embryo transfer IVF/ICSI cycles. Alternative approaches including the determination of PE dura- tion and measurement of P to follicle index ratio (PF) may prove useful to assess the role of follicular phase PE on pregnancy success, but further research is needed to confirm their clinical utility. April 2018 \| Volume 9 \| Article 201 7 PE and Pregnancy Outcomes Esteves et al. for PE might compromise the overall health of the mother and generated offspring (53). There have been reports of increased incidence of placenta accreta, preeclampsia, macrosomia, and large for gestational age as a result of the transfer of frozen-thawed embryos compared to fresh counterparts (54). Along the same lines, adoption of a freeze-all policy would require additional embryo manipulation that might induce epigenetic changes, and thus further add risk to children born from frozen-thawed embryo transfer cycles (55–61). counterparts and the possibility of small dose adjustments using the former, individualized dose adaptation can be used during COS to modulate the GCs progesterone output. FUTURE ASPECTS An analysis of the freeze all policy adopted in all the cycles with PE on the day of hCG, taking into account cost-effectiveness, patient-centeredness, and time to live-birth has to be carried out to better assess the clinical validity of such additional interventions. Furthermore, development of robust assays to measure P at low limit levels is essential to determine PE cutoff levels with clinical impact. Better identification of the patient profile at higher risk of implantation failure due to PE on the late follicular phase and the optimal algorithm for PE determination is of utmost importance for clinicians providing care to infertile couples undergoing ART. FUNDING The panel of ART experts was organized by Merck, India, repre- sented by PD. The data were assembled, analyzed, and interpreted by a panel of Indian experts in ART. These activities were organ- ized and funded by Merck, India, an affiliate of Merck KGaA, Darmstadt, Germany. Conflicting evidence still prevails concerning the effect of P eleva- tion on the day of hCG trigger in fresh embryo transfer IVF/ICSI pregnancy outcomes. Clinicians should exercise caution to adopt cutoff values of P in the clinical management of patients with PE due to limitations of exiting assays to measure progesterone at the lower levels and the lack of unequivocal evidence indicating a negative effect of PE on pregnancy success in fresh embryo transfer IVF/ICSI cycles. Gonadotropin type and regimen, as routinely used during COS, seems to have an eligible effect on late follicular phase progesterone levels. However, given the higher potency of recombinant gonadotropin preparations than urinary AUTHORS CONTRIBUTIONS GK, SG, DR, SD, KI, MP, JS, KM, and KM were involved in critical analysis of data and provided expert comments and amendments for formulation of the manuscript. PT participated in its design and coordination, primarily searched the literature, and helped to draft the manuscript. PD organized the meetings and helped to formulate the study questions, participated in its design and coor- dination, primarily searched the literature, and helped to draft the manuscript. SE designed the review, helped in coordination and literature search, provided critical analysis of data, and drafted the manuscript. All authors approved its final version. Which P Thresholds Are Best to Identify Patients at Risk of Implantation Failure due to PE in a Fresh Embryo Transfer?f A potential adverse impact of PE on pregnancy success does not seem to affect the overall patient population equally, with high responders to COS being less susceptible to the possible detrimental effect of PE. An individualized approach should be used in cases of PE, which could include fresh embryo transfers in hyper-responders with low risk of OHSS and in patients with supranumerary embryos undergoing blastocyst transfer. In normal responders with PE undergoing day 3 fresh embryo transfers, a “freeze-all” strategy might be considered. As for poor responders, the optimal strategy in the face of PE is yet to be determined. In conclusion, the current limitations of progesterone assays, the conflicting data on P thresholds, and the equivocal clinical implications of PE to pregnancy outcomes should prompt clini- cians to critically evaluate their dataset and practices to determine the usefulness of routine measurement of progesterone levels on the day of hCG trigger and adopting a freeze-all strategy to all cycles of PE. At present, the indiscriminate adoption of 1.5 ng/ml cut off to adopt an embryo freeze-all strategy is not evidence- based. Moreover, the net effect of PE on pregnancy success of ART units is likely minimal. ACKNOWLEDGMENTS Dr. Shivali Arora of Knowledge Isotopes Pvt. Ltd. (http://www. knowledgeisotopes.com/) for the medical writing support funded by Merck India, an affiliate of Merck KGaA, Darmstadt, Germany. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fendo.2018.00201/ full#supplementary-material. 4. Payne AH, Hales DB. Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr Rev (2004) 25(6):947–70. doi:10.1210/er.2003-0030 5. Seger R, Hanoch T, Rosenberg R, Dantes A, Merz WE, Strauss JF III, et al. The ERK signaling cascade inhibits gonadotropin-stimulated steroidogenesis. J Biol Chem (2001) 276(17):13957–64. doi:10.1074/jbc.M006852200 6. Leao Rde B, Esteves SC. Gonadotropin therapy in assisted reproduction: an evolutionary perspective from biologics to biotech. Clinics (Sao Paulo) (2014) 69(4):279–93. doi:10.6061/clinics/2014(04)10 7. Czyzyk A, Podfigurna A, Genazzani AR, Meczekalski B. 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Perinatal morbidity after in vitro fertilization is lower with frozen embryo transfer. Fertil Steril (2011) 95(2):548–53. doi:10.1016/j.fertnstert.2010. 05.049 The reviewer AC and handling Editor declared their shared affiliation. Copyright © 2018 Esteves, Khastgir, Shah, Murdia, Gupta, Rao, Dash, Ingale, Patil, Moideen, Thakor and Dewda. 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. 52. Li R, Qiao J, Wang L, Zhen X, Lu Y. Serum progesterone concentration on day of HCG administration and IVF outcome. Reprod Biomed Online (2008) 16(5):627–31. doi:10.1016/S1472-6483(10)60475-0 53. 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https://openalex.org/W2765867177	https://bmcmusculoskeletdisord.biomedcentral.com/track/pdf/10.1186/s12891-017-1777-9	English	null	Prognostic factors for recovery following acute lateral ankle ligament sprain: a systematic review	BMC musculoskeletal disorders	2,017	cc-by	10,782	* Correspondence: jacqueline.thompson@ndorms.ox.ac.uk 1Nuffield Department of Orthopaedics Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK Full list of author information is available at the end of the article Prognostic factors for recovery following acute lateral ankle ligament sprain: a systematic review Jacqueline Yewande Thompson1*, Christopher Byrne1,2, Mark A. Williams3, David J. Keene1, Micheal Maia Schlussel1 and Sarah E. Lamb1 © Queen's Printer and Controller of HMSO. 2017 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. Thompson et al. BMC Musculoskeletal Disorders (2017) 18:421 DOI 10.1186/s12891-017-1777-9 Thompson et al. BMC Musculoskeletal Disorders (2017) 18:421 DOI 10.1186/s12891-017-1777-9 Methods This systematic review is reported according to PRISMA guidelines [15] and details of the protocol were registered on PROSPERO and can be accessed at https.//www.crd.york.a- c.uk/PROSPERO/display_record.asp?ID=CRD42014014471. Electronic searches were performed from inception to September 2016 in AMED, EMBASE, and Psych Info via Ovid; CINAHL and SportDiscus (EBSCOHost); PubMed, and the Cochrane Register of Clinical Trials using the National Institutes of Health Medical Subject Headings where appropriate. In addition, search strings of health condition or body region were used in the Physiotherapy Evidence Database, International Foot and Ankle Biomechanics, International Ankle Symposium, and Open Grey. No language restrictions were applied in the searches. The bibliographies of all full-text articles included for data extraction were screened for further eligible articles. Details of the search strategy are avail- able in Additional file 1: Appendix A. p p A key feature of acute lateral ankle ligament sprain (ankle sprain) is that about one-third of injured individuals will experience long-term residual symptoms [5–7]. For example, in an observational study of 648 individuals with an ankle sprain, 32% reported chronic complaints of pain, swelling, or recurrent sprains at 7 years [8]. Similarly, 30% of individuals at 2.5 to 5 years post ankle sprain reported pain on activity [9] with one study reporting that 74% of individuals exhibited at least one residual symptom of either pain, swelling, weakness, or instability 1 to 4 years after an ankle sprain [10]. Furthermore, there is evidence to suggest that these long-term residual impairments of the ankle influence an individual’s level of functioning dur- ing sporting activities and activities of daily living [6, 8]. Articles were included in this review if they met the following eligibility criteria. (1) The study sample or a sep- arately analysed sub-group had a clinical diagnosis of acute (≤7 days) lateral ankle ligament sprain assembled within 7 days of injury; (2) the study had a prospective or retrospective longitudinal design, with at least one follow- up time point and; (3) the study presented data on the effect of at least one baseline prognostic factor on recov- ery outcomes which are collected at presentation. Studies that included patients with ankle fracture (excluding flake fracture <2 mm), or other recent (< 3 months) lower limb injuries and presented results using descriptive or correc- tional statistics alone were excluded. Background this review was to systematically review and identify evidence of prognostic factors associated with poor recov- ery following acute lateral ankle ligament sprain. g Ankle sprains account for the majority of ankle injuries and therefore represent one of the most common musculoskel- etal injuries. The incidence rate in the United States general population is 2.15 per 1000 person-years, with sporting activity accounting for half of all injuries [1]. In the Netherlands, an incidence rate of 37.5 and 17.5 per 1000 person-years during sporting activities and activities of daily living respectively was reported over a 10-25 year period [2]. It is estimated that ankle sprains account for up to 1.5 million visits to UK emergency departments each year [3]. A recent systematic review and meta-analysis of the ankle sprain literature, estimated an incidence rate of 11.6 per 1000 exposures and a prevalence of 11.9% [4]. Abstract Background: One-third of individuals who sustain an acute lateral ankle ligament sprain suffer significant disability due to pain, functional instability, mechanical instability or recurrent sprain after recovery plateaus at 1 to 5 years post injury. The identification of early prognostic factors associated with poor recovery may provide an opportunity for early-targeted intervention and improve outcome. Methods: We performed a comprehensive search of AMED, EMBASE, Psych Info, CINAHL, SportDiscus, PubMed, CENTRAL, PEDro, OpenGrey, abstracts and conference proceedings from inception to September 2016. Prospective studies investigating the association between baseline prognostic factors and recovery over time were included. Two independent assessors performed the study selection, data extraction and quality assessment of the studies. A narrative synthesis is presented due to inability to meta-analyse results due to clinical and statistical heterogeneity. Results: The search strategy yielded 3396 titles/abstracts after duplicates were removed. Thirty-six full text articles were then assessed, nine of which met the study inclusion criteria. Six were prospective cohorts, and three were secondary analyses of randomised controlled trials. Results are presented for nine studies that presented baseline prognostic factors for recovery after an acute ankle sprain. Age, female gender, swelling, restricted range of motion, limited weight bearing ability, pain (at the medial joint line and on weight-bearing dorsi-flexion at 4 weeks, and pain at rest at 3 months), higher injury severity rating, palpation/stress score, non-inversion mechanism injury, lower self-reported recovery, re-sprain within 3 months, MRI determined number of sprained ligaments, severity and bone bruise were found to be independent predictors of poor recovery. Age was one prognostic factor that demonstrated a consistent association with outcome in three studies, however cautious interpretation is advised. Conclusions: The associations between prognostic factors and poor recovery after an acute lateral ankle sprain are largely inconclusive. At present, there is insufficient evidence to recommend any factor as an independent predictor of outcome. There is a need for well-conducted prospective cohort studies with adequate sample size and long-term follow-up to provide robust evidence on prognostic factors of recovery following an acute lateral ankle sprain. Trial registration: Prospero registration: CRD42014014471 Keywords: Acute lateral ankle sprain, Prognostic factors, Recovery, Epidemiology, Systematic review Page 2 of 14 Thompson et al. BMC Musculoskeletal Disorders (2017) 18:421 Methods for each of the six domains as low, moderate, or high based on three-to-seven sub-item reporting prompts which were rated as “yes”, “no”, “partial” or “unsure” [17]. A consensus meeting followed during which the two assessors reached agreement upon judgements for each of the six domains and an overall risk of bias for each study. See details of the QUIPS assessment process here. for each of the six domains as low, moderate, or high based on three-to-seven sub-item reporting prompts which were rated as “yes”, “no”, “partial” or “unsure” [17]. A consensus meeting followed during which the two assessors reached agreement upon judgements for each of the six domains and an overall risk of bias for each study. See details of the QUIPS assessment process here. We employed the Quality In Prognosis Studies (QUIPS) tool to assess the risk of bias in the included articles [17]. The QUIPS tool considers six important domains affecting validity and risk of bias in studies of prognostic factors. 1) Study participation, 2) study attri- tion, 3) prognostic factor measurement, 4) confounding measurement and 5) outcome measurement, and 6) analysis and reporting [17]. The first domain - Study Participation addresses the representativeness of the study sample, i.e. whether the studies reported associa- tions that are valid estimates of the true relationship between the prognostic factor and the outcome of inter- est in the source population. Here we considered the information provided on the baseline characteristics of study participants to evaluate the risk of selection bias. The second domain - Study Attrition addresses whether participants with follow-up data represent persons enrolled in the study i.e. whether the reported associ- ation between the prognostic factor and outcome was biased by the assessment of outcomes in a selected group of participants who completed the study. We sought after reasons for loss to follow-up, and attempts to restrict attrition to ≤20% and reduce the risk of sys- tematic differences in the associations reported. The third and fourth domains respectively, were Prognostic Factor Measurement and Outcome Measurement. These domains address the adequacy of prognostic factor and outcome measurement, i.e. whether the study measured the prognostic factor or outcome in a similar, valid, and reliable way for all participants. In this domain, we sought for similarities in the methods and settings used to reduce mis-classification bias. Results Figure 1 illustrates the PRISMA flow diagram for this systematic review. The search strategy identified 4173 reports with eight reports identified from additional sources. After removing the duplicate records, the title and abstract of 3396 reports were screened for eligibility. For 3360 reports, the title or abstract clearly indicated that the topic of the report was not relevant to the topic of this review or the reports did not meet our inclusion criteria. The remaining 36 reports were assessed for eligibility as full-text articles. Twenty-seven full-text arti- cles were excluded because they did not employ multi- variate prognostic analyses such as linear or logistic regression (n = 13) [10, 18–27]; used a cohort assembled >7 days after injury (n = 4) [28–31]; used outcome mea- sures that did not meet the study eligibility criteria (n = 6) [32–37]; were conference abstracts (n = 2) [38, 39] or dissertation (n = 1) [40] of full texts already included in the review; or represented participants with ankle syndes- mosis injury (n = 1) [41]. Nine studies from nine cohorts were included in the review [42–50]. The fifth domain, Study Confounding addresses poten- tial confounding factors, i.e. whether another factor may explain the reported association. At this point, we sought after a clear definition of important potential confounding variables a prior, similarities in their measurements and appropriate adjustment for these factors in the analysis. Finally, the sixth domain, Statistical Analysis and Report- ing address the appropriateness of the study’s statistical analysis and completeness of reporting i.e. whether results are likely to be spurious or biased because of poor analyt- ical strategies or reporting standards [17]. As a part of the assessment of the adequacy of the approach used in the analysis, we paid particular attention to strategies used to develop the model. These include investigations to check that key assumptions were met, interaction tests that assess the correlation between factors, and performance measures for model diagnosis. For example, we rated studies down when reports on multi-collinearity were not performed or explicit in the study results. For each study, two independent assessors (CB, JT) judged the risk of bias Table 1 illustrates the key characteristics of the included studies. Six of the nine studies employed a pro- spective cohort design, whereas three studies [45, 47, 49] represented retrospective analyses of three randomised controlled trials [11–13]. Studies were conducted in five countries. Methods We present a narrative synthesis of prognostic factors that have demonstrated a statistically significant relation- ship with recovery outcomes following acute lateral ankle ligament sprains. We defined the quality of the evidence using set criteria. Prognostic factors were clas- sified as demonstrating strong evidence when consistent findings were identified from at least two high quality articles using different cohorts. For moderate evidence, consistent findings were sought from at least two adequate quality studies using different cohorts. Limited evidence was classified as findings identified in one adequate quality article or at least two low quality articles from different cohorts. Finally, inconclusive evidence was defined as inconsistent findings from one low quality cohort alone or insufficient research. Methods The combination of a high volume injury with poor prognosis in one-third of injuries, suggests that being able to predict those individuals with expected poor recovery would be of considerable value to injured indi- viduals and healthcare providers. However, prognostic factors associated with chronic residual symptoms from acute lateral ankle ligament sprains are poorly under- stood [7]. Understanding prognostic factors for poor recovery following an ankle sprain could help clinicians identify patients with poor prognosis and direct the provision of targeted treatment. Conversely, identifying those patients with good prognosis could have benefits for health care cost and resource use as the most effect- ive treatment for this population is unknown. The title and abstract of all records identified by the search strategy were screened by two reviewers (CB, JT) applying the eligibility criteria. A third reviewer (MW) screened 10% of the total identified records. We used the Rayyan systematic review web application during the screening process [16]. Full-text articles of all records eligible for inclusion were independently reviewed by the two reviewers (CB, JT) applying the eligibility criteria and screening for duplication. Any discrepancies between the two independent reviewers regarding eligibility were resolved by consensus or consultation with a third member of the review team (MS or MW). We also made attempts to contact the original authors via electronic mail when supplementary information was required to improve clarity. For all articles eligible for inclusion, both reviewers (CB, JT) independently completed a full data extraction form and a risk of bias assessment form. Following this, the two reviewers met to cross-validate data extraction Conventional management of ankle sprains which initially begin with instructions to protect and rest the joint, and reduce swelling, and progress to early mobilisa- tion with external support and exercises, has been shown to be beneficial [11–14]. However, studies investigating the addition of a supervised programme of physiotherapy to conventional care found no important clinical differ- ence in outcomes of recovery [13, 14]. Research into prog- nostic factors of recovery could enable patients on a good recovery trajectory to be distinguished from those who are likely to experience difficulties and better target monitor- ing and interventions after injury. Therefore, the aim of Thompson et al. BMC Musculoskeletal Disorders (2017) 18:421 Page 3 of 14 Page 3 of 14 Page 3 of 14 forms for discrepancies and to reach consensus on risk of bias assessment. Results The Netherlands (n = 3), USA (n = 3), England (n = 1), Germany (n = 1), and Northern Ireland (n = 1). Five studies employed a single site for recruitment. Settings included school or university sports medicine clinics, hos- pital emergency departments, primary care Physiotherapists Thompson et al. BMC Musculoskeletal Disorders (2017) 18:421 Page 4 of 14 Fig. 1 PRISMA flow diagram for the systematic review of prognostic factors for outcome following acute lateral ankle ligament sprain Fig. 1 PRISMA flow diagram for the systematic review of prognostic factors for outcome following acute lateral ankle ligament sprain and General Practitioners. A total of 1047 participants, with a median sample size of 33 (range 20-553), provided follow-up data over a time frame ranging from 1 day to 12 months across the studies. Three studies recruited high school or university athletes whereas the remainder recruited from the general population presenting to primary or secondary care. A single large-scale multi- centre randomised controlled trial [12], that recruited participants from eight emergency departments in hospi- tals across England and demonstrated low risk of bias, accounted for over 50% of the total participants in this review [45]. This study presented only two prognostic factors of recovery – age and female gender. studies. The main discrepancies we identified were related to the use of poor statistical methods or poor reporting standards. For example, no study reported performing a collinearity diagnostics to check for multi- collinearity between the prognostic factors presented in the final models. In addition, none of the studies included in the review explored or reported results for the performance of their models (measures of interval validity or external validation). The regression analyses employed were not reported in sufficient detail to iden- tify whether prognostic factors were eliminated due to low statistical power or poor clinical utility. The high loss to follow-up identified in two studies [46, 48] is a pointer to the risk of selection bias that may have been due to the method of recruitment employed. However, these studies did not provide information on the comparisons between participants who completed and who did not complete the final follow-up. Conse- quently, the profile of the participants lost to follow-up cannot be accurately evaluated. This further highlights the poor reporting standards employed by the studies included in the review. Figure 2 illustrates the risk of bias assessment for the nine included studies. Results See online supplementary informa- tion (Additional file 2: Appendix B) for further details. Studies were judged particularly poorly on the risk of bias domains of Study Attrition, Study Confounding, and Stat- istical Analysis and Reporting. One study was classified as having an overall low risk of bias [45], five having an over- all moderate risk of bias [42, 44, 47, 49, 50], and three studies as having an overall high risk of bias [43, 46, 48]. Fig. 2 presents the risk of bias ratings for the prognostic factors identified each time it was explored in a study. The overall quality of evidence derived was mainly from eight studies with high-to-moderate risk of bias (n = 8) and one study with low risk of bias (n = 1). Meta-analysis was inappropriate due to the heteroge- neous nature of prognostic factors, recovery outcome measures, follow-up durations, and the limited number of included studies. Prognostic factors were categorised according to the duration of follow-up employed in the study and grouped as relevant to short term (≤8 weeks), medium-term (≤4 months), and long-term (>4 months) Most of the studies rated poorly due to incomplete and/or inadequate reporting standards within individual Page 5 of 14 Thompson et al. BMC Musculoskeletal Disorders (2017) 18:421 Table 1 Key characteristics of included studies Study Design Setting Sample size Sample characteristics Time since injury Injury severity Follow-up de Bie et al. [42] Prospective cohort The Netherlands 1 x Hospital FAD N = 35 at baseline N = 33 at 2 weeks N = 31 at 4 weeks General population N = 22 M, 13 F 28 ± 10 (13-59) y NR NR 2 weeks 4 weeks Wilson & Gansneder [43] Prospective cohort USA 1 x University N = 24 at baseline N = 21 at follow-up Athletes N = 13 M, 8 F 20 ± 2 y 67.8 ± 15.2 h Grade I, II 11.9 ± 6.6 days Cross et al. [44] Prospective cohort USA 1 x University N = 20 at baseline N = 20 at follow-up Athletes N = 7 M, 13 F 19 ± 1 (18-21) y ≤24 h NR 14.7 ± 8.8 (3–40) days Akacha et al. Results BMC Musculoskeletal Disorders (2017) 18:421 Page 6 of 14 Source Study Participation Study Attrition Prognostic Factor Measurement Outcome Measurement Study Confounding Statistical Analysis & Reporting Overall Risk of Bias de Bie et al.[1] Wilson & Gansneder[2] Cross et al.[3] Akacha et al.[4] Langner et al.[5] van Middlekoop et al.[6] van der Wees et al.[7] O’Connor et al.[8] Medina McKeon et al.[9] Key: Risk of bias Low Moderate High Fig. 2 Risk of bias assessment of the nine included studies according to the Quality in Prognosis Studies (QUIPS) tool [10] ig. 2 Risk of bias assessment of the nine included studies according to the Quality in Prognosis Studies (QUIPS) tool [10] score and self-reported current athletic ability rating) as prognostic factors for greater disability duration with 33% of the variance explained [43]. The combination of impair- ment and functional limitation prognostic factors pro- duced an additive effect and explained 59% of the variance in disability duration [43]. Cross et al. [44] reported the baseline prognostic factors of lower self-reported physical function (R2 = .28), lower self-reported global function (R2 = .22), and lower objectively measured ambulation status (R2 = .27) as being associated with a greater num- ber of days to return to sport (i.e. 14.7 ± 8.8 days). When combined into a multiple regression model, the three prognostic factors explained 37% of the variance in num- ber of days to return-to-sport [44]. recovery. No factor demonstrated strong evidence of an association with recovery. Prognostic factors for short-term recovery (≤8 weeks) Five of the nine included studies reported data on prog- nostic factors for short-term recovery [42–44, 48, 49]. Table 2 summarises the analytical approach and prog- nostic factors identified from these studies. de Bie et al. [42] reported that baseline Ankle Function Score (AFS) ≤35 was a prognostic factor for non-recovery at 2 weeks with a sensitivity and specificity of 97% and 100%, respectively. Non-recovery at 4 weeks was predicted by the combination of three baseline prognostic factors (i.e. AFS ≤35, higher 0-10 severity grading by a doctor, and higher palpation / ligament stress test score) with a sen- sitivity and specificity of 81% and 80%, respectively [42]. Similarly, van der Wees et al. [48] using only patients with baseline measurements ≤5 days after injury, reported that baseline AFS ≤40 was a prognostic factor for non-recovery at 2 weeks with a sensitivity and speci- ficity of 76% and 63%, respectively. Results [45] Retrospective cohort England 8 x Hospital ED N = 584 at baseline N = 553 at 4 weeks, 12 weeks, & 9 months General population N = 321 M, 232 F 30 ± 11 (16-72) y ≤7 days Severe (NWB at 3 days) 4 weeks 12 weeks 9 months Langner et al. [46] Prospective cohort Germany 1 x Hospital ED N = 38 at baseline N = 26 at 6 months N = NR at 12 months General population N = 18 M, 20 F 38 ± 13 (20-75) y < 24 h ATFL Grade I (27%), II (27%), III (46%) 6 months 12 months van Middelkoop et al. [47] Retrospective cohort The Netherlands 32 x GP 1 x Hospital ED N = 102 at baseline N = 95 at 3 months N = 80 at 12 months General population N = 59 M, 43 F 37 ± 12 (18-60) y ≤7 days Mild (42%), moderate or severe (44%), unknown (14%) 3 months 12 months van der Wees et al. [48] Prospective cohort The Netherlands 20 x Primary care Physiotherapists N = 107 at baseline N = 33 at 2 weeks General population N = 65 M, 42 F 32 ± 14 y 8.7 ± 8.9 days ≤5 days N = 53 > 5 days N = 54 Light (50%), severe (50%) 2 weeks O’Connor et al. [49] Retrospective cohort Northern Ireland 1 x Hospital ED 1 x University sports injury clinic N = 101 at baseline N = NR at 4 weeks N = 85 at 4 months General population, athletes. N = 69 M, 31 F 27 ± 10 (16-58) y < 7 days 40 ± 36 h Grade I (26%), II (63%), II+ (11%) 4 weeks 4 months McKeon et al. [50] Prospective cohort USA 7 x High schools N = 204 sprains at baseline N = 198 sprains in analysis High school athletes ≤24 h Time to return to play. Same day (23.7%), next day (21.2%), 3 days (29.3%), 7 days (11.6%), 10 days (8.6%), >22 days (5.6%) Time to return to play. Same day, next day, 3 days, 7 days, 10 days, 21 days, >22 days. Abbreviations: FAD first aid department; N number; M males; F females; y years; NR not reported, NWB non-weight bearing status; ED emergency department; ATFL anterior talofibular ligament; GP general practice primary care Thompson et al. Results Wilson & Gansneder [43] reported that greater impairment (i.e. greater range of motion loss and greater swelling) were prognostic fac- tors for a longer disability duration (i.e. 11.9 ± 6.6 days) with 34% of the variance in disability duration explained by their combination in a regression model. They also reported that greater functional limitation (i.e. lower scores on an objective six-item weight-bearing activity Prognostic factors for short-term recovery (≤8 weeks) O’Connor et al. [49] reported that lower subjective ankle function at 4 weeks was significantly associated with the baseline prognostic factors of greater age (β = −. 32), more severe injury grade (β = −.23), and poorer weight bearing status (β = −.34). When combined in a stepwise multivariate regression model, the prog- nostic factors explained 34% of the variance in subjective ankle function at 4 weeks [49]. Finally, Medina McKeon et al. [50] reported that recurrent ankle sprain was not a prognostic factor in explaining time to return-to-play. They reported no significant difference in Kaplan-Meier time to return-to-play curves for new (median = 3 days, inter-quartile ranges = same day to 7-day return) and recurrent (median = next day, inter-quartile range = next Page 7 of 14 Thompson et al. BMC Musculoskeletal Disorders (2017) 18:421 e 2 Prognostic factors for short-term (≤8 weeks) outcome in acute lateral ankle sprain Primary outcome measure Independent variables Analysis Prognostic factors for short-term outcome e et al. [42] Healed or not healed at 2 & 4 weeks. Healed = AFS >75 (0-100) & palpation/ ligament stress test score < 2 (0-12). AFS (0-100) ≤35, doctor severity grading (0-10), palpation/ligament stress test score (0-12). Multivariate logistic regression 2 weeks: Baseline AFS ≤35 predicted recovery status. Sensitivity = 97%, specificity = 100%. 4 weeks: Combined baseline AFS ≤35, severity grading, & palpation/ ligament stress test score predicted recovery status. Sensitivity = 81%, specificity = 80%. n & Gansneder [43] Number of days to return to full sports practice or competition (11.9 ± 6.6 days). Joint swelling (ml), sagittal plane ROM loss (°), objective WB activity score (0-6), self-reported athletic ability score (VAS, 0-100). Hierarchical regression Combined swelling (β = −.02) & ROM loss (β = −.08). R2 = .34, p = .023. Combined WB activity score (β = −.55) & self-reported ability score (β = −.39). R2 = .33, p = .004. Combined swelling, ROM loss, WB activity score, & self-reported athletic ability score. Adjusted R2 = .59; p = .001. et al. [44] Number of days to return to sport (14.7 ± 8.8 days). SF36PF (0-100), Self-reported global function (0-100%), objective ambulation status (1-7). Univariate regression, stepwise multivariate regression. SF36PF. R2 = .28, p = .016. Self-reported global function. R2 = .22, p = .036. Objective ambulation status. R2 = .22, p = .019. Combined SF36PF, self-reported global function, & objective ambulation status. Discussion This systematic review provides a summary of prognos- tic factors of recovery after an acute ankle sprain. Nine- teen prognostic factors demonstrated an association with outcome in the final multivariate models presented across the included studies. These measures are mostly acknowledged in the routine management of ankle sprains (See Table 5). At short-term follow-up, we found consistent findings from at least two studies with moderate risk of bias, for weight-bearing status and injury grade, indicative of moderate evidence. There was limited evidence for age, pain reproduced by ligament stress test, and the patient reported measures of levels of physical activity. The evidence for swelling, restricted joint range of motion, and self-report athletic ability was inconclusive, due to insufficient findings from two studies with a high risk of bias. This seems to suggest that the severity of the injury and objective assessment of ability to weight-bear dem- onstrate some degree of accuracy in predicting return to pre-injury functional status. At medium term follow-up, pain, weight-bearing, mechanism of injury and functional activity score were identified as prognostic indicators of recovery; demon- strating limited evidence from only one study with moderate risk of bias. Similarly, one study [24] included in a review [7] reported high levels of athletic competi- tion, defined as ≥3 times of training per week, as a prog- nostic factor for poor recovery. However, that study [24] did not adjust for other important prognostic factors or confounding variables such as previous injury. Prognostic factors for long-term recovery (>4 months) Table 4 summarises three studies that reported data on prognostic factors for long-term recovery [45–47]. Akacha et al. [45] employed non-linear mixed modelling to re-- analyse data from a large scale RCT [12]. They demon- strated that higher age and female gender were prognostic factors for slower and incomplete recovery [45]. For example, the predicted time to attain a FAOS-S (0-100) score of 65 for 21-year-old male and female participants receiving below knee cast treatment was 2.9 (95% CI. 2.4 to 3.4) and 3.9 (95% CI. 3.0 to 4.7) weeks, respectively [45]. In contrast, 66-year-old male and female participants receiving the same treatment were predicted to attain a score of 65 in 8.3 (95% CI. 4.2 to 12.5) and 17.1 (95% CI. 4.7 to 29.5) weeks, respectively [45]. At 12 months follow-up, Langner et al. Prognostic factors for short-term recovery (≤8 weeks) Adjusted R2 = .34, p < .01. er Wees et al. [48] Global perceived effect ≥2 (1 = recovered, 2-7 = not recovered) at 2 weeks. AFS (0-100) ≤40. Sensitivity & specificity using sample with ≤5 days duration of injury (n = 53) 2 weeks: Baseline AFS ≤40 predicted recovery status. Sensitivity = 76%, specificity = 63%. nnor et al. [49] Karlsson function score (0-100) at 4 weeks. Age (years), injury grade (1, 2, 2+), WB status (FWB, FWB with pain, PWB, NWB). Univariate regression, step-wise multivariate regression. 4 weeks: Combined age (β = −.32, p = .001), injury grade (β = −.23, p = .003), & WB status (β = −.34, p = .038). Adjusted R2 = .34, p < .01. viations: AFS ankle function score; ° degrees; VAS visual analogue scale; R2 the coefficient of determination; β standardised beta; ROM range of motion; WB weight-bearing; SF36PF short form-36 physical function FWB full weight-bearing status; PWB partial weight-bearing status; NWB non-weight-bearing status Thompson et al. BMC Musculoskeletal Disorders (2017) 18:421 Page 8 of 14 Page 8 of 14 day to 7-day return) ankle sprains [50]. Only two [44, 49] studies explored univariate correlations between variables included in the model. However, overall, measures of functional ability explained larger part of the variance of recovery compared with measures of symptoms of clinical severity alone. of their potential prognostic factors measured at base- line were associated with outcome at 12 months follow-up. Further sub-group analysis of 63 non-recovered participants at 3 months revealed that having a re-sprain within 3 months (β = −1.64) and the magnitude of pain at rest at 3 months (β = −.69) were prognostic factors for poorer self-reported recovery at 12 months [47]. Prognostic factors for medium-term recovery (≤4 months) Table 3 shows data reported by one study on prognostic factors for medium term recovery. O’Connor et al. [49] reported that 20% of the variance in subjective ankle function at 4 months was explained by the combined baseline prognostic factors of age, weight bearing status, and injury mechanism. The study participants, who sus- tained a lateral ankle ligament sprain, were classified as hav- ing sustained an injury via an inversion mechanism (70%), or other mechanisms of injury in the analysis [49]. Prognostic factors for short-term recovery (≤8 weeks) Greater age (β = −.26), poorer weight bearing status (β = −.23), and non-inversion injury mechanism (β = −.25) were prognostic factors for poorer subjective function at 4 months follow- up [49]. The authors also identified medial joint line pain on palpation (β = .24) and pain on WB during ankle dorsi- flexion (β = .60) at 4 weeks as prognostic factors for poorer subjective function at 4 months [49]. These two independ- ent variables explained 49% of the variance in subjective ankle function at 4 months [49]. Only a small difference was identified in the magnitude of variance explained by measures of severity versus measures of functional ability at presentation of injury. However, at 4 weeks, the ability to weight bear explained a larger percentage of the vari- ance of the model. Discussion [46] reported that three baseline prognostic factors of more severe MRI grad- ing of injury (R2 = .45), greater number of injured lig- aments determined by MRI (R2 = .35), and presence of a bone bruise determined by MRI (R2 = .32) were associated with greater time to return to sports activ- ities. Van Middelkoop et al. [47] reported that none At long term follow-up, there was limited evidence from one study [45] showing evidence for female gender and age as a prognostic factor for recovery. However, these may be confounded by psycho-social factors such as recovery expectations, coping mechanisms or self- efficacy that have been linked to recovery in musculo- skeletal conditions [51]. Other prognostic factors with insufficient evidence for long-term outcome include injury severity, the number of injured ligaments and the presence of bone bruise as determined by magnetic resonance imaging. The observation of insufficient evidence for radiographic findings and recovery, suggests that structural pathology may not be indicative of clinical presentation. The lack of an association between structural changes in the ankle observed with imaging Page 9 of 14 Thompson et al. BMC Musculoskeletal Disorders (2017) 18:421 Table 3 Prognostic factors for medium-term (≤4 months) outcome in acute lateral ankle sprain Study Primary outcome measure Independent variable(s) Analysis Prognostic factors for medium-term outcome O’Connor et al. [49] Karlsson ankle function scores (0-100) at 4 months. Baseline. Age (years); WB status (FWB, FWB with pain, PWB, NWB); injury mechanism (inversion / other). 4 weeks. Pain on WB ankle DF; medial joint line pain (yes/no). Univariate regression, step-wise multivariate regression. 4 months: baseline combined age (β = −.26, p = .01), WB status (β = −.23, p = .25), & injury mechanism (β = −.25, p = .17). Adjusted R2 = .34, p < .01. 4 months: 4 week combined pain on WB ankle DF (β = .60, p < .001), medial joint line pain (β = .24, p = .07). Adjusted R2 = .49, p < .01. Abbreviations: WB weight-bearing; FWB full weight-bearing status; PWB partial weight-bearing status; NWB non-weight-bearing status; DF dorsiflexion, β, standardised beta; R,2 the coefficient of determination Table 3 Prognostic factors for medium-term (≤4 months) outcome in acute lateral ankle sprain Study Primary outcome measure Independent variable(s) Analysis Prognostic factors for medium-term outcome O’Connor et al. [49] Karlsson ankle function scores (0-100) at 4 months. Baseline. Discussion Age (years); WB status (FWB, FWB with pain, PWB, NWB); injury mechanism (inversion / other). 4 weeks. Pain on WB ankle DF; medial joint line pain (yes/no). Univariate regression, step-wise multivariate regression. 4 months: baseline combined age (β = −.26, p = .01), WB status (β = −.23, p = .25), & injury mechanism (β = −.25, p = .17). Adjusted R2 = .34, p < .01. 4 months: 4 week combined pain on WB ankle DF (β = .60, p < .001), medial joint line pain (β = .24, p = .07). Adjusted R2 = .49, p < .01. Abbreviations: WB weight-bearing; FWB full weight-bearing status; PWB partial weight-bearing status; NWB non-weight-bearing status; DF dorsiflexion, β, standardised beta; R,2 the coefficient of determination Page 10 of 14 Thompson et al. BMC Musculoskeletal Disorders (2017) 18:421 Table 4 Prognostic factors for long-term (> 4 months) outcome in acute lateral ankle sprain Study Primary outcome measure Independent variable(s) Analysis Prognostic factors for long-term outcome Akacha et al. [45] FAOS-S (0-100, 0 = extreme symptoms, 100 = no symptoms). Age, gender. Non-linear mixed model Greater age and female gender associated with slower and incomplete recovery. Greater age (β = −0.01, 95% CI −0.12 to −0.004) Female (β = −0.06, 95% CI −0.01 to −0.002) Langner et al. [46] Time to return to sports activities. MRI grading of ligamentous injury (1-3, 1 = stretching, 2 = partial tear, 3 = complete tear); number of injured ligaments; presence of bone bruise. Multivariate regression MRI grading of ligamentous injury, R2 = .45, p < 0.01. Number of injured ligaments, R2 = .35, p < 0.01. Bone bruise, R2 = .32, p < 0.01. Van Middelkoop et al. [47] Self-reported recovery (NRS, 0-10. 0 = not recovered; 10 = completely recovered) at 12 months. Re-sprain within 3 months; pain at rest at 3 months (NRS, 0-10). Multivariate regression 12 months. Re-sprain within 3 months (β = −1.64, 95% CI −3.11 to −.16); pain at rest at 3 months (β = −.69, 95% CI −1.08 to −.29). Abbreviations: FAOS-S foot and ankle outcome score symptoms subscale; β standardised beta; 95% CI 95% confidence interval; MRI magnetic resonance imaging; R2 the coefficient of determination; NRS numerical rating scale Page 11 of 14 Thompson et al. Discussion BMC Musculoskeletal Disorders (2017) 18:421 Table 5 Summary of number of studies reporting prognostic factors for poor outcome in acute lateral ankle sprain No Follow-up time points/ Baseline prognostic factors explored Number of studies reporting an association (n) 1. At short-term follow-up (≤8 weeks) Age 1 [49] Swelling 1 [43] Reduced range of motion 1 [43] Palpation stress test scores 1 [42] Self-reported physical limitations 1 [44] Self-reported athletic ability 1 [43] Injury severity rating 2 [42, 49] Ankle function score 2 [42, 48] Weight bearing ability / status 3 [42, 44, 49] 2. At medium-term (≤4 months) Age 1 [49] Non-inversion injury 1 [49] Pain (medial joint line) at week 4 1 [49] Pain (on WB DF) at week 4 1 [49] WB status 1 [49] 3. At long-term (>4 months) Age 1 [45] Female gender 1 [45] MRI, severity grading 1 [46] MRI, number of ligaments 1 [46] MRI, bone bruise 1 [46] Pain (at rest) at 3 months 1 [50] Re-sprain within 3 months 1 [50] Abbreviations: WB weight-bearing; DF ankle dorsiflexion; MRI magnetic resonance imaging explaining 1% and female gender 6% of the variance. In contrast, another study [43] that included only 20 partic- ipants reported combined prognostic factors of impair- ment and function that explained 60% of the variance in recovery. Overall, while the included studies in this review do not provide definite evidence of a causal link between the factors identified and recovery, they do highlight the of role biomechanical factors on recovery. Overall, a number of the selected prognostic factors identified, demonstrated some consistency across short, medium and long-term recovery time-points. We defined factors as consistent when it was explored by at least two studies or at two different time points within the same study. Measures of pain [42, 47–49], swelling [42, 43, 48], injury severity [42, 46, 49], weight-bearing status [43, 48, 49] and self-reported functional ability [42–44, 48] showed some degree of consistency, however, the evidence of an association with recovery is equivocal because of the poor quality of individual studies. Evidence for the prognostic value of age was, however, consistent according to results from one study with low risk of bias [45], and another study [49] with moderate risk of bias. Higher baseline age was associated with poor recovery at short [49], medium [49] and long term follow-up time points [45]. Discussion We observed a trend where clinical indicators of symptoms such as swelling, injury severity, or restricted range of motion (ROM) demonstrated a greater prog- nostic ability of recovery at short- and medium term, than at long-term follow-up. This may be useful to inform clinical decision making earlier on in the recov- ery pathway. Measures explored later in the course of recovery, rather than early on, seemed to have a good prognostic value. Examples of these factors include pain at rest, on palpation and on weight bearing, as well as self-reported functional ability. This may suggest that measures of functional ability may be more sensitive at identifying sensory or neuro-muscular deficits in patients experiencing functional or mechanical instabil- ity. Alternatively, this may imply that the timing of the measurements, influences association. Abbreviations: WB weight-bearing; DF ankle dorsiflexion; MRI magnetic resonance imaging techniques and persistent impairment has also been reported by previous research [52]. It seems that diag- nostic classifications may have a poor reliability in predicting recovery at long term. In this review, baseline measures of pain at rest and re-sprain at long-term also showed no association with recovery [47]. This is, however, contrary to reports of an association between recurrent sprains and chronic ankle instability noted by a previous systematic review [53]. To our knowledge, this is the first systematic review of prognostic factors specific to recovery from acute ankle sprains. Overall, results of previous reviews [53, 54] support the findings of our review, and the relevance of these factors to the prediction of recovery in the management of ankle sprains remains conflicting. We observed a substantial amount of clinical and methodological heterogeneity. There were differences in the treatments administered to the study participants, classification of an index ankle sprain (3 studies with inclusion criteria of ≤24 h since injury), injury severity, the duration of follow-up, the measurement instruments employed, and the methodological quality. Furthermore, there was little overlap in the definition of outcome variables Studies with low risk of bias and larger sample sizes tended to report conservative estimates of the associ- ation between variables and recovery. For example, the study by Akacha et al. [45] which included over 500 participants reported a β value - indicating the amount of change in the rate of improvement expected with one unit change in the prognostic factors when other variables are held constant. Discussion The study reported that the maximum achievable score on the foot and ankle outcome score (FAOS) varied over time with greater age Page 12 of 14 Thompson et al. BMC Musculoskeletal Disorders (2017) 18:421 Although we performed a comprehensive search strat- egy to reduce bias in our results, we did not perform hand searching of journals; hence, some studies that, generally, tend to be of poorer methodological quality may have been missed. We evaluated our studies using a robust quality assessment tool – QUIPS that covered all the im- portant criteria for addressing the objectives of prognostic studies, which was pilot-tested to ensure consistency. However, a possible limitation in our approach at this stage was not performing an assessment of the inter-rater reliability for evaluating the quality of the studies. and considerable variation across the potential prognostic factors explored in studies. This made the statistical pooling of the results a difficulty. It is worth noting that a significant proportion of the participants included in this review sustained grade I or II injuries, with considerably shorter duration for return to function. One of the strengths of this review is that we included a homogenous study population of acute lateral ankle ligament injuries, excluding other ligamentous injuries (i.e. peroneal tendon ruptures or high ankle sprain) that predisposes patients to longer recovery trajectory. Furthermore, two- thirds of the sample included in this review were broadly representative of the age range, severity presentations and recreational activity levels of the general population, allowing transferability to most real world acute settings. Most factors identified exhibit a good degree of accessi- bility in clinical practice (See Table 5). The vast majority of the studies included in this review were of a short-term duration when symptoms are still severe and rapidly re- solving, hence recovery at this stage is still quite variable. We identified a shortage of adequate prognostic studies evaluating predictors of recovery after acute ankle sprain at medium- (2-4 months) and long-term (≥4 months). Larger studies with adequate sample size per prognostic factor are also needed. There were considerable differences in the measure- ment of study factors, poorly defined selection procedures for potential prognostic factors, and different outcomes with little or no overlap. For example, injury severity was reported as a prognostic factor associated with recovery, however, two studies used clinical symptoms [42, 49], while a third study [46] used MRI to evaluate grade sever- ity. Discussion This made direct comparisons difficult as previous research has shown poor associations between radio- graphic findings and recovery [53]. Subjective methods increase variability in measurement errors, but objective assessments using MRI are not readily available in acute settings. A number of studies did not use validated outcome measures. For example, two studies [42, 48] used a continuous outcome measure that was dichotomised using an arbitrary cut-off point of ≤35 points to indicate recovery [42] and ≤40 points for a mild ankle injury [48]. There was no pre-specification of this cut-off point from the wider literature; hence, this threshold may not be valid and could have introduced bias. Furthermore, psychosocial and contextual factors such as recovery expectations, coping mechanisms, self efficacy, which have been implicated in recovery from musculoskeletal disorders [56] should be considered in future studies. We suggest that future studies consider the replication and confirmation of existing prognostic factors; exploring measures of internal and external val- idity; and adhere to current recommendations for con- ducting and reporting prognostic studies [57]. This will enable the translation of definitive prognostic factors into clinical practice. Overall, the existing evidence from the studies identified by this review does not allow firm conclusions to be drawn about prognostic factors of recovery from an acute ankle sprain. It has been suggested that a minimum of 10 events may not be required for each prognostic factor consid- ered in a study [55]. However, most of the studies (n = 7) included in our review had too small sample sizes in relation to the number of predictors that were explored and tended to be unreliable. Only one study [45] defined potential confounding factors (a priori) and made suitable adjustments for the treatment group and time since injury in their model. Although the treatments described in studies included in this re- view reflect current practice, most of these were not standardised and the nature of rehabilitation pro- grammes such as neuromuscular training has been found to be correlated with better outcome [51]. Only one study [45] accounted for this confounding variable in their model. Two studies [47, 49] with a cohort from a randomised trial considered the mean effect of treat- ments administered, but did include it in their model be- cause there was no difference between the groups. Conclusions At present, the associations between baseline prognostic factors and recovery are largely inconsistent. Age seems to be an independent prognostic factor identified in three studies with consistent evidence for predicting recovery in patients with acute ankle sprain. However, we suggest a cautious interpretation due to the small associations between predictors and recovery. There is still some lack of clarity on the underlying mechanisms of recovery after an ankle sprain. More research is needed to inform an accurate understanding of the prog- nosis of acute ankle sprains. Factors that may be associated with poor recovery – at short-term include: pain intensity, difficulties bearing weight, restricted joint motion and functional ability. Clinical implications Clinical implications Factors that may be associated with poor recovery – at short-term include: pain intensity, difficulties bearing weight, restricted joint motion and functional ability. Page 13 of 14 Thompson et al. BMC Musculoskeletal Disorders (2017) 18:421 At long-term: older age, female gender. At long-term: older age, female gender. Competing interests The authors declare that they have no competing interests. There is limited evidence that re-current sprain within 3 months, predicts subjective recovery at long term. Department of Health Disclaimer The views and opinions expressed therein are those of the authors and do not necessarily reflect those of the Health Technology Assessment programme, NIHR, NHS or the Department of Health. 10. Anandacoomarasamy A, Barnsley L. Long term outcomes of inversion ankle injuries. Br J Sports Med. 2005;39(Suppl 3):e14–e. 11. van Rijn RM, van Os AG, Kleinrensink GJ, Bernsen RM, Verhaar JA, Koes BW, Bierma-Zeinstra SM. Supervised exercises for adults with acute lateral ankle sprain: a randomised controlled trial. Br J Gen Pract. 2007;57(Suppl 543):793–800. Received: 14 February 2017 Accepted: 10 October 2017 Received: 14 February 2017 Accepted: 10 October 2017 Received: 14 February 2017 Accepted: 10 October 2017 Additional file 2: Appendix B. Criteria used for evaluating the quality of studies included in the systematic review using the QUIPS tool. (DOCX 17 kb) Availability of data and materials All data supporting our findings are contained within the manuscript. 9. Kemler E, Thijs KM, Badenbroek I, van de Port IG, Hoes AW, Backx FJ. Long-term prognosis of acute lateral ankle ligamentous sprains: high incidence of recurrences and residual symptoms. Fam Pract. 2016; 33(Suppl. 6):596–600. 9. Kemler E, Thijs KM, Badenbroek I, van de Port IG, Hoes AW, Backx FJ. Long-term prognosis of acute lateral ankle ligamentous sprains: high incidence of recurrences and residual symptoms. Fam Pract. 2016; 33(Suppl. 6):596–600. Abbreviations 1. Waterman BR, Owens BD, Davey S, Zacchilli MA, Belmont PJ Jr. The epidemiology of ankle sprains in the United States. The journal of bone and joint surgery. Am Volume. 2010;92(Suppl 13):2279–84. 1. Waterman BR, Owens BD, Davey S, Zacchilli MA, Belmont PJ Jr. The epidemiology of ankle sprains in the United States. The journal of bone and joint surgery. Am Volume. 2010;92(Suppl 13):2279–84. AFS: Ankle Function Score; AMED: Allied and complementary medicine database; CINAHL: Cumulative index of nursing and allied health; FAOS: Foot and ankle outcome score; MRI: Magnetic resonance imaging; PRISMA: Preferred reporting items for systematic reviews and meta-analyses; QUIPS: Quality in prognosis studies; RCT: Randomised controlled trial; ROM: Restricted range of motion; SF36PF: 36-item short form survey physical functioning sub-domian; UK: United Kingdom; USA: United States of America 2. Kemler E, van de Port I, Valkenberg H, Hoes AW, Backx FJ. Ankle injuries in the Netherlands: trends over 10-25 years. Scand J Med Sci Sports. 2015; 25(Suppl 3):331–7. 3. Cooke MW, Marsh JL, Clark M, Nakash R, Jarvis RM, Hutton JL, Szczepura A, Wilson S, Lamb SE. Treatment of severe ankle sprain: a pragmatic randomised controlled trial comparing the clinical effectiveness and cost-effectiveness of three types of mechanical ankle support with tubular bandage. The CAST trial. Health Technol Assess. 2009;13(Suppl 13):iii. ix-x, 1-121. Author details 1 1Nuffield Department of Orthopaedics Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK. 2School of Health Professions, Faculty of Health and Human Sciences, Plymouth University, Plymouth, UK 3Department of Sport, Health Sciences and Social Work, Oxford Brookes University, Oxford, UK. Publisher’s Note Factors that were not investigated to date – psychosocial factors. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. p y There is a substantial gap in the literature for prognostic factors of poor recovery. Acknowledgements The SPRAINED (Synthesising a clinical Prognostic Rule for Ankle Injuries in the Emergency Department) study group. 4. Doherty C, Delahunt E, Caulfield B, Hertel J, Ryan J, Bleakley C. The incidence and prevalence of ankle sprain injury: a systematic review and meta-analysis of prospective epidemiological studies. Sports Med (Auckland, NZ). 2014;44(Suppl 1):123–40. Funding This project was funded by the National Institute for Health Research Health Technology Assessment (project number 13/19/06). The research was supported by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care Oxford at Oxford Health NHS Foundation Trust, and supported by the NIHR Biomedical Research Centre, Oxford. The views expressed are those of the author(s) and not necessarily those of the HTA, NHS, the NIHR or the Department of Health. 5. Schaap GR, de Keizer G, Marti K. Inversion trauma of the ankle. Arch Orthop Trauma Surg. 1989;108(Suppl 5):273–5. 6. Verhagen RA, de Keizer G, van Dijk CN. Long-term follow-up of inversion trauma of the ankle. Arch Orthop Trauma Surg. 1995;114(Suppl 2):92–6. 7. van Rijn RM, van Os AG, Bernsen RM, Luijsterburg PA, Koes BW, Bierma- Zeinstra SM. What is the clinical course of acute ankle sprains? A systematic literature review. Am J Med. 2008;121(Suppl 4):324–31. e6. 8. Konradsen L, Bech L, Ehrenbjerg M, Nickelsen T. Seven years follow-up after ankle inversion trauma. Scand J Med Sci Sports. 2002;12(Suppl 3):129–35. Authors’ contributions JT – made contributions to the acquisition of data, analysis, interpretation of data and drafting of the manuscript. 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https://openalex.org/W3105960111	https://quantum-journal.org/papers/q-2019-09-30-189/pdf/	English	null	The complexity of simulating local measurements on quantum systems	Quantum	2,019	cc-by	24,409	Justin Yirka: yirka@utexas.edu, https://www.justinyirka.com/ Sevag Gharibian: sgharibian@upb.de, http://groups.uni-paderborn.de/fg-qi/people.html Sevag Gharibian: sgharibian@upb.de, http://groups.uni-paderborn.de/fg-qi/people.html Justin Yirka: yirka@utexas.edu, https://www.justinyirka.com/ The complexity of simulating local measurements on quan- tum systems Sevag Gharibian1,3 and Justin Yirka2,3 1Department of Computer Science, Paderborn University, Germany 2Department of Computer Science, The University of Texas at Austin, USA 3Department of Computer Science, Virginia Commonwealth University, USA 1Department of Computer Science, Paderborn University, Germany 2Department of Computer Science, The University of Texas at Austin, USA 3Department of Computer Science, Virginia Commonwealth University, USA 1Department of Computer Science, Paderborn University, Germany 2Department of Computer Science, The University of Texas at Austin, USA 3Department of Computer Science, Virginia Commonwealth University, USA 606.05626v5 [quant-ph] 7 Apr 2020 An important task in quantum physics is the estimation of local quantities for ground states of local Hamiltonians. Recently, [Ambainis, CCC 2014] deﬁned the complexity class PQMA[log], and motivated its study by showing that the physical task of estimating the expectation value of a local observable against the ground state of a local Hamiltonian is PQMA[log]-complete. In this paper, we continue the study of PQMA[log], obtaining the following lower and upper bounds. arXiv:1606.05626v5 [quant-ph] 7 A Lower bounds (hardness results): arXiv:1606.05626v5 [quant- • The PQMA[log]-completeness result of [Ambainis, CCC 2014] requires O(log n)-local observables and Hamiltonians. We show that simulating even a single qubit mea- surement on ground states of 5-local Hamiltonians is PQMA[log]-complete, resolving an open question of Ambainis. • We formalize the complexity theoretic study of estimating two-point correlation functions against ground states, and show that this task is similarly PQMA[log]- complete. • We identify a ﬂaw in [Ambainis, CCC 2014] regarding a PUQMA[log]-hardness proof for estimating spectral gaps of local Hamiltonians. By introducing a “query valida- tion” technique, we build on [Ambainis, CCC 2014] to obtain PUQMA[log]-hardness for estimating spectral gaps under polynomial-time Turing reductions. Upper bounds (containment in complexity classes): Upper bounds (containment in complexity classes): • PQMA[log] is thought of as “slightly harder” than QMA. We justify this formally by exploiting the hierarchical voting technique of [Beigel, Hemachandra, Wechsung, SCT 1989] to show PQMA[log] ⊆PP. This improves the containment QMA ⊆PP [Kitaev, Watrous, STOC 2000]. This work contributes a rigorous treatment of the subtlety involved in studying oracle classes in which the oracle solves a promise problem. This is particularly relevant for quantum complexity theory, where most natural classes such as BQP and QMA are deﬁned as promise classes. This work contributes a rigorous treatment of the subtlety involved in studying oracle classes in which the oracle solves a promise problem. 2Implicitly, if Hi acts on a subset Si ⊆[n] of qubits non-trivially, then more accurately one writes Hi ⊗I[n]\Si. We write H = P i Hi for simplicity. Contents Contents 1 Introduction 2 1.1 Background on Quantum Hamiltonian Complexity . . . . . . . . . . . . . . . . . . . 2 1.2 Simulating local measurements on low-temperature quantum systems . . . . . . . . . 4 1.3 Oracle complexity classes, PQMA[log], and PNP[log] . . . . . . . . . . . . . . . . . . . . 4 1.4 Our results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1.5 Proof techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.6 Discussion and open questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2 Preliminaries 10 3 Ambainis’s Query Hamiltonian 13 4 Lower bounds (hardness results) 16 4.1 PQMA[log]-completeness of APX-SIM . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 4.2 PQMA[log]-completeness of APX-2-CORR . . . . . . . . . . . . . . . . . . . . . . . . 21 4.3 PUQMA[log]-hardness of SPECTRAL-GAP . . . . . . . . . . . . . . . . . . . . . . . 23 5 Upper bounds (containment in PP) 28 A Additional proofs 38 1More precisely, QMA is Merlin-Arthur (MA) with a quantum proof and quantum veriﬁer. 2Implicitly, if Hi acts on a subset Si ⊆[n] of qubits non-trivially, then more accurately one writes Hi ⊗I[n]\Si. We write H = P i Hi for simplicity. 1More precisely, QMA is Merlin-Arthur (MA) with a quantum proof and quantum veriﬁer. The complexity of simulating local measurements on quan- tum systems This is particularly relevant for quantum complexity theory, where most natural classes such as BQP and QMA are deﬁned as promise classes. 1 Accepted in Quantum 2019-04-24, click title to verify 3Since these surveys were published, the study of ground spaces through the ﬁeld of Quantum Hamiltonian Com- plexity has continued to grow. For example, recent variants on circuit-to-Hamiltonian mappings such as the space-time construction of Breuckmann and Terhal [14], of Bausch and Crosson [6] for considering complex weights and branch- ing transitions, of Bausch, Cubitt and Ozols [7] for embedding QMAexp computations into 1D translation invariant systems of local dimension 42, or of Caha, Landau, and Nagaj [17] for achieving an arbitrarily high success probability of extracting a computation result from a history state while only needing to increase the clock size logarithmically, have been given. Bravyi and Hastings [13] have shown that the local Hamiltonian problem for the quantum Ising model is StoqMA-complete, completing the complexity classiﬁcation scheme of Cubitt and Montanaro [22]. Gilyén and Sattath [35] have given a constructive Quantum Lovasz Local Lemma which eﬃciently prepares a frustration-free Hamiltonian’s ground state under the assumption that the system is “uniformly” gapped. (This list of results is a small sample of recent work.) 1.1 Background on Quantum Hamiltonian Complexity The use of computational complexity theory to study the inherent diﬃculty of computational problems has proven remarkably fruitful over the last decades. For example, the theory of NP- completeness [21, 42, 46] has helped classify the worst-case complexity of hundreds of computa- tional problems which elude eﬃcient classical algorithms. In the quantum setting, the study of a quantum analogue of NP, known as Quantum Merlin Arthur1 (QMA), was started in 1999 by the seminal “quantum Cook-Levin theorem” of Kitaev [45], which showed that estimating the ground state energy of a given k-local Hamiltonian is QMA-complete for k ≥5. Here, a k-local Hamiltonian H can be thought of as a quantum constraint satisfaction system in which each quantum clause acts non-trivially on k qubits. More formally, H ∈C2n×2n is an exponentially large Hermitian matrix acting on n qubits, but with a succinct description2 H = P i Hi, where each local clause Hi ∈C2k×2k acts non-trivially on k qubits. The “largest total weight of satisﬁable clauses” is given by the ground state energy of H, i.e. the smallest eigenvalue of H. Physically, the ground state energy and its corresponding eigenvector, the ground state, are motivated in that they represent the energy level and state of a given quantum system at low temperature, respectively. For this reason, since Kitaev’s work [45], a number of physically motivated problems have been shown complete for QMA (this has given rise to the ﬁeld of Quantum Hamiltonian Complexity, see, e.g., [52], [10] 2 and [31] for surveys3). Many of these QMA-complete problems focus on estimating ground state energies of local Hamiltonians. Beyond ground state energies. In recent years, however, new directions in quantum complexity theory involving other physical properties of local Hamiltonians have appeared. We attempt to survey a number of such results here. Brown, Flammia and Schuch [15] (also Shi and Zhang [59]) introduced a quantum analogue of #P, denoted #BQP, and showed that computing the ground state degeneracy or density of states of local Hamiltonians is #BQP-complete. Gharibian and Sikora [30] showed that determining whether the ground space of a local Hamiltonian has an “energy barrier” is QCMA-complete, where QCMA [2] is Merlin-Arthur (MA) with a classical proof and quantum prover. This was strengthened by Gosset, Mehta, and Vidick [38], who showed that QCMA-completeness holds even for commuting local Hamiltonians. 1.1 Background on Quantum Hamiltonian Complexity the number of qubits n goes to inﬁnity), in our setting the number of qubits n (expressed in unary) is part of the input to the problem, as is standard in complexity theory. Perez-Garcia [8]. Note that both the current paper and [5] also study spectral gaps, but from a complexity theory perspective; in particular, in contrast to the undecidability studies listed above, which consider the thermodynamic limit (i.e. the number of qubits n goes to inﬁnity), in our setting the number of qubits n (expressed in unary) is part of the input to the problem, as is standard in complexity theory. 1.2 Simulating local measurements on low-temperature quantum systems In Section 1.1, we listed a number of results studying properties of local Hamiltonians beyond ground state energies. We intentionally omitted one particular problem, however, which is extremely well motivated physically and which is the starting point of this work. Suppose one cools a many- body quantum system to low temperature in the lab and a priori does not know the system’s properties (such as its ground state energy, ground space, etc); this is a natural assumption, as many of these properties are QMA-complete to estimate to begin with. The most “basic” action an experimenter can now take is to perform a local measurement (typically on a constant number of qubits) in an attempt to extract information about the system. The question is: How diﬃcult is it to computationally simulate this “basic” action? This computational task was formalized by Ambainis [5] (formal deﬁnitions in Section 2) and designated Approximate Simulation (APX-SIM): Given a k-local Hamiltonian H and an l-local observable A, estimate the expectation value of the measurement A against the ground state of H, i.e. estimate ⟨A⟩:= ⟨ψ\| A \|ψ⟩for \|ψ⟩a ground state of H. It turned out that not only is this problem “hard”, but that it is in fact harder then even QMA. To formalize this, Ambainis introduced [5] the complexity class PQMA[log], which intuitively is “slightly harder” than QMA, and showed that APX-SIM is PQMA[log]-complete when the Hamilto- nian H and observable A are both O(log n)-local. To help set context before stating our results, we now discuss PQMA[log] and its classical analogue PNP[log] in further depth. 1.3 Oracle complexity classes, PQMA[log], and PNP[log] 1.1 Background on Quantum Hamiltonian Complexity Bravyi and Gosset [12] studied the complexity of quantum impurity problems, which involve a bath of free fermions coupled to an interacting subsystem dubbed an “impurity”. Cubitt, Montanaro, and Piddock [24] have shown that certain simple spin-lattice models are “universal”, in the sense that they can replicate the entire physics of any other quantum many-body system. From a hardness of approximation perspective, Gharibian and Kempe [29] introduced cq-Σ2, a quantum generalization of Σp 2, and showed that determining the smallest subset of interaction terms of a given local Hamiltonian which yields a frustrated ground space is cq-Σ2-complete (and additionally, cq-Σ2-hard to approximate). Aharonov and Zhou [3] studied the task of “Hamiltonian sparsiﬁcation” or “degree-reduction”, in which one attempts to simulate an input local Hamiltonian with a new local Hamiltonian with an interaction graph of bounded degree while preserving only the ground space and spectral gap (in general, [3] show this is impossible). This was in pursuit of answering whether classical proof techniques for the classical PCP theorem carry over to the quantum setting. Finally, various authors have studied spectral gaps of local Hamiltonians from a computability theory perspective. (In computability theory, one asks whether a decision or promise problem can be decided by a Turing machine running in a ﬁnite number of steps. In the current paper, our focus is instead on complexity theory, in which problems are typically known to be computable; the question is rather to obtain an estimate of the resources the Turing machine requires to solve the problem.) Gosset and Mozgunov [37] and Bravyi and Gosset [11] have studied spectral gaps for frustration- free 1D translation-invariant systems (the latter, in particular, shows that distinguishing between gapped and gapless phases is decidable in the spin-1/2 chains studied). Cubitt, Perez-Garcia and Wolf [23] have shown undecidability of estimating spectral gaps in the thermodynamic limit for translation-invariant, nearest-neighbor Hamiltonians on a 2D square lattice. This has very recently been improved to undecidability for 1D translation invariant systems by Bausch, Cubitt, Lucia, and 3 Perez-Garcia [8]. Note that both the current paper and [5] also study spectral gaps, but from a complexity theory perspective; in particular, in contrast to the undecidability studies listed above, which consider the thermodynamic limit (i.e. 1.3 Oracle complexity classes, PQMA[log], and PNP[log] k As far as we are aware, whether PNP[k] (i.e. k ∈O(1) NP queries), PNP[logk n] (i.e. O(logk n) NP queries for k ≥1), and PNP (i.e. polynomially many NP queries) coincide remains open. Notably, if PNP[1] = PNP[2], then PNP[1] = PNP[log] and PH collapses [39]. But, it is known is that if the queries to the NP oracle are non-adaptive, meaning they are all made in parallel, then the resulting class P\|\|NP equals PNP[log] [16, 40]. (The analogous statement P\|\|QMA = PQMA[log] has recently been shown [33].) Similarly, a P machine making O(logk n) adaptive NP queries is equivalent in power to a P machine making O(logk+1 n) non-adaptive queries for all k ≥1 [18]. In terms of complete problems, determining the election winner in Lewis Carroll’s 1876 voting system is PNP[log]-complete [41], and model checking for certain branching-time temporal logics is PNP[log2 n]-complete [57]. Finally, it is important to note that NP is a class of decision problems. Formally, this means any NP problem is speciﬁed by a language L ⊆{ 0, 1 }∗with the corresponding decision problem: Given input x ∈{ 0, 1 }∗, is x ∈L? In the context of PNP[log], this means any string x ∈{ 0, 1 }∗ is a valid problem instance or query to an NP oracle for language L, since any such x is either in the language or not. Unfortunately, an analogous statement cannot be made about PQMA[log], complicating its study; this brings us to our third, crucial remark about PQMA[log] from earlier. Oracles for promise classes and the issue of invalid queries. In contrast to NP, QMA is a class of promise problems. Formally, this means the space of all inputs { 0, 1 }∗is partitioned into three sets, A, B, C, where A and C are YES and NO instances, respectively, and B is the set of “invalid” instances. (Classes of decision problems are a special case of this in which A = L, B = ∅, and C = { 0, 1 }∗\ L.) Why might this pose a problem for studying PQMA[log]? Recall that we assume all calls by the PQMA[log] machine to the QMA oracle Q are for instances (H, a, b) of 2-LH: Is the ground state energy of H at most a (YES case), or at least b (NO case), for b −a ≥1/poly(n)? 1.3 Oracle complexity classes, PQMA[log], and PNP[log] Formally, PQMA[log] is the class of decision problems which can be decided by a polynomial-time deterministic Turing machine making O(log n) queries to an oracle for QMA. Thus, it is an example of an oracle complexity class. Let us make three remarks. First, by “oracle for QMA”, one typically means an oracle for a QMA-complete problem Π (since any other problem in QMA may be reduced in polynomial-time to Π); in this paper, we shall set Π as the QMA-complete 2-local Hamiltonian problem (2-LH) [43], an instance (H, a, b) of which asks: Is the ground state energy of H at most a (YES case), or at least b (NO case), for b −a ≥1/poly(n)? Second, although PQMA[log] uses a QMA oracle, it in fact also contains the complementary class co-QMA. This is because to solve any co-QMA problem, a PQMA[log] machine can plug the co-QMA problem instance into the QMA oracle, and subsequently ﬂip the oracle’s answer in polynomial-time to solve the co-QMA problem. Thus, PQMA[log] ̸= QMA unless co-QMA ⊆QMA (which is unlikely), and so PQMA[log] is likely strictly harder than QMA. The third remark is that PQMA[log] uses an oracle for a class of promise problems; this is a subtle but crucial point, which we discuss shortly; ﬁrst, let us set the stage by reviewing the analogous classical class PNP[log]. The class PNP[log]. PNP[log] is deﬁned analogously to PQMA[log] except it utilizes an NP oracle. Analogous to PQMA[log], PNP[log] contains both NP and co-NP and thus is likely strictly harder than 4 NP. As an upper bound, PNP[log] ⊆NPNP = Σp 2, where an NPNP machine is an NP machine which nondeterministically makes up to a polynomial number of calls to an NP oracle, and Σp 2 is the second level of the Polynomial-Time Hierarchy (PH). In contrast, it is unlikely for PQMA[log] to be in PH, as even BQP ⊆QMA ⊆PQMA[log] is generally not believed to be in PH [1, 20, 26, 55, 56]. A natural question is whether PQMA[log] might instead be contained in an appropriate quantum analogue of PH. The answer is not clear, since unlike the fact that Σp 2 = NPNP, it is not clear that “quantum Σp 2” should equal (say) QMAQMA; see [32] for a discussion and treatment of quantum analogues of PH. Additional references on “quantum PH” are [29, 48, 63]. 4By deﬁnition, the P machine can only execute polynomial-time computations. Thus, even if we assume without loss of generality that the P machine uses speciﬁc circuit-to-Hamiltonian constructions (for preparing queries to the QMA oracle) whose promise gaps are precisely known (e.g. [6, 17]), it is not clear how the machine should know whether the quantum circuit it feeds into said construction satisﬁes a promise gap to begin with — where such circuit-to-Hamiltonian constructions require the input circuit to satisfy an inverse polynomial promise gap, which is unlikely to be relaxed to more easily veriﬁed, say, inverse exponentially small promise gaps, since it would imply PSPACE ⊆QMA, which follows since QMA with exponentially small gap equals PSPACE [25]. 1.4 Our results Our results fall into two categories: Lower bounds (hardness results) and upper bounds (containment in complexity classes). Our results fall into two categories: Lower bounds (hardness results) and upper bounds (containment in complexity classes). Lower bounds (hardness results). We begin by showing three hardness results; two focus on computational problems introduced in [5] (APX-SIM and SPECTRAL-GAP) and one introduces a new problem (APX-2-CORR). 1. PQMA[log]-completeness of APX-SIM for O(1)-local Hamiltonians and single-qubit observables. Recall that in [5], Ambainis introduced PQMA[log] and showed that APX-SIM (i.e. given k-local Hamiltonian H and l-local observable A, simulate measurement A on the ground state of H) is PQMA[log]-complete. This proof required both the Hamiltonian H and observable A to be O(log n)- local. From a physical standpoint, however, it is typically desirable to have O(1)-local Hamiltonians and observables — whether PQMA[log]-hardness holds in this regime was left as an open question in [5]. We thus ﬁrst ask: Is APX-SIM still hard for O(1)-local Hamiltonians and 1-local observables? 1. PQMA[log]-completeness of APX-SIM for O(1)-local Hamiltonians and single-qubit observables. Recall that in [5], Ambainis introduced PQMA[log] and showed that APX-SIM (i.e. given k-local Hamiltonian H and l-local observable A, simulate measurement A on the ground state of H) is PQMA[log]-complete. This proof required both the Hamiltonian H and observable A to be O(log n)- local. From a physical standpoint, however, it is typically desirable to have O(1)-local Hamiltonians and observables — whether PQMA[log]-hardness holds in this regime was left as an open question in [5]. We thus ﬁrst ask: Is APX-SIM still hard for O(1)-local Hamiltonians and 1-local observables? Let us develop some intuition before answering this question. Typically, computational problems (such as estimating ground state energies) of 1-local Hamiltonians are easy, since the qubits do not interact. This intuition does not, however, carry over to the setting of simulating 1-local measurements. For example, by embedding a 3-SAT instance φ into a 3-local Hamiltonian and then using the ability to repeatedly measure observable Z against single qubits of the ground state, we can extract a solution to φ! Thus, the 3-local Hamiltonian and 1-local observable case is at least NP-hard. Indeed, here we show it is much harder, resolving Ambainis’s open question. Let us develop some intuition before answering this question. Typically, computational problems (such as estimating ground state energies) of 1-local Hamiltonians are easy, since the qubits do not interact. 1.3 Oracle complexity classes, PQMA[log], and PNP[log] Unfortunately, a P machine cannot in general tell4 whether the instance (H, a, b) it feeds to Q satisﬁes the promise conditions of LH; in particular, the ground state energy may lie in the interval (a, b). We call any such instance or query (H, a, b) violating the 2-LH promise “invalid” (these instances belong in the set B of instances above). For any invalid query, the oracle Q is allowed to accept or reject arbitrarily. This raises a potential issue: if the oracle is allowed to respond arbitrarily to invalid queries, how does one ensure a YES instance (or NO instance) of a PQMA[log] problem is well-deﬁned (normally, the P machine conditions its future actions on 5 the response the oracle returns for each query)? To do so, we stipulate (see, e.g., Deﬁnition 3 of Goldreich [36]) that the P machine must output the same answer regardless of how any invalid queries are answered by the oracle. We view the issue of formally handling invalid queries as one of the central themes and contributions of this work. 3. PUQMA[log]-hardness of estimating spectral gaps. A third well-motivated problem involving Hamil- tonians is SPECTRAL-GAP [5]: Given a k-local Hamiltonian H, estimate λ1(H) −λ2(H), where 1.4 Our results Recall now that, as discussed in Section 1.3, a central theme of this work is the subtlety involved in the study of oracle classes in which the oracle solves a promise problem (such as PQMA[log]), as opposed to a decision problem (such as PNP[log]). In particular, for 2-LH queries to the QMA oracle which violate the promise gap for 2-LH, the oracle is allowed to give an arbitrary answer. We observe that this point appears to have been missed in [5]’s claimed proofs of PQMA[log]-hardness of APX-SIM and of PUQMA[log]-hardness of SPECTRAL-GAP, rendering the proofs incorrect. Though, as shown by our ﬁrst result, we are able to correct and improve on the proof that APX-SIM is PQMA[log]-hard. In our last result, we also overcome this diﬃculty to recover PUQMA[log]-hardness of SPECTRAL- GAP, with the tradeoﬀthat we obtain a “slightly weaker” hardness claim, in the sense that it follows from a Turing reduction as opposed to a Karp or mapping reduction5 ([5] claimed hardness under the latter). In the process, we also improve the locality of H to O(1). Theorem 1.3. Given a 4-local Hamiltonian H, estimating its spectral gap (i.e. SPECTRAL-GAP) is PUQMA[log]-hard under polynomial-time Turing reductions. Thus, the ability to solve PUQMA[log] problems in polynomial time would imply a polynomial-time algorithm for SPECTRAL-GAP. Note that whether UQMA = QMA remains open. For the cases of NP [60] or MA and QCMA [4], it is known that Unique NP, Unique MA, and Unique QCMA reduce to NP, MA, and QCMA, respectively, under randomized reductions. Upper bounds (containment in complexity classes). As mentioned earlier, since both QMA and co-QMA are contained in PQMA[log], it is likely that PQMA[log] is strictly more powerful than both QMA and co-QMA. This raises the question: What is an upper bound on the power of PQMA[log]? Two points of reference are worth mentioning here. First, the classical analogue of PQMA[log], PNP[log], is known to be upper bounded by PP [9]. Here, PP is the set of promise problems solvable in probabilistic polynomial time with unbounded error; in other words, a PP machine accepts YES instances with probability strictly larger than 1/2 and accepts NO instances with probability less than or equal to 1/2. The second point of reference is that QMA is bounded by PP [44, 49] ([61] actually shows the slightly stronger containment QMA ⊆A0PP). 1.4 Our results This intuition does not, however, carry over to the setting of simulating 1-local measurements. For example, by embedding a 3-SAT instance φ into a 3-local Hamiltonian and then using the ability to repeatedly measure observable Z against single qubits of the ground state, we can extract a solution to φ! Thus, the 3-local Hamiltonian and 1-local observable case is at least NP-hard. Indeed, here we show it is much harder, resolving Ambainis’s open question. Theorem 1.1. Given a 5-local Hamiltonian H on n qubits and a 1-local observable A, estimating ⟨A⟩(i.e. APX-SIM) is PQMA[log]-complete. Theorem 1.1. Given a 5-local Hamiltonian H on n qubits and a 1-local observable A, estimating ⟨A⟩(i.e. APX-SIM) is PQMA[log]-complete. Thus, measuring just a single qubit of the ground state of a local Hamiltonian H with a single- qubit observable A (in fact, A is ﬁxed, independent of H in our construction) is harder than QMA (assuming QMA ̸= PQMA[log], which is likely as otherwise co-QMA ⊆QMA). 2. PQMA[log]-completeness of APX-2-CORR for O(1)-local Hamiltonians and single-qubit observ- ables. We next introduce a second natural problem related to APX-SIM, denoted APX-2-CORR. APX-2-CORR is deﬁned similarly to APX-SIM except one is given Hamiltonian H and observ- ables A and B and asked to estimate the two-point correlation function ⟨A ⊗B⟩−⟨A⟩⟨B⟩(recall ⟨A⟩:= ⟨ψ\| A \|ψ⟩for \|ψ⟩a ground state of H). By modifying the construction behind the proof of Theorem 1.1, we also show APX-2-CORR is PQMA[log]-complete. Theorem 1.2. Given a 5-local Hamiltonian H on n qubits and a pair of 1-local observables A and B, estimating ⟨A ⊗B⟩−⟨A⟩⟨B⟩(i.e. APX-2-CORR) is PQMA[log]-complete. 3. PUQMA[log]-hardness of estimating spectral gaps. A third well-motivated problem involving Hamil- tonians is SPECTRAL-GAP [5]: Given a k-local Hamiltonian H, estimate λ1(H) −λ2(H), where 3. PUQMA[log]-hardness of estimating spectral gaps. A third well-motivated problem involving Hamil- tonians is SPECTRAL-GAP [5]: Given a k-local Hamiltonian H, estimate λ1(H) −λ2(H), where 6 λi(H) denotes the i-th smallest eigenvalue of H. (For clarity, if the ground space of H is degener- ate, we deﬁne its spectral gap as 0.) In [5], it is shown that SPECTRAL-GAP ∈PQMA[log], and a claimed proof is given that SPECTRAL-GAP for O(log n)-local Hamiltonians H is PUQMA[log]- hard. Here, PUQMA[log] is PQMA[log] except with a Unique QMA oracle, and Unique QMA is roughly QMA with a unique accepting quantum witness in the YES case (see Section 4.3 for formal deﬁni- tions). 5A Karp, many-one, or mapping reduction from a decision problem A to a decision problem B maps any input ΠA for A into an input ΠB for B such that ΠA is a YES (NO) instance of A if and only if ΠB is a YES (NO) instance for B. A Turing reduction generalizes the notion of a Karp reduction; here, one is given access to an oracle solving B, and the goal is to solve ΠA using potentially multiple calls to the oracle. In this paper, both notions of reduction are assumed to be deterministic polynomial-time. 1.4 Our results We thus ask whether, like its classical analogue, PQMA[log] can bounded by PP, and so is “slightly harder” than QMA. Indeed, we show this is the case. Theorem 1.4. PQMA[log] ⊆PP. 1.5 Proof techniques w outline our proof techniques for both our upper bound and lower bound results. We now outline our proof techniques for both our upper bound and lower bound res We now outline our proof techniques for both our upper bound and lower bound results. Lower bounds. We begin with proof techniques techniques for our lower bounds involving APX- SIM (Theorem 1.1), APX-2-CORR (Theorem 1.2), and SPECTRAL-GAP (Theorem 1.3). Lower bounds. We begin with proof techniques techniques for our lower bounds involving APX- SIM (Theorem 1.1), APX-2-CORR (Theorem 1.2), and SPECTRAL-GAP (Theorem 1.3). 1. PQMA[log]-hardness of APX-SIM. To show Theorem 1.1, our intuition is simple: To design our local Hamiltonian H so that its ground state encodes a so-called history state6 \|ψ⟩for a given PQMA[log] instance such that measuring observable Z on the designated “output qubit” of \|ψ⟩reveals the answer of the computation. At a high level, this is achieved by combining a variant of Kitaev’s circuit-to-Hamiltonian construction [45] (which forces the ground state to follow the P circuit) with Ambainis’s “query Hamiltonian” [5] (which forces the ground state to encode correctly answered queries to the QMA oracle). Making this rigorous requires a careful analysis of the ground space of Ambainis’s query Hamiltonian when queries violating the promise gap of the oracle are allowed (Lemma 3.1), showing a simple but useful extension of Kempe, Kitaev, and Regev’s Projection Lemma [43] (Lemma 4.1) that any low energy state of H must be close to a valid history state, which thus allows us to use just a single-qubit observable, and applying Kitaev’s unary encoding trick [45] to bring the locality of the Hamiltonian H down to O(1) (Lemma 3.3). 2. Containment of APX-2-CORR in PQMA[log]. The hardness proof for APX-2-CORR is similar to that of APX-SIM, so we focus instead on the containment proof of APX-2-CORR ∈PQMA[log], for which a trick is required. For containment, the naive approach would be to run Ambainis’s PQMA[log] protocol [5] for APX-SIM independently for each term ⟨A ⊗B⟩, ⟨A⟩, and ⟨B⟩. However, if a cheating prover does not send the same ground state \|ψ⟩for each of these measurements, soundness of the protocol can be violated. To address this, we exploit a trick of Chailloux and Sattath [19] from the setting of QMA(2): we observe that the correlation function requires only knowledge of the two-body reduced density matrices { ρij } of \|ψ⟩. 6A history state can be seen as a quantum analogue of the “tableau” which appears in the proof of the Cook-Levin theorem, i.e. a history state encodes the history of a quantum computation. In contrast to tableaus, however, the history encodes information in quantum superposition. Theorem 1.4. PQMA[log] ⊆PP. Theorem 1.4. PQMA[log] ⊆PP. 7 Thus, QMA ⊆PQMA[log] ⊆PP, which rigorously justiﬁes the intuition that PQMA[log] should be thought of as “slightly harder” than QMA. Thus, QMA ⊆PQMA[log] ⊆PP, which rigorously justiﬁes the intuition that PQMA[log] should be thought of as “slightly harder” than QMA. 1.5 Proof techniques A prover can send classical descriptions of the { ρij } (which is possible since each ρij is of constant size), along with a “consistency proof” for the QMA-complete Consistency problem [47] to ensure the { ρij } indeed correspond to some state. The veriﬁer can then freely copy each ρij and thus can calculate each term. 3. PUQMA[log]-hardness of estimating spectral gaps. As mentioned in Sections 1.3 and 1.4, a central theme of this work is the fact that a PQMA[log] machine can make invalid queries to the QMA oracle, and this point appears to have been missed in [5], where all queries were assumed to satisfy the LH promise. This results in the proofs of two key claims of [5] being incorrect. The ﬁrst claim was used in the proof of PQMA[log]-completeness for APX-SIM (Claim 1 in [5]); we provide a corrected 8 statement and proof in Lemma 3.1 (which suﬃces for the PQMA[log]-hardness results in [5] regarding APX-SIM to hold). The error in the second claim (Claim 2 of [5]), wherein PUQMA[log]-hardness of determining the spectral gap of a local Hamiltonian is shown, appears arguably more serious. The construction of [5] requires a certain “query Hamiltonian” to have a spectral gap, which indeed holds if the PUQMA[log] machine makes no invalid queries. However, if the machine makes invalid queries, this gap can close, and it is not clear how one can recover PUQMA[log]-hardness under mapping reductions. To overcome this, we introduce a technique of “query validation”: given a query to the UQMA oracle, we would like to determine if the query is valid or “far” from valid. While it is not clear how a P machine alone can perform such “query validation”, we show how to use a SPECTRAL-GAP oracle to do so, allowing us to eliminate “suﬃciently invalid” queries. Combining this idea with Ambainis’s original construction [5], we show Theorem 1.3, i.e. PUQMA[log]-hardness for SPECTRAL-GAP, with the additional improvement that the input Hamiltonians are now O(1)-local, versus O(log n)- local as in [5], via the same unary encoding trick from our previous results. Since our “query validation” requires a polynomial number of calls to the SPECTRAL-GAP oracle, this result requires a polynomial-time Turing reduction. Whether this can be improved to a mapping reduction is left as an open question. Upper bounds. We now move to our upper bound result, which is the most technically involved. 1.6 Discussion and open questions 1.6 Discussion and open questions The problems studied here explore the line of research recently initiated by Ambainis [5] on PQMA[log] and focus on central problems for local Hamiltonian systems. The complexity theoretic study of such problems is appealing in that it addresses the original motivation of physicist Richard Feyn- man in proposing quantum computers [27], who was interested in avenues for simulating quantum systems. Indeed, hardness results, such as Kitaev’s quantum Cook-Levin theorem, rigorously justify Feynman’s intuition that such simulation problems are hard. Our work (e.g. Theorem 1.1) strongly supports this view by demonstrating that even some of the simplest and most natural simulation tasks, such as measuring a single qubit (!) of a ground state, can be harder than QMA. Our study of spectral gaps (Theorem 1.3) further highlights another theme: the subtleties which must be care- fully treated when studying oracle classes for promise problems (such as PQMA[log]). As quantum complexity theory commonly focuses on such promise problems, we believe this theme could be of interest to a broader computer science audience. Moving to open questions, although we resolve one of the open questions from [5], there are others we leave open, along with some new ones. Do our results for APX-SIM and APX-2-CORR hold for more restricted classes of Hamiltonians, such as 2-local Hamiltonians, local Hamiltonians on a 2D lattice, or speciﬁc Hamiltonian models of interest (see e.g. [22, 53] for QMA-completeness results for estimating ground state energies of the spin-1/2 Heisenberg anti-ferromagnet)? Is SPECTRAL- GAP PUQMA[log]-complete or PQMA[log]-complete (recall SPECTRAL-GAP ∈PQMA[log] and that [5] and our work together show PUQMA[log]-hardness)? What is the relationship between PQMA[log] and PUQMA[log]? Finally, what is the complexity of other physical tasks “beyond” estimating ground state energies? Remark added later: Since the original preprint of this paper appeared, the present authors, together with Stephen Piddock, have partially answered the ﬁrst open question above by showing [33] that APX-SIM remains PQMA[log]-complete for any family of local Hamiltonians which can simulate “spatially sparse” [51] Hamiltonians. This, in turn, implies (e.g. using [53], [58], [54]) that APX-SIM remains PQMA[log]-complete even on physically motivated models, such as the Heisenberg interaction on a 2D square lattice. Organization. This paper is organized as follows: In Section 2, we give notation and formal deﬁnitions. In Section 3, we show various lemmas regarding Ambainis’s query Hamiltonian. 1.6 Discussion and open questions In Section 4.1, Section 4.2, and Section 4.3 we show Theorem 1.1, Theorem 1.2, and Theorem 1.3, respectively. Section 5 proves Theorem 1.4. A result used in Section 3 is proved in Appendix A. 1.5 Proof techniques To show PQMA[log] ⊆PP (Theorem 1.4), we exploit the technique of hierarchical voting, used by Beigel, Hemachandra, and Wechsung [9] to show PNP[log] ⊆PP, in conjunction with the QMA strong ampliﬁcation results of Marriott and Watrous [49]. To give some intuition as to how the technique works, we sketch its application in the classical case of PNP[log] [9]. There, we have a P machine making queries to an NP oracle for SAT (i.e. the i-th query feeds the oracle some SAT formulae φi), and the goal is to simulate this by a PP machine. Since the PP machine does not have access to an NP oracle, the best it can do is guess the answer to each query; so, it begins by guessing the answers to each NP query by picking a random assignment xi for each formula φi in the hope that φi(xi) = 1. Of course, such a guess xi can be satisfying only if φi is satisﬁable to begin with; with a bit of thought, this implies that the “correct” query string y∗ (i.e. the i-th bit of y∗equals 1 if and only if φi is satisﬁable) is the lexicographically largest string y∗attainable by this guessing process. Unfortunately, the PP machine might have a very small probability of guessing y∗; thus, it next “boosts” this probability via several rounds of “hierarchical voting”. Conceptually, this technique does not actually increase the probability of outputting y∗but rather decreases the probability of outputting other, lexicographically smaller query strings y′ ̸= y∗. This works because the machine is a PP machine (i.e. it can have unbounded error), and so it suﬃces to reduce the probability of obtaining any such y′ being output to be strictly smaller than the probability of y∗being output, in which case the PP machine is more likely to output the correct answer in its simulation of the PNP[log] computation than not, as needed. We develop a quantum variant of this scheme which is quite natural. However, its analysis is markedly more involved than the classical setting due to both the probabilistic nature of QMA and the possibility of “invalid queries” violating the QMA promise gap. To give a sense of the problems which arise, for PQMA[log] it is no longer necessarily true that the lexicographically largest obtainable y∗is a correct query string. 9 2 Preliminaries Notation. For x ∈{ 0, 1 }n, \|x⟩∈(C2)⊗n denotes the computational basis state labeled by x. Let X be a complex Euclidean space. Then, L (X) and D (X) denote the sets of linear and density operators acting on X, respectively. For subspace S ⊆X, S⊥denotes the orthogonal complement of S. For Hermitian operator H, λ(H) and λ(H\|S) denote the smallest eigenvalue of H and the smallest eigenvalue of H restricted to space S, respectively. The spectral and trace norms are deﬁned ∥A∥∞:= max{∥A \|v⟩∥2 : ∥\|v⟩∥2 = 1} and ∥A∥tr := Tr √ A†A, respectively, where := denotes a deﬁnition. We set [m] := { 1, . . . , m }. 10 Deﬁnitions and lemmas. PP and PQP. The class PP [34] is the set of decision problems for which there exists a polynomial- time probabilistic Turing machine which accepts any YES instance with probability > 1/2 and accepts any NO instance with probability ≤1/2. The quantum analogue of PP, denoted PQP, is deﬁned analogously to BQP except in the YES case the veriﬁer accepts with probability > 1/2 and in the NO case the veriﬁer accepts with probability ≤1/2. PQMA[log]. Deﬁned by Ambainis [5], PQMA[log] is the set of decision problems decidable by a polynomial-time deterministic Turing machine with the ability to query an oracle for a QMA- complete problem O(log n) times, where n is the size of the input. For clarity, without loss of generality we assume that for any QMA problem instance Π the P machine wishes to solve, it ap- plies a polynomial-time Karp/many-one reduction to map Π to an instance of the QMA-complete problem 2-LH [43] which it then feeds to the QMA oracle. 2-LH is deﬁned as: Given a 2-local Hamiltonian H and inverse polynomially separated thresholds a, b ∈R, decide whether λ(H) ≤a (YES-instance) or λ(H) ≥b (NO-instance). Note that the P machine is allowed to make queries which violate the promise gap of 2-LH, i.e. with λ(H) ∈(a, b); in this case, the oracle can output YES or NO arbitrarily. The P machine is nevertheless required to output the same ﬁnal answer (i.e. accept or reject) regardless of how such “invalid” queries are answered [36]. For any P machine M making m queries to a QMA oracle, we use the following terminology throughout this article. A valid (invalid) query satisﬁes (violates) the promise gap of the QMA oracle. A correct query string y ∈{ 0, 1 }m encodes a sequence of correct answers to all of the m queries. Note that for any invalid query by M, any answer is considered correct, yielding the possible existence of multiple correct query strings. An incorrect query string is one which contains at least one incorrect query answer. The problem APX-SIM. Deﬁnition 2.1 (APX-SIM(H, A, k, l, a, b, δ) (Ambainis [5])). Given a k-local Hamiltonian H, an l-local observable A, and real numbers a, b, and δ such that b −a ≥n−c and δ ≥n−c′, for n the number of qubits H acts on and c, c′ > 0 some constants, decide: Deﬁnition 2.1 (APX-SIM(H, A, k, l, a, b, δ) (Ambainis [5])). Given a k-local Hamiltonian H, an l-local observable A, and real numbers a, b, and δ such that b −a ≥n−c and δ ≥n−c′, for n the number of qubits H acts on and c, c′ > 0 some constants, decide: • If H has a ground state \|ψ⟩satisfying ⟨ψ\| A \|ψ⟩≤a, output YES. • If for all \|ψ⟩satisfying ⟨ψ\| H \|ψ⟩≤λ(H) + δ, it holds that ⟨ψ\| A \|ψ⟩≥b, output NO. • If for all \|ψ⟩satisfying ⟨ψ\| H \|ψ⟩≤λ(H) + δ, it holds that ⟨ψ\| A \|ψ⟩≥b, output NO. Kitaev’s circuit-to-Hamiltonian construction. Next, we brieﬂy review Kitaev’s circuit-to-Hamiltonian construction from the “quantum Cook-Levin theorem” [45]. Given a quantum circuit U = UL · · · U1 consisting of 1- and 2-qubit gates Ui and acting on registers Q (proof register) and W (workspace register), this construction maps U to a 5-local Hamiltonian H = Hin + Hout + Hprop + Hstab acting on registers Q (proof register), W (workspace register), and C (clock register). Each of H’s terms 11 11 are deﬁned below: are deﬁned below: Hin := p X i=1 \|1⟩⟨1\|Wi ! ⊗\|0⟩⟨0\|C Hout := \|0⟩⟨0\|W1 ⊗\|L⟩⟨L\|C Hprop := L X j=1 Hj, where Hj is deﬁned as −1 2Uj ⊗\|j⟩⟨j −1\|C −1 2U† j ⊗\|j −1⟩⟨j\|C + 1 2I ⊗(\|j⟩⟨j\| + \|j −1⟩⟨j −1\|)C Hstab := L−1 X i=1 \|01⟩⟨01\|Ci,Ci+1 . Above, the notation Ri refers to the i-th qubit of any given register R. For any candidate proof \|ψ⟩to be evaluated in expression Tr(H \|ψ⟩⟨ψ\|), each penalty term of H forces a structure on any minimizing \|ψ⟩: at time zero, Hin ensures the workspace register W is set to zero; Hout checks that at the end of the computation, i.e. at time step L, the output qubit is close to \|1⟩; the propagation Hamiltonian Hprop ensures all steps of U appear in superposition in \|ψ⟩with equal weights. The problem APX-SIM. Finally, although we have labeled (for ease of exposition) the clock register in Hout and Hprop in binary, note that in Kitaev’s construction it is actually encoded in unary. In other words, time t in clock register C is encoded as \|1t0L−t⟩(note for Hstab above, register C is already written in unary); Hstab’s role is thus to prevent invalid encodings of time steps. (For the interested reader, we remark that instead of working explicitly with Kitaev’s construction, one can also consider the more general Quantum Thue System framework [7].) In this paper, we shall use two key properties of Hin + Hprop + Hstab (note Hout is omitted). First, the null space of Hin + Hprop + Hstab is spanned by history states, which for any proof \|ψ⟩ have form \|ψhist⟩= 1 √ L + 1 L X t=0 Ut · · · U1 \|ψ⟩Q \|0 · · · 0⟩W \|t⟩C , (1) (1) where C is a clock register keeping track of time [45], and each Ui acts on at most two qubits from registers Q and W. Second, we use the following lower bound7 on the smallest non-zero eigenvalue of Hin + Hprop + Hstab: Lemma 2.2 (Lemma 3 (Gharibian, Kempe [29])). The smallest non-zero eigenvalue of ∆(Hin + Hprop + Hstab) is at least π2∆/(64L3) ∈Ω(∆/L3), for ∆∈R+ and L ≥1. Lemma 2.2 (Lemma 3 (Gharibian, Kempe [29])). The smallest non-zero eigenvalue of ∆(Hin + Hprop + Hstab) is at least π2∆/(64L3) ∈Ω(∆/L3), for ∆∈R+ and L ≥1. Lemma 2.2 (Lemma 3 (Gharibian, Kempe [29])). The smallest non-zero eigenvalue of ∆(Hin + Hprop + Hstab) is at least π2∆/(64L3) ∈Ω(∆/L3), for ∆∈R+ and L ≥1. Miscellaneous useful facts. Let V denote a QMA veriﬁcation circuit acting on M proof qubits with completeness c and soundness s. If one runs V on “proof” ρ = I/2M, then for a YES instance, V accepts with probability ≥c/2M (since I/2M can be viewed as “guessing” a correct proof with probability ≥1/2M), and in a NO instance, V accepts with probability ≤s (see, e.g., [49, 62]). A useful fact for complex unit vectors \|v⟩and \|w⟩is (see e g Equation 1 33 of [28]) A useful fact for complex unit vectors \|v⟩and \|w⟩is (see, e.g., Equation 1.33 ∥\|v⟩⟨v\| −\|w⟩⟨w\|∥tr = 2 q 1 −\|⟨v\|w⟩\|2 ≤2 ∥\|v⟩−\|w⟩∥2 . 3 Ambainis’s Query Hamiltonian We now show various results regarding Ambainis’s “query Hamiltonian” [5], which intuitively aims to have its ground space contain correct answers to a sequence of QMA queries. Let U be a PQMA[log] computation8, and let Hi,y1···yi−1 Yi be the 2-local Hamiltonian9 corresponding to the i-th query made by U given that the answers to the previous i −1 queries are given by y1 · · · yi−1 ∈{ 0, 1 }0,1. (Without loss of generality, we may assume Hi,y1···yi−1 Yi ⪰0 by adding multiples of the identity and rescaling.) The oracle query made at step i corresponds to an input (Hi,y1···yi−1 Yi , ϵ, 3ϵ) to 2-LH, with (as in [5]) completeness/“YES case” and soundness/“NO case” parameters ϵ and 3ϵ, respectively, for ϵ > 0 a ﬁxed inverse polynomial. Then, Ambainis’s [5] O(log(n))-local query Hamiltonian H acts on X ⊗Y, where X = (Xi)⊗m = (C2)⊗m and Y = ⊗m i=1Yi, such that Xi is intended to encode the answer to query i with Yi encoding the ground state of the corresponding query Hamiltonian Hi,y1···yi−1 Yi . Speciﬁcally, H = m X i=1 1 4i−1 X y1,...,yi−1 i−1 O j=1 \|yj⟩⟨yj\|Xj ⊗ 2ϵ \|0⟩⟨0\|Xi ⊗IYi + \|1⟩⟨1\|Xi ⊗Hi,y1···yi−1 Yi =: m X i=1 1 4i−1 X y1,...,yi−1 My1···yi−1. (3) (3) Recall from Section 2 that a sequence of query answers y = y1 · · · ym ∈{ 0, 1 }m is correct if it corresponds to a possible execution of U. Since U can make queries to its QMA oracle which violate the QMA promise gap, the set of correct y is generally not a singleton. However, we henceforth assume without loss of generality that U makes at least one valid query (i.e. which satisﬁes the QMA promise gap). (Assume to the contrary that U makes only invalid queries. Then, since recall we stipulate [36] that the oracle’s answer on any invalid query must not aﬀect the ﬁnal output of U, it follows that for all possible query strings, U outputs the same answer. Moreover, since m ∈O(log n), we can eﬃciently verify this property — simply iterate via brute force through all possible polynomially many query strings, and check that U outputs the same answer for each one. 9While each query is encoded as a 2-local Hamiltonian, our hardness results in Section 4 will be for 5- and 4-local Hamiltonians, since the query Hamiltonians are just components of our larger, ﬁnal Hamiltonian constructions. The problem APX-SIM. (2) (2) d as Ω(∆/L3) in [29]; the constant π2/64 can be derived from the analysis therein . 12 8In this work, we let U denote a uniformly generated classical circuit, as opposed to a Turing machine; this is to ease the transition to quantum circuits and corresponding application of Kitaev’s circuit-to-Hamiltonian construction later. 10At a high level, in an exchange argument, one begins with a solution y to some problem instance, where y is lacking some additional desired property P. One then shows how to “exchange” y for another solution y′ such that y′ is at least as “good” as y for the problem at hand, and so that y′ now has the desired property P. In our application here, we will exchange query string y for a query string y′ satisfying λy′ < λy. The property P in this case will be whether the query string correct. 3 Ambainis’s Query Hamiltonian Therefore, we can assume that any reduction includes such a check as an implicit subroutine, prepended to the start of the reduction, which will handle any case in which U makes only invalid queries.) We now prove the following about H: Lemma 3.1. Deﬁne for any x ∈{ 0, 1 }m the space Hx1···xm := Nm i=1 \|xi⟩⟨xi\| ⊗Yi. Then, there exists a correct query string x ∈{ 0, 1 }m such that the ground state of H lies in Hx1···xm. Moreover, suppose this space has minimum eigenvalue λ. Then, for any incorrect query string y1 · · · ym, any state in Hy1···ym has energy at least λ + ϵ 4m . Lemma 3.1. Deﬁne for any x ∈{ 0, 1 }m the space Hx1···xm := Nm i=1 \|xi⟩⟨xi\| ⊗Yi. Then, there exists a correct query string x ∈{ 0, 1 }m such that the ground state of H lies in Hx1···xm. Moreover, suppose this space has minimum eigenvalue λ. Then, for any incorrect query string y1 · · · ym, any state in Hy1···ym has energy at least λ + ϵ 4m . As discussed in Section 1, Claim 1 of [5] proved a similar statement under the assumption that the correct query string x is unique. In that setting, [5] showed the ground state of H is in Hx, and that for all query strings y ̸= x, the space Hy has energy at least λ + ϵ 4m−1 . However, in general invalid 13 queries must be allowed, and in this setting this claim no longer holds — two distinct correct query strings can have eigenvalues which are arbitrarily close if they contain queries violating the promise gap. The key observation we make here is that even in the setting of non-unique x, a spectral gap between the ground space and all incorrect query strings can be shown. Proof of Lemma 3.1. Observe ﬁrst that H in Equation (3) is block-diagonal with respect to register X, i.e. to understand the spectrum of H, it suﬃces to understand the eigenvalues in each of the blocks corresponding to ﬁxing Xi to some string y ∈{ 0, 1 }m. Thus, we can restrict our attention to spaces Hy for y ∈{ 0, 1 }m. To begin, let x ∈{ 0, 1 }m denote a correct query string which has lowest energy among all correct query strings against H, i.e. 3 Ambainis’s Query Hamiltonian the block corresponding to x has the smallest eigenvalue among such blocks. (Note that x is well-deﬁned, though it may not be unique; in this latter case, any such x will suﬃce for our proof.) For any y ∈{ 0, 1 }m, deﬁne λy as the smallest eigenvalue in block Hy. We show that for any incorrect query string y = y1 · · · ym, λy ≥λx + ϵ/(4m). We use proof by contradiction, via an exchange argument10. Suppose there exists an incorrect query string y = y1 · · · ym such that λy < λx + ϵ/(4m). Since y is an incorrect query string, there exists an i ∈[m] such that yi is the wrong answer to a valid query Hi,y1···yi−1 Yi . Let i denote the ﬁrst such position. Now, consider operator My1···yi−1, which recall is deﬁned as My1···yi−1 = i−1 O j=1 \|yj⟩⟨yj\|Xj ⊗ 2ϵ \|0⟩⟨0\|Xi ⊗IYi + \|1⟩⟨1\|Xi ⊗Hi,y1···yi−1 Yi and let λy1···yi−1yi denote the smallest eigenvalue of My1···yi−1 restricted to space Hy1···yi−1yi, where yi denotes the complement of bit yi; for clarity, here we deﬁne and let λy1···yi−1yi denote the smallest eigenvalue of My1···yi−1 restricted to space Hy1···yi−1yi, where yi denotes the complement of bit yi; for clarity, here we deﬁne Hy1···yi−1yi := \|y1⟩⟨y1\|X1 ⊗· · · ⊗\|yi⟩⟨yi\|Xi ⊗   m O j=i+1 Xj  ⊗Y. Note that string y1 · · · yi−1yi corresponds to i correct query bits, with yi the correct answer to query i. Then, we claim that any state \|φ⟩∈Hy1···yi (i.e. \|φ⟩is of the same dimension as H, with registers X1 through Xi ﬁxed to state \|y1⟩⊗· · · ⊗\|yi⟩) satisﬁes the bound of the following lemma. Lemma 3.2. For \|φ⟩, My1···yi−1, and λy1···yi−1yi as deﬁned above, we have Lemma 3.2. For \|φ⟩, My1···yi−1, and λy1···yi−1yi as deﬁned above, we have ⟨φ\| My1···yi−1 \|φ⟩≥λy1···yi−1yi + ϵ. Here, for clarity, My1···yi−1 is of the same dimension as \|φ⟩, since we assume My1···yi−1 has an implicit tensor product with term IXi+1⊗Yi+1···⊗Xm⊗Ym. (This is intuitively because My1···yi−1 corresponds to making query i given responses to queries 1 through i −1, and queries i + 1 through m are yet to be determined.) 14 14 Proof. Constrained to space Hy1···yi−1, My1···yi−1 reduces to operator M′ := 2ϵ \|0⟩⟨0\|Xi ⊗IYi + \|1⟩⟨1\|Xi ⊗Hi,y1···yi−1 Yi , which is block-diagonal in the standard basis with respect to Xi. which implies the claim. With Lemma 3.2 in hand, we return to our exchange argument. Let c My1···yt denote the set of terms from Equation (3) which are consistent with some preﬁx y1 . . . yt (e.g. My1...yt, My1...yt0, My1...yt1, etc). Recall that the bits yi+1 · · · ym form the tail of the string y following the incorrect query answer yi. Set y′ i+1 · · · y′ m so that y′ := y1 · · · yiy′ i+1 · · · y′ m is a correct query string. Care is now required; the new query bits y′ i+1 · · · y′ m may lead to diﬀerent energy penalties than the previous string yi+1 · · · ym against the Hamiltonian terms in set c My1···yi. In other words, we must upper bound any possible energy penalty increase when exchanging y1 · · · yiyi+1 · · · ym for y′. To do so, recall that all Hamiltonian terms in Equation (3) are positive semideﬁnite. Thus, for any state \|ψ⟩in space Hy1···yi, the energy obtained by \|ψ⟩against terms in c My1···yi is at least 0. Conversely, in the worst case, since each term in c My1···yi has minimum eigenvalue at most 2ϵ, the eigenvector \|ψ⟩of smallest eigenvalue in block Hy′ incurs an additional penalty against H for queries i + 1 through m of at most (taking into account the normalization factor 1/4i−1 in Equation (3)) 2ϵ ∞ X k=i 1 4k = 2ϵ 3 · 4i−1 . We conclude that (again taking into account the normalization factor 1/4i−1 in Equation (3)) We conclude that (again taking into account the normalization factor 1/4i−1 in Equation (3)) λy′ ≤λy − ϵ 4i−1 + 2ϵ 3 · 4i−1 < λx + ϵ 4m − ϵ 4i−1 + 2ϵ 3 · 4i−1 < λx nd inequality follows by the assumption λy < λx +ϵ/4m. This is a contradiction. where the second inequality follows by the assumption λy < λx +ϵ/4m. This is a contradiction. where the second inequality follows by the assumption λy < λx +ϵ/4m. This is a contradiction. The next lemma converts H from an O(log n)-local Hamiltonian to an O(1)-local one. where the second inequality follows by the assumption λy < λx +ϵ/4m. This is a contradiction. The next lemma converts H from an O(log n)-local Hamiltonian to an O(1)-local one. Lemma 3.3. 3 Ambainis’s Query Hamiltonian Thus, if query i is a YES instance, the smallest eigenvalue of M′ lies in the block corresponding to setting Xi to (the correct query answer) \|1⟩, and is at most ϵ (since we are assuming each query has YES and NO energy thresholds ϵ and 3ϵ). On the other hand, the block with Xi set to \|0⟩by deﬁnition reduces to 2ϵIYi, and hence has all eigenvalues equaling 2ϵ. Thus, setting Xi to the correct query answer \|1⟩yields an energy penalty which is at least ϵ less than the penalty obtained for setting Xi to the wrong query answer, \|0⟩. Analogously, when query i is a NO instance (i.e. the correct query answer is \|0⟩), a similar argument shows the \|0⟩-block again has eigenvalues equaling 2ϵ and now the \|1⟩-block has eigenvalues at least 3ϵ. We conclude that in both YES and NO cases, setting Xi to the correct query answer achieves an energy penalty at least ϵ less than setting Xi to the wrong query answer. As a result, λy1···yi−1yi ≤λy1···yi −ϵ, 1. The ground state of H′ lies in subspace \|ˆx⟩⟨ˆx\| ⊗Y. which implies the claim. We now replace register X1 ⊗· · ·⊗Xm with register X ′ = (C2)⊗2m−1 and encode each binary string x ∈{ 0, 1 }m as the unary string ˆx = \|1⟩⊗\|x\| \|0⟩⊗2m−\|x\|−1, where \|x\| is the non-negative integer corresponding to string x. In other words, for Mx1···xi−1, we replace each string \|x⟩⟨x\|X1⊗···⊗Xm with \|1⟩⟨1\|X1⊗···⊗X\|x\| ⊗\|0⟩⟨0\|X\|x\|+1⊗···⊗X2m−1. Denote the resulting Hamiltonian as H1. \| \| \| \|+ To ensure states in X ′ follow this encoding, add a weighted version of Kitaev’s [45] penalty Hamiltonian, introduced in Section 2, Hstab = 3ϵ 2m−2 X j=1 \|0⟩⟨0\|j ⊗\|1⟩⟨1\|j+1 , i.e., our ﬁnal Hamiltonian is H′ = H1 + Hstab. To show that H′ satisﬁes the same properties as H as stated in the claim, we follow the analysis of Kitaev [45]. Namely, partition the space X ′ ⊗Y into orthogonal spaces S and S⊥corresponding to the space of valid and invalid unary encodings of X ′, respectively. Since H1 and Hstab act invariantly on S and S⊥, we can consider S and S⊥ separately. In S, H′ is identical to H, implying the claim. In S⊥, the smallest non-zero eigenvalue of Hstab is at least 3ϵ. Thus, since H1 ⪰0, if we can show that the smallest eigenvalue of H is at most 3ϵ −ϵ/4m, we have shown the claim (since, in particular, we will have satisﬁed statement 2 of our claim). To show this bound on the smallest eigenvalue, suppose x is all zeros, i.e. set register X1 ⊗· · · ⊗Xm for H to all zeros. Then, each term M01···0i−1 yields an energy penalty of exactly 2ϵ, yielding an upper bound on the smallest eigenvalue of H of 2ϵ Pm−1 k=0 1 4k ≤8 3ϵ = 3ϵ −ϵ/3. i.e., our ﬁnal Hamiltonian is H′ = H1 + Hstab. To show that H′ satisﬁes the same properties as H as stated in the claim, we follow the analysis of Kitaev [45]. Namely, partition the space X ′ ⊗Y into orthogonal spaces S and S⊥corresponding to the space of valid and invalid unary encodings of X ′, respectively. Since H1 and Hstab act invariantly on S and S⊥, we can consider S and S⊥ separately. In S, H′ is identical to H, implying the claim. In S⊥, the smallest non-zero eigenvalue of Hstab is at least 3ϵ. which implies the claim. Thus, since H1 ⪰0, if we can show that the smallest eigenvalue of H is at most 3ϵ −ϵ/4m, we have shown the claim (since, in particular, we will have satisﬁed statement 2 of our claim). To show this bound on the smallest eigenvalue, suppose x is all zeros, i.e. set register X1 ⊗· · · ⊗Xm for H to all zeros. Then, each term M01···0i−1 yields an energy penalty of exactly 2ϵ, yielding an upper bound on the smallest eigenvalue of H of 2ϵ Pm−1 k=0 1 4k ≤8 3ϵ = 3ϵ −ϵ/3. which implies the claim. For any x ∈{ 0, 1 }m, let ˆx denote its unary encoding. Then, for any PQMA[log] circuit U acting on n bits and making m ≥1 queries to a QMA oracle, there exists a mapping to a 4-local Hamiltonian H′ acting on space (C2)⊗2m−1 ⊗Y such that there exists a correct query string x = x1 · · · xm satisfying: 15 2. For any state \|ψ⟩in subspace \|ˆx′⟩⟨ˆx′\| ⊗Y where either ˆx′ is not a unary encoding of a binary string x′ or x′ is an incorrect query string, one has ⟨ψ\| H′ \|ψ⟩≥λ(H′) + ϵ/4m, for inverse polynomial ϵ. 2. For any state \|ψ⟩in subspace \|ˆx′⟩⟨ˆx′\| ⊗Y where either ˆx′ is not a unary encoding of a binary string x′ or x′ is an incorrect query string, one has ⟨ψ\| H′ \|ψ⟩≥λ(H′) + ϵ/4m, for inverse polynomial ϵ. 2. For any state \|ψ⟩in subspace \|ˆx′⟩⟨ˆx′\| ⊗Y where either ˆx′ is not a unary encoding of a binary string x′ or x′ is an incorrect query string, one has ⟨ψ\| H′ \|ψ⟩≥λ(H′) + ϵ/4m, for inverse polynomial ϵ. 3. For all strings x′ ∈{ 0, 1 }m, H′ acts invariantly on subspace \|ˆx′⟩⟨ˆx′\| ⊗Y. 3. For all strings x′ ∈{ 0, 1 }m, H′ acts invariantly on subspace \|ˆx′⟩⟨ˆx′\| ⊗Y. 4. The mapping can be computed in time polynomial in n (recall m ∈O(log n)). 4. The mapping can be computed in time polynomial in n (recall m ∈O(log n)) Proof. We show how to improve the O(log n)-local construction H of Lemma 3.1 to 4-local H′. Speciﬁcally, recall that H from Equation (3) acts on (X ⊗Y). Using a trick of Kitaev [45], we encode the X = X1 ⊗· · · ⊗Xm register in unary. Speciﬁcally, we can write My1···yi−1 = X yi+1,...,ym 2ϵ i−1 O j=1 \|yj⟩⟨yj\|Xj ⊗\|0⟩⟨0\|Xi m O k=i+1 \|yk⟩⟨yk\|Xk ⊗IY + i−1 O j=1 \|yj⟩⟨yj\|Xj ⊗\|1⟩⟨1\|Xi m O k=i+1 \|yk⟩⟨yk\|Xk ⊗Hi,y1···yi−1 Yi . We now replace register X1 ⊗· · ·⊗Xm with register X ′ = (C2)⊗2m−1 and encode each binary string x ∈{ 0, 1 }m as the unary string ˆx = \|1⟩⊗\|x\| \|0⟩⊗2m−\|x\|−1, where \|x\| is the non-negative integer corresponding to string x. In other words, for Mx1···xi−1, we replace each string \|x⟩⟨x\|X1⊗···⊗Xm with \|1⟩⟨1\|X1⊗···⊗X\|x\| ⊗\|0⟩⟨0\|X\|x\|+1⊗···⊗X2m−1. Denote the resulting Hamiltonian as H1. 4.1 PQMA[log]-completeness of APX-SIM 4.1 PQMA[log]-completeness of APX-SIM In this section, we restate and prove Theorem 1.1. For this, we ﬁrst show an extension of the Projection Lemma of Kempe, Kitaev, Regev [43]. 16 16 Lemma 4.1 (Extended Projection Lemma (cf. [43])). Let H = H1 + H2 be the sum of two Hamiltonians operating on some Hilbert space H = S + S⊥. The Hamiltonian H1 is such that S is a zero eigenspace and the eigenvectors in S⊥have eigenvalue at least J > 2 ∥H2∥∞. Let K := ∥H2∥∞. Then, for any δ ≥0 and \|ψ⟩satisfying ⟨ψ\| H \|ψ⟩≤λ(H) + δ, there exists a \|ψ′⟩∈S such that: Lemma 4.1 (Extended Projection Lemma (cf. [43])). Let H = H1 + H2 be the sum of two Hamiltonians operating on some Hilbert space H = S + S⊥. The Hamiltonian H1 is such that S is a zero eigenspace and the eigenvectors in S⊥have eigenvalue at least J > 2 ∥H2∥∞. Let K := ∥H2∥∞. Then, for any δ ≥0 and \|ψ⟩satisfying ⟨ψ\| H \|ψ⟩≤λ(H) + δ, there exists a \|ψ′⟩∈S such that: • (Ground state energy bound) λ(H2\|S) − K2 J −2K ≤λ(H) ≤λ(H2\|S), where λ(H2\|S) denotes the smallest eigenvalue of H2 restricted to space S. where λ(H2\|S) denotes the smallest eigenvalue of H2 restricted to space S. • (Ground state deviation bound) • (Ground state deviation bound) \|⟨ψ\|ψ′⟩\|2 ≥1 − K + p K2 + δ(J −2K) J −2K !2 . • (Energy obtained by perturbed state against H) • (Energy obtained by perturbed state against H) • (Energy obtained by perturbed state against H) ⟨ψ′\| H \|ψ′⟩≤λ(H) + δ + 2K K + p K2 + δ(J −2K) J −2K . Proof. The ﬁrst claim is the original Projection Lemma [43] (which we do not require in this work, but which we also state above for completeness). The second and third claims we prove here. Consider arbitrary \|ψ⟩such that ⟨ψ\| H \|ψ⟩≤λ(H) + δ. We can write \|ψ⟩= α1 \|ψ1⟩+ α2 \|ψ2⟩for unit vectors \|ψ1⟩∈S, \|ψ2⟩∈S⊥, α1, α2 ∈R, α1, α2 ≥0, and α2 1 + α2 2 = 1. Following the proof of the Projection Lemma of [43], we have Proof. The ﬁrst claim is the original Projection Lemma [43] (which we do not require in this work, but which we also state above for completeness). The second and third claims we prove here. Consider arbitrary \|ψ⟩such that ⟨ψ\| H \|ψ⟩≤λ(H) + δ. We can write \|ψ⟩= α1 \|ψ1⟩+ α2 \|ψ2⟩for unit vectors \|ψ1⟩∈S, \|ψ2⟩∈S⊥, α1, α2 ∈R, α1, α2 ≥0, and α2 1 + α2 2 = 1. Following the proof of the Projection Lemma of [43], we have ⟨ψ\| H \|ψ⟩ ≥ ⟨ψ\| H2 \|ψ⟩+ Jα2 2 = (1 −α2 2) ⟨ψ1\| H2 \|ψ1⟩+ 2α1α2 Re ⟨ψ1\| H2 \|ψ2⟩+ α2 2 ⟨ψ2\| H2 \|ψ2⟩+ Jα2 2 ≥ ⟨ψ1\| H2 \|ψ1⟩−K(α2 2 + 2α2 + α2 2) + Jα2 2 = ⟨ψ1\| H \|ψ1⟩+ (J −2K)α2 2 −2Kα2, (4) 2 ⟨ψ2\| 2 \|ψ2⟩+ 2 ≥ ⟨ψ1\| H2 \|ψ1⟩−K(α2 2 + 2α2 + α2 2) + Jα2 2 = ⟨ψ1\| H \|ψ1⟩+ (J −2K)α2 2 −2Kα2, (4) (4) where the last equality follows since \|ψ1⟩∈S. Since by assumption ⟨ψ\| H \|ψ⟩≤λ(H) + δ, Equation (4) implies λ(H) + δ ≥⟨ψ1\| H \|ψ1⟩+ (J −2K)α2 2 −2Kα2. Combined with the fact λ(H) ≤⟨ψ1\| H \|ψ1⟩, we have 0 ≥λ(H) −⟨ψ1\| H \|ψ1⟩≥(J −2K)α2 2 −2Kα2 −δ, which holds only if \|α2\| ≤K+√ K2+δ(J−2K) J−2K . Thus, setting \|ψ′⟩:= \|ψ1⟩yields the ﬁrst claim. To see that \|ψ′⟩also satisﬁes the second claim, by Equation (4) we have which holds only if \|α2\| ≤K+√ K2+δ(J−2K) J−2K . Thus, setting \|ψ′⟩:= \|ψ1⟩yields the ﬁrst claim. To see that \|ψ′⟩also satisﬁes the second claim, by Equation (4) we have λ(H) + δ ≥⟨ψ\| H \|ψ⟩≥⟨ψ1\| H \|ψ1⟩+ (J −2K)α2 2 −2Kα2. The assumption J > 2K implies (J −2K)α2 2 ≥0, and so ⟨ψ1\| H \|ψ1⟩≤λ(H) + δ + 2K \|α2\| . • (Energy obtained by perturbed state against H) ⟨ψ1\| H \|ψ1⟩≤λ(H) + δ + 2K \|α2\| . Plugging in our upper bound on \|α2\| now yields the second claim. Plugging in our upper bound on \|α2\| now yields the second claim. Plugging in our upper bound on \|α2\| now yields the second claim. 17 With Lemma 4.1 in hand, we now prove the main result of this section. Theorem 1.1. APX-SIM is PQMA[log]-complete for k = 5 and l = 1, i.e., for 5-local Hamiltonian H and 1-local observable A. Proof. Containment in PQMA[log] was shown11 for k, l ∈O(log n) in [5], for n the input size. We show PQMA[log]-hardness of APX-SIM via a polynomial-time mapping/Karp reduction. Let U′ be an arbitrary PQMA[log] circuit12 for instance Π, such that U′ acts on workspace register W and query result register Q. Suppose U′ consists of L′ gates and makes m = c log(n) queries, for c ∈O(1). Without loss of generality, U′ can be simulated with a similar unitary U which treats Q as a proof register which it does not alter at any point: Namely, U does not have access to a QMA oracle, but rather reads bit Qi whenever it desires the answer to the i-th query. Thus, if a correct query string y1 · · · ym corresponding to an execution of U′ on input x is provided in Q as a “proof”, then the output statistics of U′ and U are identical. We can also assume that Q is encoded not in binary, but in unary. Thus, Q consists of 2m −1 ∈poly(n) bits. For simplicity, however, in our discussion we will speak of m-bit query strings y = y1 · · · ym in register Q. The construction. We now construct our instance of APX-SIM. First, we map U to a 5-local Hamiltonian H1 via a modiﬁcation of the circuit-to-Hamiltonian construction of Kitaev [45], such that H1 acts on registers W (workspace register), Q (proof register), and C (clock register). Recall (Section 2) that Kitaev’s construction outputs Hamiltonian terms Hin + Hprop + Hstab + Hout. Set H1 = ∆(Hin + Hprop + Hstab) for ∆to be set as needed. In contrast to [45], it is crucial that Hout be omitted from H1, as we require our ﬁnal Hamiltonian H to enforce a certain structure on the ground space regardless of whether the computation should accept or reject. 11Intuitively, the idea is as follows. First, the P machine uses O(log n) queries to perform a version of binary search tailored to promise problems to estimate the ground state energy of the given Hamiltonian H to within additive inverse polynomial error; call this estimate g. Then, the P machine makes one ﬁnal QMA query: Does H have a ground state \|ψ⟩with ground state energy approximately g and with small expectation against the given observable A? 12As noted in Section 3, the base class P is normally deﬁned using a polynomial-time deterministic Turing machine. In this paper, we assume the action of the P machine is, without loss of generality, instead expressed via a polynomial- time uniform family of quantum circuits. • (Energy obtained by perturbed state against H) pair of lemmas together show this. They both assume the terminology set up in this proof thus far. Lemma 4.2. Suppose Π is a YES instance of a PQMA[log] problem. Then, (H, A, k, l, a, b, δ) is a YES instance of APX-SIM. Proof. By Lemma 3.3, the ground space of H2 is spanned by states of the form \|ˆx⟩Q ⊗\|φ⟩Q′ where ˆx is a correct query string encoded in unary. Fix an arbitrary such ground state \|ˆx⟩Q ⊗\|φ⟩Q′. Since Π is a YES instance, setting Q to ˆx in this manner causes U = UL · · · U1 to accept with certainty. Consider a version of the history state \|ψhist⟩from Equation (1) on registers W (workspace), C (clock), Q, and Q′ (Q and Q′ together are the “proof register”, where recall U reads query answers from Q but does not access Q′ at any point), \|ψhist⟩= 1 √ L + 1 L X t=0 Ut · · · U1 \|ˆx⟩Q \|φ⟩Q′ \|0 · · · 0⟩W \|ˆt⟩C , which by construction lies in the ground space of H1. Since U can read but does not alter the contents of Q, the history state has the tensor product form which by construction lies in the ground space of H1. Since U can read but does not alter the contents of Q, the history state has the tensor product form \|ψhist⟩= \|ψ′ hist(x)⟩W,C ⊗\|ˆx⟩Q ⊗\|φ⟩Q′ for appropriately deﬁned \|ψ′ hist(x)⟩W,C. Since the state encodes a correct query string in register Q, \|ψhist⟩lies in the ground space of H2. We conclude that \|ψhist⟩is in the joint ground space of H1 and H2, and hence in the ground space of H = H1 + H2. Finally, by assumption, the output qubit W1 of the construction is set to \|0⟩in timesteps 0 to L −1. In timestep L, since U accepts with certainty given the correct query string ˆx, W1 is set to \|1⟩. It follows that, as desired, ⟨ψhist\| A \|ψhist⟩= 1 2 ⟨ψhist\| (I + Z) \|ψhist⟩= 1 − 1 L + 1 = a. Lemma 4.3. Suppose Π is a NO instance of a PQMA[log] problem. Then, (H, A, k, l, a, b, δ) is a NO instance of APX-SIM. Lemma 4.3. Suppose Π is a NO instance of a PQMA[log] problem. Then, (H, A, k, l, a, b, δ) is a NO instance of APX-SIM. Lemma 4.3. • (Energy obtained by perturbed state against H) The job of “checking the output” will instead be delegated to the observable A. Formally, following the discussion in Section 2 regarding Equation (1), H1 has a non-trivial null space, which is its ground space, consisting of history states \|ψhist⟩(see Equation (1)) which simulate U on registers W and Q. These history states correctly simulate U′ assuming that Q is initialized to a correct proof. To thus enforce that Q is initialized to a correct proof, let H2 be our variant H′ of Ambainis’s query Hamiltonian from Lemma 3.3, such that H2 acts on registers Q and Q′, where for clarity Q = (C2)⊗2m−1 for m ∈O(log n); moreover, setting Q′ = Y from Lemma 3.3 enforces the ground space of H2 to contain unary encodings of valid strings of query answers y1 · · · ym for input Π in register Q, as desired. Our ﬁnal Hamiltonian is H = H1 + H2, which is 5-local since H1 is 5-local and H2 is 4-local. Suppose without loss of generality that U’s output qubit is W1, which is set to \|0⟩until the ﬁnal time step, in which the correct output is copied to it. Then, set observable A = (I + Z)/2 such that A acts on qubit W1. Set a = 1 −1/(L + 1), and b = 1 −1/2L for L the number of gates in U. Fix η ≥max(∥H2∥∞, 1) (such an η can be eﬃciently computed by applying the triangle 18 inequality and summing the spectral norms of each term of H2 individually). Set ∆= 128L3ηγ for γ a monotonically increasing polynomial function of L to be set as needed. Finally, set δ = 1/∆. This completes the construction. inequality and summing the spectral norms of each term of H2 individually). Set ∆= 128L3ηγ for γ a monotonically increasing polynomial function of L to be set as needed. Finally, set δ = 1/∆. This completes the construction. Correctness. We now prove correctness, meaning that if Π is a YES (NO) instance of a PQMA[log] problem, then APX-SIM(H, A, 5, 1, a, b, δ) is a YES (NO) instance of APX-SIM. The following pair of lemmas together show this. They both assume the terminology set up in this proof thus far. Lemma 4.2. Suppose Π is a YES instance of a PQMA[log] problem. Then, (H, A, k, l, a, b, δ) is a YES instance of APX-SIM. • (Energy obtained by perturbed state against H) Suppose Π is a NO instance of a PQMA[log] problem. Then, (H, A, k, l, a, b, δ) is a NO instance of APX-SIM. Proof. Consider any low energy state \|ψ⟩satisfying ⟨ψ\| H \|ψ⟩≤λ(H) + δ. By Lemma 2.2, the smallest non-zero eigenvalue of H1 is at least J = π2∆/(64L3) = π2ηγ/64. Recalling that δ = 1/∆, apply Lemma 4.1 to obtain that there exists a valid history state \|ψ′⟩on W, C, Q, and Q′ such that \|⟨ψ\|ψ′⟩\|2 ≥1 −O(γ−2) (see Lemma A.1 in Appendix A for a proof), which by Equation (2) implies \|ψ⟩⟨ψ\| −\|ψ′⟩⟨ψ′\| tr ≤c γ (5) (5) 19 for some constant c > 0. By deﬁnition, such a history state \|ψ′⟩simulates U given “quantum proof” \|φ⟩Q,Q′ in registers Q and Q′, i.e. \|ψ′⟩= X t Ut · · · U1 \|φ⟩Q,Q′ \|0 · · · 0⟩W \|t⟩C . Lemma 4.1 and the proof of Lemma A.1 also give that Lemma 4.1 and the proof of Lemma A.1 also give that ⟨ψ′\| H \|ψ′⟩≤λ(H) + δ + O η γ2 =: λ(H) + δ + γ′. Unfortunately, a priori the state \|φ⟩in the proof register (Q, Q′) of \|ψ′⟩is arbitrary. Let us now approximate \|ψ′⟩with another history state \|ψ′′⟩, which contains a string of correct query answers in Q. This is accomplished by the following lemma, which assumes the deﬁnitions introduced in this proof thus far. Unfortunately, a priori the state \|φ⟩in the proof register (Q, Q′) of \|ψ′⟩is arbitrary. Let us now approximate \|ψ′⟩with another history state \|ψ′′⟩, which contains a string of correct query answers in Q. This is accomplished by the following lemma, which assumes the deﬁnitions introduced in this proof thus far. There exists a history state \|ψ′′⟩such that Lemma 4.4. There exists a history state \|ψ′′⟩such that \|ψ⟩⟨ψ\| −\|ψ′′⟩⟨ψ′′\| tr ≤c γ + 2 s 4m(δ + γ′) ϵ (6) (6) and where register Q of \|ψ′′⟩contains only correct query answers when written in the standard basis. Proof. By Lemma 3.3, the ground space G of H2 is contained in the span of states of the form \|ˆx⟩Q ⊗\|φ′⟩Q′ where ˆx is a correct query string encoded in unary. Since the ground spaces of H1 and H2 have non-empty intersection, i.e. history states acting on “quantum proofs” from G (which lie in the null space of H1 and obtain energy λ(H2) against H2), we know λ(H) = λ(H2). • (Energy obtained by perturbed state against H) Thus, since H1 ⪰0, ⟨ψ′\| H2 \|ψ′⟩≤⟨ψ′\| H \|ψ′⟩≤λ(H2) + (δ + γ′). (7) (7) Write \|φ⟩= α \|φ1⟩+ β \|φ2⟩for \|φ1⟩∈Span { \| ˆx⟩Q ⊗\| φ′⟩Q′ \| correct query string x } and \|φ2⟩∈ Span { \| ˆx⟩Q ⊗\| φ′⟩Q′ \| incorrect query string x } (\|φ1⟩, \|φ2⟩normalized), α, β ∈C, \|α\|2 +\|β\|2 = 1. Since any history state \|ψ′⟩, for any amplitudes αx and unit vectors \|φ′ x⟩, has form X t,x αxUt · · · U1 \|0 · · · 0⟩W \|t⟩C \|ˆx⟩Q \|φ′ x⟩Q′ = X x αx \|ψ′ hist(x)⟩W,C \|ˆx⟩Q \|φ′ x⟩Q′ (i.e. for any ﬁxed x, \|ˆx⟩Q is not altered), and since H2 is block-diagonal with respect to the standard basis in Q, by Equation (7) and Lemma 3.3 we have (i.e. for any ﬁxed x, \|ˆx⟩Q is not altered), and since H2 is block-diagonal with respect to the standard basis in Q, by Equation (7) and Lemma 3.3 we have λ(H2) + (δ + γ′) ≥ ⟨ψ′\| H2 \|ψ′⟩= \|α\|2 ⟨φ1\| H2 \|φ1⟩+ \|β\|2 ⟨φ2\| H2 \|φ2⟩ ≥ \|α\|2 λ(H2) + \|β\|2 λ(H2) + ϵ 4m , which implies \|β\|2 ≤4m(δ + γ′)/ϵ. Thus, deﬁning \|ψ′′⟩as the history state for “proof” \|φ1⟩Q,Q′, we have that ∥\|ψ⟩⟨ψ\| −\|ψ′′⟩⟨ψ′′\|∥tr is at most which implies \|β\|2 ≤4m(δ + γ′)/ϵ. Thus, deﬁning \|ψ′′⟩as the history state for “proof” \|φ1⟩Q,Q′, we have that ∥\|ψ⟩⟨ψ\| −\|ψ′′⟩⟨ψ′′\|∥tr is at most \|ψ⟩⟨ψ\| −\|ψ′⟩⟨ψ′\| tr + ∥\|φ⟩⟨φ\| −\|φ1⟩⟨φ1\|∥tr ≤c γ + 2 s 4m(δ + γ′) ϵ , which follows from the triangle inequality, Equation (5), and the structure of the history state. which follows from the triangle inequality, Equation (5), and the structure of the history state. from the triangle inequality, Equation (5), and the structure of the history state. 20 With Lemma 4.4 in hand, we are ready to complete the proof of Lemma 4.3. Observe that increasing γ by a polynomial factor decreases γ′ by a polynomial factor,13 so δ + γ′ in Equation (6) also decreases by a polynomial factor. Thus, set γ as a large enough polynomial in L such that c γ + 2 s 4m(δ + γ′) ϵ ≤1 2L. 13In a previous version of this article, γ′ ∈Θ(∥H∥∞/γ). But this was circularly deﬁned (recall H = H1 + H2), since ∥H1∥∞scales with ∆, which in turn scales with γ. In the present version of the article, γ′ ∈O η/γ2 , where η depends only on ∥H2∥∞, and is thus independent of γ. Hence, increasing γ now correctly decreases γ′. • (Energy obtained by perturbed state against H) (8) (8) Since U rejects any correct query string (with certainty) in the NO case, and since \|ψ′′⟩is a valid history state whose Q register is a superposition over correct query strings (all of which must lead to reject), we conclude that ⟨ψ′′\| A \|ψ′′⟩= 1. Moreover, we have that Tr(A \|ψ⟩⟨ψ\|) −Tr(A \|ψ′′⟩⟨ψ′′\|) ≤∥A∥∞ \|ψ⟩⟨ψ\| −\|ψ′′⟩⟨ψ′′\| tr ≤1 2L, where the ﬁrst inequality follows from Hölder’s inequality, and the second by Equations (6) and (8). We conclude that ⟨ψ\| A \|ψ⟩≥1 −1/(2L), completing the proof. ( ) 4.2 PQMA[log]-completeness of APX-2-CORR Similar to the proof of Theorem 1.1, set ∆= 128L3ηγ and δ = 1/∆, for γ large enough so that s c γ + 2 s 4m(δ + γ′) ϵ ≤ 1 2(L + 13), (9) (9) for γ′ as deﬁned in the proof of Theorem 1.1. Set a = 3/(L + 13) and b = 1/(L + 13). This completes the construction. To set up the correctness proof, consider history state \|ψhist⟩for V given quantum proof \|φ⟩Q,Q′, and deﬁne for brevity \|φt⟩:= Vt · · · V1 \|φ⟩Q,Q′ \|0 · · · 0⟩W \|00⟩W2W3. Then, ⟨ψhist\| ZW2 ⊗ZW3 \|ψhist⟩= 1 L + 13 L+12 X t=0 Tr (\|φt⟩⟨φt\|Q,Q′,W ⊗\|t⟩⟨t\|C)ZW2 ⊗ZW3 , (10) (10) since ZW2 ⊗ZW3 acts invariantly on the clock register. Deﬁning \|v⟩:= PL+12 t=L+1 \|φt⟩Q,Q′,W \|t⟩C, we have that since W2W3 is set to \|00⟩for times 0 ≤t ≤L, Equation (10) simpliﬁes to since ZW2 ⊗ZW3 acts invariantly on the clock register. Deﬁning \|v⟩:= PL+12 t=L+1 \|φt⟩Q,Q′,W \|t⟩C, we have that since W2W3 is set to \|00⟩for times 0 ≤t ≤L, Equation (10) simpliﬁes to 1 L + 13 (L + 1 + ⟨v\| ZW2 ⊗ZW3 \|v⟩) . 1 L + 13 (L + 1 + ⟨v\| ZW2 ⊗ZW3 \|v⟩) . Thus, via similar reasoning Thus, via similar reasoning f(\|ψhist⟩, ZW2, ZW3) = 1 L + 13 [(L + 1) + ⟨v\| ZW2 ⊗ZW3 \|v⟩] − 1 (L + 13)2 [(L + 1) + ⟨v\| ZW2 \|v⟩] [(L + 1) + ⟨v\| ZW3 \|v⟩)] . (11) = 1 L + 13 [(L + 1) + ⟨v\| ZW2 ⊗ZW3 \|v⟩] − 1 (L + 13)2 [(L + 1) + ⟨v\| ZW2 \|v⟩] [(L + 1) + ⟨v\| ZW3 \|v⟩)] . (11) (11) Suppose now that Π is a YES instance. Then there exists a history state \|ψhist⟩in the ground space of H (i.e. with quantum proof \|φ⟩Q,Q′ = \|ˆx⟩Q ⊗\|φ′⟩Q′ for a correct query string x) for which W2W3 is set to \|φ+⟩in the ﬁnal seven timesteps (since U′ is deterministic). Since ⟨φ+\| Z ⊗Z \|φ+⟩= 1 and ⟨φ+\| Z ⊗I \|φ+⟩= 0, via Equation (11) we have f(\|ψhist⟩, ZW2, ZW3) ≥(L + 1) −5 + 7 L + 13 −((L + 1) + 5)2 (L + 13)2 = 1 L + 13 4 − 49 L + 13 , where the ±5 terms correspond to timesteps t = L + 1, . . 4.2 PQMA[log]-completeness of APX-2-CORR We now deﬁne APX-2-CORR and show that it is PQMA[log]-complete using similar techniques to Section 4.1. For brevity, deﬁne f(\|ψ⟩, A, B) := ⟨ψ\| A ⊗B \|ψ⟩−⟨ψ\| A \|ψ⟩⟨ψ\| B \|ψ⟩. We now deﬁne APX-2-CORR and show that it is PQMA[log]-complete using similar techniques to Section 4.1. For brevity, deﬁne f(\|ψ⟩, A, B) := ⟨ψ\| A ⊗B \|ψ⟩−⟨ψ\| A \|ψ⟩⟨ψ\| B \|ψ⟩. Deﬁnition 4.5 (APX-2-CORR(H, A, B, k, l, a, b, δ)). Given a k-local Hamiltonian H, l-local ob- servables A and B, and real numbers a, b, and δ such that a −b ≥n−c and δ ≥n−c′, for n the number of qubits H acts on and c, c′ ≥0 some constants, decide: • If H has a ground state \|ψ⟩satisfying f(\|ψ⟩, A, B) ≥a, output YES. or any \|ψ⟩satisfying ⟨ψ\| H \|ψ⟩≤λ(H) + δ it holds that f(\|ψ⟩, A, B) ≤b, output N We now prove Theorem 1.2 by showing PQMA[log]-hardness in Lemma 4.6 and containment in PQMA[log] in Lemma 4.7. We now prove Theorem 1.2 by showing PQMA[log]-hardness in Lemma 4.6 and containment in PQMA[log] in Lemma 4.7. Lemma 4.6. APX-2-CORR is PQMA[log]-hard for k = 5 and l = 1, i.e., for 5-local Hamiltonian H and 1-local observables A and B. Proof. For an arbitrary PQMA[log] circuit U′, deﬁne U as in the proof of Theorem 1.1, consisting of L one- and two-qubit gates. We modify U as follows. Let U’s output qubit be denoted W1. We add two ancilla qubits, W2 and W3, which are set to \|00⟩throughout U’s computation. We then append to U a sequence of six 2-qubit gates which, controlled on W1, map \|00⟩in W2W3 to \|φ+⟩= (\|00⟩+\|11⟩)/ √ 2, e.g. apply a controlled Hadamard gate and the 5-gate Toﬀoli construction from Figure 4.8 of [50]. Appending six identity gates on W1, we obtain a circuit V = VL+12 · · · V1 which has L+12 gates. Finally, we construct H = H1+H2 as in the proof of Theorem 1.1, mapping V to a 5-local Hamiltonian H1 on registers W, Q, and C, and we set A = ZW2 and B = ZW3 for 21 Pauli Z. Lemma 4.7. APX-2-CORR is in PQMA[log]. Lemma 4.7. APX-2-CORR is in PQMA[log]. Proof. The proof combines ideas from Ambainis’s original proof of APX-SIM ∈PQMA[log] [5] (see Theorem 6 therein) and a trick of Chailloux and Sattath [19] from the study of QMA(2). We give a proof sketch here. Speciﬁcally, let Π = (H, A, B, k, l, a, b, δ) be an instance of APX-2-CORR. Similar to [5], the PQMA[log] veriﬁcation procedure proceeds, at a high level, as follows: 1. Use logarithmically many QMA oracle queries to perform a binary search to obtain an esti- mate γ ∈R satisfying λ(H) ∈[γ, γ + δ 2]. 1. Use logarithmically many QMA oracle queries to perform a binary search to obtain an esti- mate γ ∈R satisfying λ(H) ∈[γ, γ + δ 2]. 2. Use a single QMA oracle query to verify the statement: “There exists \|ψ⟩satisfying (1) ⟨ψ\| H \|ψ⟩≤λ(H) + δ and (2) f(\|ψ⟩, A, B) ≥a”. 2. Use a single QMA oracle query to verify the statement: “There exists \|ψ⟩satisfying (1) ⟨ψ\| H \|ψ⟩≤λ(H) + δ and (2) f(\|ψ⟩, A, B) ≥a”. The ﬁrst of these steps is performed identically to the proof of APX-SIM ∈PQMA[log] [5]; we do not elaborate further here. The second step, however, diﬀers from [5] for the following reason. Intuitively, [5] designs a QMA protocol which takes in many copies of a proof \|ψ⟩, performs phase estimation on each copy, postselects to “snap” each copy of \|ψ⟩into a low-energy state \|ψi⟩of H, and subsequently uses states { \| ψi⟩} to estimate the expectation against an observable A. If the ground space of H is degenerate, the states { \| ψi⟩} may not all be identical. This does not pose a problem in [5], as there soundness of the protocol is guaranteed since all low energy states have high expectation against A. In our setting, however, if we use this protocol to individually estimate each of the terms ⟨ψ\| A ⊗B \|ψ⟩, ⟨ψ\| A \|ψ⟩, and ⟨ψ\| B \|ψ⟩, soundness can be violated if each of these three terms are not estimated using the same state \|ψi⟩, since the promise gap of the input does not necessarily say anything about the values of each of these three terms individually. Lemma 4.7. APX-2-CORR is in PQMA[log]. To circumvent this, we observe that to evaluate f(\|ψ⟩, A, B), we do not need the ground state \|ψ⟩itself, but only a classical description of its local reduced density matrices (a similar idea was used in [19] to verify the energy of a claimed product state proof against a local Hamiltonian in the setting of QMA(2)). Speciﬁcally, suppose Π consists of a k-local Hamiltonian H acting on n qubits. Then, the prover sends classical descriptions of k-qubit density matrices { ρS } for each subset S ⊆[n] of size \|S\| = k, along with a QMA proof that the states { ρS } are consistent with a global n-qubit pure state \|ψ⟩(recall the problem of verifying consistency is QMA-complete [47]). The veriﬁer runs the QMA circuit for consistency, and assuming this check passes it uses the classical { ρS } to classically verify that (1) ⟨ψ\| H \|ψ⟩≤λ(H) + δ and (2) f(\|ψ⟩, A, B) ≥a (since both of these depend only on the local states { ρS }). 4.2 PQMA[log]-completeness of APX-2-CORR . , L + 5 and use the fact that ∥Z∥∞= 1. Conversely, suppose Π is a NO instance, and consider any \|ψ⟩satisfying ⟨ψ\| H \|ψ⟩≤λ(H) + δ. Then, as argued in the proof of Theorem 1.1, there exists a history state \|ψ′′⟩on “proof” \|φ1⟩Q,Q′ (consisting of a superposition of correct query strings) satisfying \|ψ⟩⟨ψ\| −\|ψ′′⟩⟨ψ′′\| tr ≤ 1 2(L + 13), (12) (12) by Equations (6), (8) and (9). Since the history state \|ψ′′⟩has W2W3 set to \|00⟩in all time steps, we know because Z ⊗Z \|00⟩= \|00⟩that f(\|ψ′′⟩, ZW2, ZW3) = 0. Thus, using Equation (11) and applying the Hölder inequality to the second term of f(\|ψ⟩, ZW2, ZW3) yields f(\|ψ⟩, ZW2, ZW3) ≤1 − 1 − 1 2(L + 13) 2 = 1 L + 13 1 − 1 4(L + 13) . 22 4.3 PUQMA[log]-hardness of SPECTRAL-GAP We now restate and prove Theorem 1.3. We begin by deﬁning SPECTRAL-GAP and UQMA. Deﬁnition 4.8 (SPECTRAL-GAP(H, α) (Ambainis [5])). Given a Hamiltonian H and a real number α ≥n−c for n the number of qubits H acts on and c > 0 some constant, decide: • If λ2 −λ1 ≤α, output YES. • If λ2 −λ1 ≥2α, output NO. where λ2 and λ1 denote the second and ﬁrst smallest eigenvalues of H, respectively. where λ2 and λ1 denote the second and ﬁrst smallest eigenvalues of H, respectively. For clarity, if the ground space of H is degenerate, then we deﬁne its spectral gap as 0. arity, if the ground space of H is degenerate, then we deﬁne its spectral gap as 0. 23 Deﬁnition 4.9 (Unique QMA (UQMA) (Aharonov et al. [4])). We say a promise problem A = (Ayes, Ano) is in Unique QMA if and only if there exist polynomials p, q and a polynomial-time uniform family of quantum circuits { Qn }, where Qn takes as input a string x ∈Σn, a quantum proof \|y⟩∈(C2)⊗p(n), and q(n) ancilla qubits in state \|0⟩⊗q(n), such that: Deﬁnition 4.9 (Unique QMA (UQMA) (Aharonov et al. [4])). We say a promise problem A = (Ayes, Ano) is in Unique QMA if and only if there exist polynomials p, q and a polynomial-time uniform family of quantum circuits { Qn }, where Qn takes as input a string x ∈Σn, a quantum proof \|y⟩∈(C2)⊗p(n), and q(n) ancilla qubits in state \|0⟩⊗q(n), such that: • (Completeness) If x ∈Ayes, then there exists a proof \|y⟩∈(C2)⊗p(n) such that Qn accepts (x, \|y⟩) with probability at least 2/3, and for all \|ˆy⟩∈(C2)⊗p(n) orthogonal to \|y⟩, Qn accepts (x, \|ˆy⟩) with probability at most 1/3. • (Soundness) If x ∈Ano, then for all proofs \|y⟩∈(C2)⊗p(n), Qn accepts (x, \|y⟩) with probability at most 1/3. The main theorem of this section is the following. Theorem 1.3. SPECTRAL-GAP is PUQMA[log]-hard for 4-local Hamiltonians H under polynomial- time Turing reductions (i.e. Cook reductions). Theorem 1.3. SPECTRAL-GAP is PUQMA[log]-hard for 4-local Hamiltonians H under polynomial- time Turing reductions (i.e. Cook reductions). We remark that Ambainis [5] showed that SPECTRAL-GAP ∈PQMA[log], and gave a claimed proof that SPECTRAL-GAP is PUQMA[log]-hard for O(log)-local Hamiltonians under mapping reductions. Proof. We begin by giving the construction of H. 4.3 PUQMA[log]-hardness of SPECTRAL-GAP (PUQMA[log] is deﬁned as PQMA[log], except with a UQMA oracle in place of a QMA oracle.) As discussed in Section 1, however, Ambainis’ proof of the latter result does not hold if the PUQMA[log] machine makes invalid queries (which in general is the case). Here, we build on Ambainis’ approach [5] to show PUQMA[log]-hardness of SPECTRAL-GAP under Turing reductions even when invalid queries are allowed, and we also improve the hardness to apply to O(1)-local Hamiltonians. ( ) We begin by showing the following modiﬁed version of Lemma 3.3 tailored to UQMA (instead of QMA). It is crucial to observe that the mapping of Lemma 3.3 which produces Hamiltonian H is eﬃcient, i.e. H can be computed in polynomial time. However, in Lemma 4.10 below, our mapping to a Hamiltonian H is not clearly eﬃcient, meaning the lemma only shows the existence of H. Roughly, this is because the proof of Lemma 4.10 proceeds by replacing invalid queries with “dummy” NO queries to obtain the desired spectral gap. But a polynomial-time machine alone cannot identify such invalid queries, and hence cannot simulate this mapping. Henceforth, we assume that all calls to the UQMA oracle Q are for an instance (H, a, b) of the Unique-Local Hamiltonian Problem (U-LH) (which is complete for UQMA [5]): Is the ground state energy of H at most ϵ with all other eigenvalues at least 3ϵ (YES case), or is the ground state energy at least 3ϵ (NO case), for ϵ ≥1/poly(n)? Lemma 4.10. For any x ∈{ 0, 1 }m, let ˆx denote its unary encoding. Then, for any PUQMA[log] circuit U acting on n bits and making m queries to a UQMA oracle, there exists a 4-local Hamil- tonian H acting on space (C2)⊗2m−1 ⊗Y such that there exists a correct query string x = x1 · · · xm such that: 1. The unique ground state of H lies in subspace \|ˆx⟩⟨ˆx\| ⊗Y. 2. The spectral gap of H is at least (ϵ−δ)/4m for inverse polynomial ϵ, δ with ϵ−δ ≥1/poly(n). 2. The spectral gap of H is at least (ϵ−δ)/4m for inverse polynomial ϵ, δ with ϵ−δ ≥1/poly(n). 3. For all strings x′ ∈{ 0, 1 }m, H acts invariantly on subspace \|ˆx′⟩⟨ˆx′\| ⊗Y. P f W b i b i i th t ti f H 3. 3. For all strings x′ ∈{ 0, 1 }m, H acts invariantly on subspace \|ˆx′⟩⟨ˆx′\| ⊗Y. 4.3 PUQMA[log]-hardness of SPECTRAL-GAP For all strings x′ ∈{ 0, 1 }m, H acts invariantly on subspace \|ˆx′⟩⟨ˆx′\| ⊗Y. 24 Construction. As done in [5], we begin with O(log n)-local Hamiltonian H′ = m X i=1 1 4i−1 X y1,...,yi−1 i−1 O j=1 \|yj⟩⟨yj\|Xj ⊗G′ y1···yi−1, (13) (13) where we deﬁne G′ y1···yi−1 := \|0⟩⟨0\|Xi ⊗AYi + \|1⟩⟨1\|Xi ⊗Hi,y1···yi−1 Yi (14) (14) with A any ﬁxed 2-local Hermitian operator with unique ground state of eigenvalue 2ϵ and spectral gap ϵ. Our approach is intuitively now as follows. We ﬁrst run a query validation phase, in which we modify H′ to obtain a new Hamiltonian H′′ by replacing “suﬃciently invalid” queries Hi,y1···yi−1 Yi with high-energy dummy queries. This creates the desired spectral gap. We then apply the technique of Lemma 3.3 to reduce the locality of H′′, obtaining a 4-local Hamiltonian H, as desired. Note that our proof shows existence of H; unlike Lemma 3.3, however, it is not clear how to construct H in polynomial-time given H′, as detecting invalid UQMA queries with a P machine seems diﬃcult. The query validation runs as follows. Fix any 1/poly(n) < δ < ϵ such that ϵ −δ ≥1/poly(n); for example, set δ = ϵ/2. For any G′ y1···yi−1 whose spectral gap is at most ϵ −δ, replace it with Gy1···yi−1 := \|0⟩⟨0\|Xi ⊗AYi + \|1⟩⟨1\|Xi ⊗3ϵI (15) Gy1···yi−1 := \|0⟩⟨0\|Xi ⊗AYi + \|1⟩⟨1\|Xi ⊗3ϵI (15) (15) in H′, denoting the new Hamiltonian as H′′. Two remarks: By Case 2 of Lemma 4.11 below, the validation phase does not catch all invalid queries. Second, setting 1/poly(n) < δ and ϵ −δ ≥ 1/poly(n) (as opposed to, say, 1/ exp(n)) is required for our proof of Theorem 1.3 later. Correctness. Intuitively, the query validation phase roughly replaces each “suﬃciently invalid” query i with a valid NO query. This is formalized by Lemma 4.11, shown subsequently, which should be roughly interpreted as saying a “suﬃciently invalid” query i is one for which the spectral gap of G′ y1···yi−1 is at most ϵ −δ. For any such replaced query i, henceforth in this proof, a query string which answers YES (i.e. \|1⟩) to query i is considered incorrect with respect to H′′ (since i is now a valid NO query in H′′). Crucially, if a string x is a correct query string for H′′, then it is also a correct query string for H′. 4.3 PUQMA[log]-hardness of SPECTRAL-GAP Thus, the 0-block of Equation (14) has minimum eigenvalue 2ϵ by construction, whereas the 1-block of Equation (14) has minimum eigenvalue at most ϵ. It follows from our previous analysis that whichever of x or y has a 0 as bit i has energy at least λx + ϵ/4m against H′′. (This, in particular, implies xi = 1 and yi = 0, by the minimality of x.) Yi Yi Equation (14) has minimum eigenvalue 2ϵ by construction, whereas the 1-block of Equation (14) has minimum eigenvalue at most ϵ. It follows from our previous analysis that whichever of x or y has a 0 as bit i has energy at least λx + ϵ/4m against H′′. (This, in particular, implies xi = 1 and yi = 0, by the minimality of x.) Case 2: Eigenvalues from the same query string. Let us next consider eigenvalues in the minimal block Hx with correct query string x. In this block, H′′ is equivalent to operator M := m X i=1 1 4i−1 Bx1···xi−1, M := m X i=1 1 4i−1 Bx1···xi−1, where Bx1···xi−1 = A if xi = 0 and Bx1···xi−1 can equal either Hi,y1···yi−1 Yi or 3ϵI (depending on how the validation phase proceeded) if xi = 1. To show our claim, it suﬃces to show that M has spectral gap (ϵ −δ)/4m. where Bx1···xi−1 = A if xi = 0 and Bx1···xi−1 can equal either Hi,y1···yi−1 Yi or 3ϵI (depending on how the validation phase proceeded) if xi = 1. To show our claim, it suﬃces to show that M has spectral gap (ϵ −δ)/4m. Recall now that each Bx1···xi−1 acts non-trivially only on space Yi. Therefore, the minimum eigenvalue of M is obtained by setting each space Yi to the ground state vector \|ψi⟩of Bx1···xi−1. If we can now show that each Bx1···xi−1 has a spectral gap of ϵ −δ, it will hence follow that swapping \|ψi⟩for any excited state \|ψ⊥ i ⟩orthogonal to \|ψi⟩on Yi yields an additive increase in energy of at least ϵ −δ against any Bx1···xi−1. It would then follow that the spectral gap of M is at least (ϵ −δ)/4m, as desired. So let us argue that each Bx1···xi−1 has spectral gap at least ϵ −δ. There are three cases to consider: • If Bx1···xi−1 = A, it has spectral gap ϵ by deﬁnition of A. This completes the correctness proof. 4.3 PUQMA[log]-hardness of SPECTRAL-GAP The converse is false; nevertheless, H′′ has at least one correct query string (since any invalid query would have allowed both \|0⟩and \|1⟩as answers), which suﬃces for our purposes. As in the proof of Lemma 3.1, observe that H′′ is block-diagonal with respect to register Nm i=1 Xi. Let x ∈{ 0, 1 }m denote a correct query string which has minimal energy among all correct query strings against H′′, and for any y ∈{ 0, 1 }m, deﬁne λy as the smallest eigenvalue in block Hy. A similar analysis to that of Lemma 3.1 shows that for any incorrect query string y, λy ≥λx + ϵ/4m. This is because replacing the term 2ϵI in My1···yi−1 from Lemma 3.1 with A in Gy1···yi−1 here preserves the property that answering NO on query i yields minimum energy 2ϵ. We now argue that x is in fact unique, and all other eigenvalues of H′′ corresponding to correct query strings have energy at least λx + (ϵ −δ)/4m. There are two cases to consider: eigenvalues arising from diﬀerent query strings and eigenvalues arising from the same query string. Case 1: Eigenvalues from diﬀerent query strings. Let y = y1 · · · ym ̸= x be a correct query string for H′′. Since both x and y are correct strings, there must exist an invalid query i where xi ̸= yi. 25 First consider the case where G′ y1···yi−1 has spectral gap at most ϵ−δ. Then, after the validation phase, query i is replaced with a valid NO query Gy1···yi−1. Thus, whichever of x or y has a 1 as bit i is an incorrect string for H′′, and from our previous analysis has energy at least λx + ϵ/4m against H′′. (This, in particular, implies xi = 0 and yi = 1, by the minimality of x.) Alternatively, suppose G′ y1···yi−1 = Gy1···yi−1 has spectral gap strictly larger than ϵ −δ. By Lemma 4.11, the invalid query i must be a “YES instance violating the uniqueness promise” with corresponding query Hamiltonian Hi,y1···yi−1 Yi satisfying λ(Hi,y1···yi−1 Yi ) ≤ϵ. Thus, the 0-block of y, pp y1 yi−1 y1 yi 1 p g p y g y Lemma 4.11, the invalid query i must be a “YES instance violating the uniqueness promise” with corresponding query Hamiltonian Hi,y1···yi−1 Yi satisfying λ(Hi,y1···yi−1 Yi ) ≤ϵ. 4.3 PUQMA[log]-hardness of SPECTRAL-GAP • If Bx1···xi−1 = Hi,y1···yi−1 Yi , then consider ﬁrst the case where query i was valid. Since Bx1···xi−1 = Hi,y1···yi−1 Yi only if xi = 1, it follows that the correct answer to query i is 1. Thus, Hi,y1···yi−1 Yi is a valid YES instance of U-LH, and so has a spectral gap of at least 2ϵ by deﬁnition. If instead query i was invalid, then since Bx1···xi−1 = Hi,y1···yi−1 Yi and not Bx1···xi−1 = 3ϵI, it must be that the query validation phase did not catch this invalid query, implying the spectral gap of G′ y1···yi−1 was strictly larger than ϵ−δ. But then Lemma 4.11 implies that the spectral gap of Hi,y1···yi−1 must be at least ϵ δ as desired • If Bx1···xi−1 = Hi,y1···yi−1 Yi , then consider ﬁrst the case where query i was valid. Since Bx1···xi−1 = Hi,y1···yi−1 Yi only if xi = 1, it follows that the correct answer to query i is 1. Thus, Hi,y1···yi−1 Yi is a valid YES instance of U-LH, and so has a spectral gap of at least 2ϵ by deﬁnition. If instead query i was invalid, then since Bx1···xi−1 = Hi,y1···yi−1 Yi and not Bx1···xi−1 = 3ϵI, it must be that the query validation phase did not catch this invalid query, implying the spectral gap of G′ y1···yi−1 was strictly larger than ϵ−δ. But then Lemma 4.11 implies that the spectral gap of Hi,y1···yi−1 Yi must be at least ϵ −δ, as desired. • Suppose Bx1···xi−1 = 3ϵI; this happens only when the query validation phase replaced an invalid query i with a valid NO query 3ϵI. But since it also holds that Bx1···xi−1 = 3ϵI only if xi = 1, this implies that x is not a correct query string with respect to H′′ (since it answered 1 on query i instead of 0), which is a contradiction. This completes the correctness proof. 26 Getting H′′ down to 4-local H. Finally, the approach of Lemma 3.3 allows us to convert O(log n)-local H′′ to 4-local H. Getting H′′ down to 4-local H. Finally, the approach of Lemma 3.3 allows us to convert O(log n)-local H′′ to 4-local H. The following lemma is used in the proof of Lemma 4.10 above, and assumes the notation introduced therein. Lemma 4.11. In Equation (14), suppose Hi,y1···yi−1 Yi is invalid. Precisely one of the following cases holds. 1. 4.3 PUQMA[log]-hardness of SPECTRAL-GAP (Neither YES or NO case) If there exists 0 < δ ≤ϵ such that λ(Hi,y1···yi−1 Yi ) = ϵ + δ or λ(Hi,y1···yi−1 Yi ) = 3ϵ −δ, then the spectral gap of G′ y1···yi−1 is at most ϵ −δ. 1. (Neither YES or NO case) If there exists 0 < δ ≤ϵ such that λ(Hi,y1···yi−1 Yi ) = ϵ + δ or λ(Hi,y1···yi−1 Yi ) = 3ϵ −δ, then the spectral gap of G′ y1···yi−1 is at most ϵ −δ. 2. (YES case violating uniqueness promise) If λ(Hi,y1···yi−1 Yi ) ≤ϵ and λ2(Hi,y1···yi−1 Yi ) < 3ϵ, then for any 0 < δ < ϵ, either the spectral gap of G′ y1···yi−1 is at most ϵ −δ, or the spectral gap of Hi,y1···yi−1 Yi is least ϵ −δ. 2. (YES case violating uniqueness promise) If λ(Hi,y1···yi−1 Yi ) ≤ϵ and λ2(Hi,y1···yi−1 Yi ) < 3ϵ, then for any 0 < δ < ϵ, either the spectral gap of G′ y1···yi−1 is at most ϵ −δ, or the spectral gap of Hi,y1···yi−1 Yi is least ϵ −δ. Proof. Observe that G′ y1···yi−1 is block-diagonal with respect to register Xi; we will refer to each of these blocks as the 0- and 1-block, respectively. For clarity, when we refer to λ2(X) for an operator X below, if the ground space of X is degenerate then we deﬁne λ2(X) = λ(X). Case 1. Suppose ﬁrst that λ(Hi,y1···yi−1 Yi ) = ϵ + δ. Then, since the smallest eigenvalue in the 0-block is 2ϵ, we have that λ(G′ y1···yi−1) = λ(Hi,y1···yi−1 Yi ), and so the spectral gap of G′ y1···yi−1 equals min(2ϵ, λ2(Hi,y1···yi−1 Yi )) −(ϵ + δ) ≤ϵ −δ. Case 1. Suppose ﬁrst that λ(Hi,y1···yi−1 Yi ) = ϵ + δ. Then, since the smallest eigenvalue in the 0-block is 2ϵ, we have that λ(G′ y1···yi−1) = λ(Hi,y1···yi−1 Yi ), and so the spectral gap of G′ y1···yi−1 equals min(2ϵ, λ2(Hi,y1···yi−1 Yi )) −(ϵ + δ) ≤ϵ −δ. min(2ϵ, λ2(Hi,y1···yi−1 Yi )) −(ϵ + δ) ≤ϵ −δ. Suppose next that λ(Hi,y1···yi−1 Yi ) = 3ϵ −δ. Then, λ(G′ y1···yi−1) = 2ϵ by the 0-block. Since the spectral gap of the 0-block is ϵ by construction, we thus have that λ2(G′ y1···yi−1) = λ(Hi,y1···yi−1 Yi ), and so the spectral gap of G′ y1···yi−1 equals (3ϵ −δ) −2ϵ = ϵ −δ. Suppose next that λ(Hi,y1···yi−1 Yi ) = 3ϵ −δ. 4.3 PUQMA[log]-hardness of SPECTRAL-GAP Then, λ(G′ y1···yi−1) = 2ϵ by the 0-block. Since the spectral gap of the 0-block is ϵ by construction, we thus have that λ2(G′ y1···yi−1) = λ(Hi,y1···yi−1 Yi ), and so the spectral gap of G′ y1···yi−1 equals (3ϵ −δ) −2ϵ = ϵ −δ. Case 2. Since the smallest eigenvalue of the 0-block is 2ϵ, we have λ(G′ y1···yi−1) = λ(Hi,y1···yi−1 Yi ). Therefore, the spectral gap of G′ y1···yi−1 equals Case 2. Since the smallest eigenvalue of the 0-block is 2ϵ, we have λ(G′ y1···yi−1) = λ(Hi,y1···yi−1 Yi ). Therefore, the spectral gap of G′ y1···yi−1 equals min(2ϵ, λ2(Hi,y1···yi−1 Yi )) −λ(Hi,y1···yi−1 Yi ). (16) (16) For any δ as deﬁned in the claim, suppose now that the spectral gap of G′ y1···yi−1 is at least ϵ−δ. Then if in Equation (16) min(2ϵ, λ2(Hi,y1···yi−1 Yi )) = 2ϵ, since λ(Hi,y1···yi−1 Yi ) ≤ϵ by assumption, we conclude the spectral gap of Hi,y1···yi−1 Yi is at least ϵ. Otherwise, if min(2ϵ, λ2(Hi,y1···yi−1 Yi )) = λ2(Hi,y1···yi−1 Yi ), then the smallest two eigenvalues of Hi,y1···yi−1 Yi and G′ y1···yi−1 coincide, and so the spectral gap of the former is at least ϵ −δ. Thus, we conclude that either the spectral gap of G′ y1···yi−1 is at most ϵ −δ, or the spectral gap of Hi,y1···yi−1 Yi is least ϵ −δ. We now prove the main theorem of this section. 27 Proof of Theorem 1.3. As done in [5], we start with the Hamiltonian H′ from Equation (13). In [5], it was shown (Section A.3, Claim 2) that if all query Hamiltonians Hi,y1···yi−1 Yi correspond to valid UQMA queries, H′ has a unique ground state and spectral gap at least ϵ/4m. When invalid queries are allowed, however, the spectral gap of H′ can vanish, invalidating the PUQMA[log]-hardness proof of [5]. Thus, we require a technique for identifying invalid queries and “removing them” from H′. Unfortunately, it is not clear how a P machine alone can achieve such a “property testing” task of checking if a query is suﬃciently invalid. However, the key observation is that an oracle Q for SPECTRAL GAP can help. We proceed as follows. Given an arbitrary PUQMA[log] circuit U acting on n bits, construct O(log n)-local H′ from Equation (13). 4.3 PUQMA[log]-hardness of SPECTRAL-GAP For each term G′ y1···yi−1 appearing in H′, perform binary search using O(log n) queries to Q to obtain an estimate ∆for the spectral gap of G′ y1···yi−1 to within suﬃciently small but ﬁxed additive error δ ∈1/poly(n). (A similar procedure involving a QMA oracle is used in Ambainis’ proof of containment of APX-SIM ∈PQMA[log] to estimate the smallest eigenvalue of a local Hamiltonian; we hence omit further details here.) As done in the proof of Lemma 4.10, if ∆≤ϵ −δ, we conclude Hi,y1···yi−1 Yi is “suﬃciently invalid”, and replace G′ y1···yi−1 with Gy1···yi−1 from Equation (15). Following the construction of Lemma 4.10, we hence can map H′ to a 4-local Hamiltonian H such that H has a unique ground state and spectral gap (ϵ −δ)/4m, and the ground state of H corresponds to a correct query string for H′. Note that implementing the mapping from H′ to H requires polynomially many queries to the oracle, hence yielding a polynomial-time Turing reduction. Next, following [5], let T := P y1...ym Nm i=1 \|yi⟩⟨yi\| ∈L (Y), where we sum over all query strings y1...ym which cause U to output 0. Unlike [5], as done in Lemma 3.3, we apply Kitaev’s unary encoding trick [45] and implicitly encode the query strings in T in unary. (We remark the term Hstab contained in H will enforce the correct unary encoding in register X). Finally, introduce a single-qubit register B, and deﬁne Hﬁnal := IB ⊗HX,Y + 4ϵ \|0⟩⟨0\|B ⊗TX ⊗IY. The claim now follows via an analysis similar to [5]. Let \|ψ⟩X,Y denote the unique ground state of H, whose X register contains the (unary encoding of) a correct query string for U. If U accepts, then \|i⟩B ⊗\|ψ⟩X,Y for i ∈{ 0, 1 } are degenerate ground states of Hﬁnal, implying Hﬁnal has no spectral gap. Conversely, if U rejects, observe that the smallest eigenvalue of Hﬁnal lies in the \|1⟩B block of Hﬁnal. This is because Hﬁnal is block-diagonal with respect to register X, and we have from the proof of Lemma 3.3 that λ(H) < 3ϵ. Restricted to this \|1⟩B block, the spectral gap of Hﬁnal is at least (ϵ −δ)/4m by Lemma 4.10. 4.3 PUQMA[log]-hardness of SPECTRAL-GAP Alternatively, restricted to the \|0⟩B block, any correct query string in X leads to spectral gap at least 4ϵ (by construction of T, since U outputs 0 in this case), and any incorrect query string in X leads to spectral gap at least (ϵ−δ)/4m by Lemma 4.10. Hence, Hﬁnal has an inverse polynomial spectral gap, as desired. 14Under the assumptions that V has zero error and Π makes only valid queries, y∗= 1m can only be obtained by this procedure if all queries are for YES instances of 2-LH. If, on the other hand, query i is a NO query, then a correct proof cannot be guessed (since it does not exist), and so y∗ i = 0 necessarily. 5 Upper bounds (containment in PP) We now restate and prove Theorem 1.4. Our approach is to use the strong error reduction technique of Marriott and Watrous [49] to develop a variant of the hierarchical voting scheme used in the proof of PNP[log] ⊆PP [9]. We also require a more involved analysis than present in [9], since QMA is a class of promise problems, not decision problems. 28 Theorem 1.4. PQMA[log] ⊆PP. Proof. Let Π be a P machine which makes m = c log n queries to an oracle for 2-LH, for c ∈O(1) and n the input size. Without loss of generality, we assume all queries involve Hamiltonians on M qubits (M some ﬁxed polynomial in n). Deﬁne q := (M + 2)m. We give a PQP computation simulating Π (i.e. the PQP computation, which does not have access to a 2-LH oracle, will accept if and only if Π is a YES instance). Since PQP = PP [62], this yields the claim. Let \|y\| denote the non-negative integer with binary encoding y, and let V denote the veriﬁcation circuit for 2-LH. The PQP computation is (intuition to follow): 1. For i from 1 to m: (a) Prepare ρ = I/2M ∈D (C2)⊗M . (a) Prepare ρ = I/2M ∈D (C2)⊗M . (b) Run V on the i-th query Hamiltonian Hi,y1···yi−1 Yi (see Equation (3)) and proof ρ, and measure the output qubit in the standard basis. Set bit yi to the result. (b) Run V on the i-th query Hamiltonian Hi,y1···yi−1 Yi (see Equation (3)) and proof ρ, and measure the output qubit in the standard basis. Set bit yi to the result. 2. Let y = y1 · · · ym be the concatenation of bits set in Step 1(b) 2. Let y = y1 · · · ym be the concatenation of bits set in Step 1(b). 3. For i from 1 to nc −1: 3. For i from 1 to nc −1: 3. For i from 1 to nc −1: (a) If \|y\| < i, then with probability 1 −2−q, set y = #, and with probability 2−q, leave y unchanged. (a) If \|y\| < i, then with probability 1 −2−q, set y = #, and with probability 2−q, leave y unchanged. 4. If y = #, output a bit uniformly at random. Else, run Π on query string y and output Π’s answer. 4. 5 Upper bounds (containment in PP) If y = #, output a bit uniformly at random. Else, run Π on query string y and output Π’s answer. Intuition. In Step 1, one tries to determine the correct answer to query i by guessing a satisfying quantum proof for veriﬁer V . Suppose for the moment that V has zero error, i.e. has completeness 1 and soundness 0, and that Π only makes valid queries. Then, if Step 1(b) returns yi = 1, one knows with certainty that the query answer should be 1. And, if the correct answer to query i is 0, then Step 1(b) returns yi = 0 with certainty. Thus, analogous to the classical case of an NP oracle (as done in [9]), it follows that the lexicographically largest query string y∗obtainable by this procedure must be the (unique) correct query string (note that y∗̸= 1m necessarily14). Thus, ideally one wishes to obtain y∗, simulate Π on y∗, and output the result. To this end, Step 3 ensures that among all values of y ̸= #, y∗is more likely to occur than all other y ̸= y∗combined. We now make this intuition rigorous (including in particular the general case where V is not zero-error and Π makes invalid queries). Correctness. To analyze correctness of our PQP computation, it will be helpful to reﬁne our partition of the set of query strings { 0, 1 }m into three sets: • (Correct query strings) Let A ⊆{ 0, 1 }m denote the set of query strings which correspond to correctly answering each of the m queries. Note we may have \|A\| > 1 if invalid queries are made. • (Correct query strings) Let A ⊆{ 0, 1 }m denote the set of query strings which correspond to correctly answering each of the m queries. Note we may have \|A\| > 1 if invalid queries are made. 29 • (Incorrect query strings) Let B ⊆{ 0, 1 }m \ A denote the set of query strings such that for any y ∈B, all bits of y which encode an incorrect query answer are set to 0 (whereas the correct query answer would have been 1, i.e. we failed to “guess” a good proof for this query in Step 1). 5 Upper bounds (containment in PP) • (Strongly incorrect query strings) Let C = { 0, 1 }m \ (A ∪B) denote the set of query strings such that for any y ∈C, at least one bit corresponding to an incorrect query answer is set to 1 (whereas the correct query answer would have been 0). Such an error can only arise due to the bounded-error of our QMA veriﬁer in Step 1(b). Let Y be a random variable corresponding to the query string y obtained at the end of Step 3. To show correctness, we claim that it suﬃces to show that ∆:= Pr[Y ∈A] −Pr[Y ∈B ∪C] > 0. To see this, let p1, p2, and p3 denote the probability that after Step 3, y = #, y ∈A, and y ∈B ∪C, respectively. Then, p1 + p2 + p3 = 1, and let p2 −p3 = ∆> 0. Suppose now that the input to Π is a YES instance. Then, our protocol outputs 1 with probability at least p1 2 + p2 = 1−p2−p3 2 + p2 = 1+∆ 2 > 1 2. If the input is a NO instance, the protocol outputs 1 with probability ≤p1 2 + p3 = 1−∆ 2 < 1 2. We hence have a PQP computation, as desired. We thus now show that ∆> 0. To ease the presentation, we begin by making two assumptions (to be removed later): (i) V is zero-error and (ii) Π makes only valid queries. In this case, assumption (i) implies C = ∅(i.e. all incorrect query strings belong to B), and (ii) implies A is a singleton (i.e. there is a unique correct query string y∗). Thus, here ∆= Pr[Y ∈A] −Pr[Y ∈B]. ) [ ] [ ] To begin, note that for any y ∈{ 0, 1 }m, we have at for any y ∈{ 0, 1 }m, we have Pr[Y = y] = Pr[y chosen in Step 2 ] · 1 2q (nc−1)−\|y\| . (17) (17) Let HW(x) denote the Hamming weight of x ∈{ 0, 1 }m. Since each query corresponds to a veriﬁer on M proof qubits, we have for (the unique) y∗∈A that Let HW(x) denote the Hamming weight of x ∈{ 0, 1 }m. 5 Upper bounds (containment in PP) Since each query corresponds to a veriﬁer on M proof qubits, we have for (the unique) y∗∈A that Pr[y∗chosen in Step 2 ] ≥2−M·HW(y∗) ≥2−Mm (18) (18) (recall from Section 2 that setting ρ = I/2M simulates “guessing” a correct proof with probability at least 1/2M). It follows by Equations (17) and (18) that (recall from Section 2 that setting ρ = I/2M simulates “guessing” a correct proof with probability at least 1/2M). It follows by Equations (17) and (18) that ∆ ≥ 1 2q (nc−1)−\|y∗\|   1 2Mm − X y∈B Pr[y chosen in Step 2 ] · 1 2q \|y∗\|−\|y\|   ≥ 1 2q (nc−1)−\|y∗\| 1 2Mm −(2m) 1 2q ≥ 1 2q (nc−1) 1 2Mm 1 −1 2m , (19) (19) where the second inequality follows from the trivial bound Pr[y chosen in Step 2 ] ≤1 and since y ∈B if and only if \|y\| < \|y∗\|, and the third since q = (M + 2)m. Thus, ∆> 0 as desired. Removing assumption (i). We now remove the assumption that V is zero error. In this case, A is still a singleton; let y∗∈A. We can now also have strongly incorrect query strings, i.e. C ̸= ∅ necessarily. Assume without loss of generality that V acts on M proof qubits, and by strong error 30 reduction [49] has completeness c := 1 −2−p(n) and soundness s := 2−p(n), for p a polynomial to be chosen as needed. Then, since V can err, Equation (18) becomes Pr[y∗chosen in Step 2 ] ≥ c 2M HW(y∗) (1 −s)m−HW(y∗) = 1 2M HW(y∗) em ln(1−1 2p ) ≥ 1 2Mm 1 − m 2p −1 , (20) (20) where the equality follows by the deﬁnitions of c and s, and the second inequality by applying the Maclaurin series expansion ln(1 + x) = P∞ n=1(−1)n+1 xn n for \|x\| < 1 and the fact that et ≥1 + t for all t ∈R. Thus, the analysis of Equation (19) yields that Pr[Y ∈A] −Pr[Y ∈B] ≥ 1 2q (nc−1) 1 2Mm 1 −1 2m − m 2p −1 , (21) (21) i.e. the additive error introduced when assumption (i) is dropped scales as ≈2−p. 5 Upper bounds (containment in PP) Crucially, Equa- tion (21) holds for all y ∈B even with assumption (i) dropped since the analysis of Equation (19) used only the trivial bound Pr[y chosen in Step 2 ] ≤1 for any y ∈B. Next, we upper bound the probability of obtaining y ∈C in Step 2. Next, we upper bound the probability of obtaining y ∈C in Step 2. Pr(Y ∈C) ≤2m 2p . Lemma 5.1. Pr(Y ∈C) ≤2m 2p . Lemma 5.1. Pr(Y ∈C) ≤2m 2p . py,i denotes the probability that at divergence point i, we go in the direction of bit yi. We deﬁne the divergence probability of y ∈{ 0, 1 }m as py = Πi∈Iypy,i, i.e. py is the probability of answering all invalid queries as y did. Proof. For any string y ∈{ 0, 1 }m, let Iy ⊆{ 1, . . . , m } denote the indices of all bits of y set by invalid queries. We call each such i ∈Iy a divergence point. (This name is chosen since on any invalid query, the computation can diverge into either of two correct computation paths.) Let py,i denote the probability that (invalid) query i (deﬁned given answers to queries 1 through i −1) outputs bit yi, i.e. py,i denotes the probability that at divergence point i, we go in the direction of bit yi. We deﬁne the divergence probability of y ∈{ 0, 1 }m as py = Πi∈Iypy,i, i.e. py is the probability of answering all invalid queries as y did. The proof now proceeds by giving for each 1 ≤i ≤\|A\|, which denotes the iteration number, a 3-tuple (y∗ i−1, y∗ i , By∗ i ) ∈{ 0, 1 }m × { 0, 1 }m × P(B), where P(X) denotes the power set of set X. S t ∆′ i := Pr[Y ∈{ y∗ 1, . . . , y∗ i }] −Pr[Y ∈By∗ 1 ∪· · · ∪By∗ i ], where it will be the case that { By∗ i }\|A\| i=1 is a partition of B. Thus, we have ∆′ ≥∆′ \|A\|, implying that a lower bound on ∆′ \|A\| suﬃces to prove our claim. We hence prove via induction on the iteration number i that for all 1 ≤i ≤\|A\|, where it will be the case that { By∗ i }\|A\| i=1 is a partition of B. Thus, we have ∆′ ≥∆′ \|A\|, implying that a lower bound on ∆′ \|A\| suﬃces to prove our claim. We hence prove via induction on the iteration number i that for all 1 ≤i ≤\|A\|, ∆′ i ≥ 1 2q (nc−1) 1 2Mm 1 −1 2m − m 2p −1 . Base case (i=1). In this case y∗ 0 is undeﬁned. Lemma 5.1. Pr(Y ∈C) ≤2m 2p . Set y∗ 1 to any string in A with divergence probability at least p∗ 1 = Y i∈Iy∗ 1 py∗ 1,i ≥2 − Iy∗ 1 . (23) (23) Such a string must exist, since at each divergence point i, at least one of the outcomes in { 0, 1 } occurs with probability at least 1/2. (Note that queries are not being made to a QMA oracle in hierarchical voting (since our PQP machine does not have access to a QMA oracle), but to a QMA veriﬁer V with a maximally mixed proof as in Step 1(a). Whereas in the former case the output of the oracle on an invalid query does not have to consistently output a value with any particular probability, in the latter case, there is some ﬁxed probability p with which V outputs 1 each time it is run on a ﬁxed proof.) Finally, deﬁne By∗ 1 := { y ∈B \| \|y\| < \|y∗ 1\| } (recall \|y\| is the non-negative integer with binary encoding y). Such a string must exist, since at each divergence point i, at least one of the outcomes in { 0, 1 } occurs with probability at least 1/2. (Note that queries are not being made to a QMA oracle in hierarchical voting (since our PQP machine does not have access to a QMA oracle), but to a QMA veriﬁer V with a maximally mixed proof as in Step 1(a). Whereas in the former case the output of the oracle on an invalid query does not have to consistently output a value with any particular probability, in the latter case, there is some ﬁxed probability p with which V outputs 1 each time it is run on a ﬁxed proof.) Finally, deﬁne By∗ 1 := { y ∈B \| \|y\| < \|y∗ 1\| } (recall \|y\| is the non-negative integer with binary encoding y). Let k∗denote the number of divergence points of y∗ 1 (i.e. k∗= Iy∗ 1 ), and k0 (k1) the number of zeros (ones) of y∗ 1 arising from valid queries. Thus, k∗+k0 +k1 = m. Lemma 5.1. Pr(Y ∈C) ≤2m 2p . Proof. Any y ∈C must have a bit j incorrectly set to 1, whereas the correct query answer (given bits 1 through j −1 of y) should have been 0. The probability of this occurring for bit j in Step 1(b) is at most 2−p, by the soundness property of V . Since \|C\| ≤2m, the claim follows. Lemma 5.1 now implies w implies Lemma 5.1 now implies ∆≥ 1 2q (nc−1) 1 2Mm 1 −1 2m − m 2p −1 −2m 2p . (22) (22) We conclude that setting p to a suﬃciently large ﬁxed polynomial ensures ∆> 0 We conclude that setting p to a suﬃciently large ﬁxed polynomial ensures ∆> 0, as desired. lude that setting p to a suﬃciently large ﬁxed polynomial ensures ∆> 0, as desired We conclude that setting p to a suﬃciently large ﬁxed polynomial ensures ∆> 0, as desired. Removing assumption (ii). We now remove the assumption that Π only makes valid queries, which is the most involved step. Here, A is no longer necessarily a singleton. The naive approach would be to let y∗denote the lexicographically largest string in A, and attempt to run a similar analysis as before. Unfortunately, this no longer necessarily works for the following reason. For any invalid query i, we do not have strong bounds on the probability that V accepts in Step 1(b); in principle, this value can lie in the range (2−p, 1−2−p). Thus, running the previous analysis with the lexicographically largest y∗∈A may cause Equation (22) to yield a negative quantity. We hence require a more delicate analysis. ng the following lower bound. We begin by showing the following lower bound. We begin by showing the following lower bound. Lemma 5.2. Deﬁne ∆′ := Pr[Y ∈A] −Pr[Y ∈B]. Then, ∆′ ≥ 1 2q (nc−1) 1 2Mm 1 −1 2m − m 2p −1 . 31 Proof. For any string y ∈{ 0, 1 }m, let Iy ⊆{ 1, . . . , m } denote the indices of all bits of y set by invalid queries. We call each such i ∈Iy a divergence point. (This name is chosen since on any invalid query, the computation can diverge into either of two correct computation paths.) Let py,i denote the probability that (invalid) query i (deﬁned given answers to queries 1 through i −1) outputs bit yi, i.e. Lemma 5.1. Pr(Y ∈C) ≤2m 2p . (26) (26) That such a y∗ i ∈Sk∗ i exists follows from an argument similar to Equation (23): By deﬁnition, q∗ i denotes the bounded divergence probability for all invalid queries up to and including query k∗ i , and the term exponential in − Iy∗ i + I ≤k∗ i y∗ i is obtained by greedily choosing, for all invalid queries of y∗ i after query k∗ i , the outcome which occurs with probability at least 1/2. Set By∗ i := { y ∈B \| y∗ i−1 < \|y\| < \|y∗ i \| }. The following lemma will be useful. That such a y∗ i ∈Sk∗ i exists follows from an argument similar to Equation (23): By deﬁnition, q∗ i denotes the bounded divergence probability for all invalid queries up to and including query k∗ i , and the term exponential in − Iy∗ i + I ≤k∗ i y∗ i is obtained by greedily choosing, for all invalid queries of y∗ i after query k∗ i , the outcome which occurs with probability at least 1/2. Set By∗ i := { y ∈B \| y∗ i−1 < \|y\| < \|y∗ i \| }. The following lemma will be useful. Lemma 5.3. For any y ∈By∗ i , Pr[y chosen in Step 2] ≤q∗ i , where recall q∗ i is the probability from Equation (25). Proof. Fix any y ∈By∗ i . Since \|y\| > y∗ i−1 , there must be an index k such that the k-th bit of y is 1 and that of y∗ i−1 is 0. Let k denote the ﬁrst such index. Since y ̸∈C (because By∗ i ∩C = ∅), it must be that query k (deﬁned given bits y1 · · · yk−1) is invalid. Thus, bit k is a divergence point of y∗ i−1, and there exists a correct query string y′ ∈Sk. By Equation (25), q∗ i was chosen as the maximum over all bounded diverge probabilities. Thus, q∗ i ≥qi(k), where recall qi(k) is the bounded divergence probability for Sk, where y′ ∈Sk. But since y and y′ agree on bits 1 through k inclusive, we have Pr[y chosen in Step 2] ≤Q t∈I≤k y py,t = qi(k), from which the claim follows. To continue with the inductive step, again consider k∗, k0, and k1, now corresponding to y∗ i . Lemma 5.1. Pr(Y ∈C) ≤2m 2p . Then, Equation (20) becomes Pr[y∗ 1 in Step 2 ] ≥ c 2M k1 (1 −s)k0 p∗ 1 ≥ 1 2M k1 1 2 k∗ 1 −m −k∗ 2p −1 ≥ 1 2Mm 1 − m 2p −1 , (24) where the second inequality follows from Equation (23), and the third since k∗≥0 and k1+k∗≤m. Thus, ∆′ 1 is lower bounded by the expression in Equation (21) via an analogous analysis for y∗ 1 and By∗ 1. Inductive step. Assume the claim holds for 1 ≤i −1 < \|A\|. We show it holds for i. Let y∗ i−1 be the choice of y∗in the previous iteration i −1. Deﬁne Ay∗ i := { y ∈A \| \|y\| > y∗ i−1 }. Partition Inductive step. Assume the claim holds for 1 ≤i −1 < \|A\|. We show it holds for i. Let y∗ i−1 be the choice of y∗in the previous iteration i −1. Deﬁne Ay∗ i := { y ∈A \| \|y\| > y∗ i−1 }. Partition 32 Ay∗ i into sets Sk for k ∈[m], such that Sk is the subset of strings in Ay∗ i which agrees with y∗ i−1 on the ﬁrst k −1 bits, but disagrees on bit k. Note that if Sk ̸= ∅, then bit k of y∗ i−1 is 0 and bit k of any string in Sk is 1. For each Sk ̸= ∅, choose arbitrary representative zk ∈Sk, and deﬁne bounded divergence probability qi(k) := Y t∈I≤k zk pzk,t where I≤k zk := { t ∈Izk \| t ≤k }. Note that qi(k) > 0 (since Sk ̸= ∅). Else if Sk = ∅, set qi(k) = 0. Let q∗ i be the max such bounded divergence probability: where I≤k zk := { t ∈Izk \| t ≤k }. Note that qi(k) > 0 (since Sk ̸= ∅). Else if Sk = ∅, set qi(k) = 0. Let q∗ i be the max such bounded divergence probability: q∗ i = max k∈[m] qi(k) and k∗ i = arg max k∈[m] qi(k). (25) (25) graphically largest query string in Sk∗ i with divergence probability p∗ i s.t.: Let y∗ i be the lexicographically largest query string in Sk∗ i with divergence probabili p∗ i ≥q∗ i · 2 − Iy∗ i + I ≤k∗ i y∗ i . Acknowledgments We thank Xiaodi Wu for stimulating discussions which helped motivate this project, including suggesting to think about two-point correlation functions (which arose via discussions with Aram Harrow, whom we also thank). We also thank Andris Ambainis and Norbert Schuch for helpful discussions, and remark they independently conceived of some of the ideas behind Lemma 3.3 and Theorem 1.1, respectively (private communication). We thank an anonymous referee for pointing out a minor error in the proof of Lemma 5.2 in a previous version of this draft. Part of this work was completed while SG was supported by a Government of Canada NSERC Banting Postdoctoral Fellowship and the Simons Institute for the Theory of Computing at UC Berkeley. SG acknowl- edges support from NSF grants CCF-1526189 and CCF-1617710. JY was supported by a Virginia Commonwealth University Presidential Scholarship. Lemma 5.1. Pr(Y ∈C) ≤2m 2p . Then, an argument similar to Equation (24) says Pr[y∗ i chosen in Step 2 ] is at least c 2M k1 (1 −s)k0 p∗ i ≥ 1 2M k1 1 −m −k∗ 2p −1 q∗ i 1 2 Iy∗ i − I ≤k∗ i y∗ i ≥ q∗ i 2Mm 1 − m 2p −1 , (27) (27) where the ﬁrst inequality follows from Equation (26), and the second since Iy∗ i − I ≤k∗ i y∗ i ≤k∗. Now, deﬁne ζi := Pr[Y = y∗ i ] −Pr[Y ∈By∗ i ]. Applying the argument of Equation (19) yields ζi ≥ 1 2q (nc−1)−\|y∗ i \|  q∗ i 2Mm 1 − m 2p −1 −q∗ i X y∈By∗ i 1 2q \|y∗ i \|−\|y\|  , 33 where the ﬁrst q∗ i is due to Equation (27), and the second q∗ i to Lemma 5.3. Thus, similar to Equation (21), ( c 1) ζi ≥ 1 2q (nc−1) q∗ i 2Mm 1 −1 2m − m 2p −1 > 0. Observing the recurrence that for all i, ∆′ i ≥∆′ i−1 + ζi, unrolling this recurrence yields ∆′ i ≥∆1, which by the base case yields the claim. Observing the recurrence that for all i, ∆′ i ≥∆′ i−1 + ζi, unrolling this recurrence yields ∆′ i ≥∆1, which by the base case yields the claim. Finally, combining Lemmas 5.1 and 5.2 (recall Lemma 5.1 shows Pr(Y ∈C) ≤2m 2p ; note the proof of Lemma 5.1 still applies after assumption (ii) is dropped) yields that Pr[Y ∈A] −Pr[Y ∈B ∪C] is lower bounded by Pr[Y ∈A] −Pr[Y ∈B] −Pr[Y ∈C] ≥ 1 2q (nc−1) 1 2Mm 1 −1 2m −m 2p −2m 2p . 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In Baeza-Yates R., Montanari U., and Santoro N., editors, Foundations of Information Technology in the Era of Network and Mobile Computing, volume 96 of IFIP — The International Federation for Information Processing, pages 323–336. Springer, Boston, MA, 2002. DOI: 10.1007/978-0-387-35608-2_27. A Additional proofs Lemma A.1. Assume the terminology of Lemma 4.3. Then, there exists a valid history state \|ψ′⟩ on W, C, Q, and Q′ such that \|⟨ψ\|ψ′⟩\|2 ≥1 −O(γ−2). Lemma A.1. Assume the terminology of Lemma 4.3. Then, there exists a valid history state \|ψ′⟩ on W, C, Q, and Q′ such that \|⟨ψ\|ψ′⟩\|2 ≥1 −O(γ−2). Proof. Recall that the smallest non-zero eigenvalue of H1 = ∆(Hin + Hprop + Hstab) is at least J := π2∆/(64L3) (by Lemma 2.2), δ = 1/∆, and η ≥max(∥H2∥∞, 1) =: K. Lemma 4.1 now implies there exists a valid history state \|ψ′⟩satisfying \|⟨ψ\|ψ′⟩\|2 ≥ 1 − K + p K2 + δ(J −2K) J −2K !2 ≥ 1 −  η + q η2 + π2 64L3 π2∆ 64L3 −2η   2 ≥ 1 − (1 + √ 2)η π2∆ 64L3 −2η !2 = 1 − (1 + √ 2)64L3η π2∆−128L3η !2 ≥ 1 −Θ 1 γ2 , where the second inequality follows since 0 ≤K ≤η, the third since π2/(64L3) ≤1 for L ≥1 and 1 ≤η2, and the last since ∆= 128L3ηγ and γ ≥1. where the second inequality follows since 0 ≤K ≤η, the third since π2/(64L3) ≤1 for L ≥1 and 1 ≤η2, and the last since ∆= 128L3ηγ and γ ≥1. 38
https://openalex.org/W4365808497	https://hal.science/hal-03908777/document	English	null	Amazon Expedition Magazine - 1st. Edition \| Manaus-Brazil (2022, Jun)	HAL (Le Centre pour la Communication Scientifique Directe)	2,022	public-domain	25,154	To cite this version: Newton Silva De Lima, Alfredo Tadeu Oliveira Coimbra, Alan dos Santos Ferreira, Aldemir Malveira De Oliveira, André Luiz Willerding, et al.. Amazon Expedition Magazine - 1st. Edition \| Manaus- Brazil (2022, Jun). 1, 2022, 10.29327/5235895. hal-03908777 Amazon Expedition Magazine - 1st. Edition \| Manaus-Brazil (2022, Jun) Newton Silva De Lima, Alfredo Tadeu Oliveira Coimbra, Alan dos Santos Ferreira, Aldemir Malveira De Oliveira, André Luiz Willerding, Daniel Valentim Barros Mansur Da Silva, Eriberto Barroso Façanha Filho, Fábio Raphael Moreira Cáuper, Fernanda Cabral Cidade, Guilherme Vilagelim De Souza, et al. To cite this version: Newton Silva De Lima, Alfredo Tadeu Oliveira Coimbra, Alan dos Santos Ferreira, Aldemir Malveira De Oliveira, André Luiz Willerding, et al.. Amazon Expedition Magazine - 1st. Edition \| Manaus- Brazil (2022, Jun). 1, 2022, 10.29327/5235895. hal-03908777 Amazon Expedition Magazine - 1st. Edition \| Manaus-Brazil (2022, Jun) Newton Silva De Lima, Alfredo Tadeu Oliveira Coimbra, Alan dos Santos Ferreira, Aldemir Malveira De Oliveira, André Luiz Willerding, Daniel Valentim Barros Mansur Da Silva, Eriberto Barroso Façanha Filho, Fábio Raphael Moreira Cáuper, Fernanda Cabral Cidade, Guilherme Vilagelim De Souza, et al. To cite this version: Newton Silva De Lima, Alfredo Tadeu Oliveira Coimbra, Alan dos Santos Ferreira, Aldemir Malveira De Oliveira, André Luiz Willerding, et al.. Amazon Expedition Magazine - 1st. 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Public Domain amazon Expedition MAGAZINE amazonexpedition.zohosites.com 2022, September\|1st Year – V. 1 N. 1 amazon Expedition MAGAZINE amazonexpedition.zohosites.com 2022, September\|1st Year – V. 1 N. 1 Black River in the La Niña Period (2020/2022) and the Climate of the Amazon: Hydrology FRACTUS Experiment Visit to MUSA Observations of Gravity Wave propagation in the Equatorial Ionosphere after the Hunga Tonga–Hunga Ha'apai eruption with Ionosonde data and HYSPLIT Modeling Reduction in water levels and regional warming of the Amazon River from Peru to the Atlantic Ocean in Brazil due to the effects of the 2016 ENSO amazonexpedition.zohosites.com Experiment Amazon Expedition Magazine SCIENTIFIC COMMITTEE PRESIDENT  Aldemir Malveira de Oliveira SCIENTIFIC COORDINATION  Liliam Greicy de Souza Oliveira MEMBERS  Alfredo Tadeu Oliveira Coimbra.  Alan dos Santos Ferreira.  Aldemir Malveira de Oliveira.  Daniel V. B. Mansur da Silva.  Eriberto Barroso Façanha Filho.  Evandro Barbosa Brandão.  Fábio Raphael Moreira Cáuper.  Fernanda Cabral Cidade.  Guilherme Vilagelim de Souza.  João Marcelo Silva Lima.  José Cavalcante Lacerda Júnior.  Kedma Cristine Yamamoto.  Kleitson José Lima Tenório.  Liliam Greicy de Souza Oliveira.  Luciana Lima de Brito Cáuper.  Madalena da Rocha Pietzsch  Manoel Feitosa Jeffreys.  Maryana Antônia Braga Batalha Souza.  Newton Silva de Lima.  Ricardo Figueiredo Monteiro.  Robson Matos Calazães.  Rodrigo de Oliveira Félix.  Roseilson Souza do Vale. CHIEF-EDITOR  Newton Silva de Lima JOURNALISTIC EDITOR  Daniel Valentim de Barros Mansur da Silva First Edition: Special Edition (September 2022) Website: (Know More) amazonexpedition.zohosites.com Consequences La Niña phenomenon in Manaus City – Brazil 2021-2022, J Photos by Niels Lima: Building (imported from England in the 19th century) and Manaus Harbour  Liliam Greicy de Souza Oliveira MEMBERS nsequences La Niña phenomenon in Manaus City – Brazil 2021-2022, June Photos by Niels Lima: Building (imported from England in the 19th century) and Manaus Harbour Map of the Black River (Rio Negro) according to Sir Alfred Russel Wallace, published in the Journal of the Geographical Society by J. M. Albermarte (London, 1895). (Copyright of Fishes of the Black River, edited by Mônica de Toledo-Piza Ragazzo, Edusp, 2002). With Climate Observations, Geological, Natural and Historical of the native peoples of the Black River, from Manaus to the Uaupés River on the border with Colombia. Map of the Black River (Rio Negro) according to Sir Alfred Russel Wallace, published in the Journal of the Geographical Society by J. M. Albermarte (London, 1895). (Copyright of Fishes of the Black River, edited by Mônica de Toledo-Piza Ragazzo, Edusp, 2002). With Climate Observations, Geological, Natural and Historical of the native peoples of the Black River, from Manaus to the Uaupés River on the border with Colombia. September, 2022 CLIMATE 4. Black River in the La Niña Period (2020-2022) and the Climate of the Amazon: Hidrology CLIMATE 15. FRACTUS Experiment African dust scattering, between Sahel and Central Amazon CLIMATE 33. Visit to MUSA (Amazon Natural Museum) SPACE WEATHER 36. Observations of Gravity Wave propagation in the Equatorial Ionosphere after the Hunga Tonga–Hunga Ha'apai eruption with Ionosonde data and HYSPLIT Modeling CLIMATE 47. Reduction in water levels and regional warming of the Amazon River from Peru to the Atlantic Ocean in Brazil due to the effects of the 2016 ENSO. ENVIRONMENT 59. A brief observation of geoindicators in the Madeira River between Novo Aripuanã and Manicoré in 2018. connections SPACE WEATHER \| CLIMATE 70. Observations with operational research on the connection of the 11-year Solar Cycle and the El Niño and La Niña phenomena and rainfall in the Amazon (1980-2030). ENVIRONMENT 59. A brief observation of geoindicators in the Madeira River between Novo Aripuanã and Manicoré in 2018. CLIMATE It also brings a brief description of the FRACTUS Experiment, which involves 5 different sites and the modeling of Sahel dust in the Amazon. The research team pays a visit to the Natural Museum of the Amazon. In an unprecedented aspect, it shows the Gravity Wave phenomenon on the east coast of South America after the eruption of the Hunga Tonga–Hunga Ha'apai volcano in the central pacific. Discusses the ENSO phenomenon of 2016 from the Peruvian city of Iquitos to the mouth of the river in the Atlantic Ocean during the dry period, it constitutes a recurring and interesting topic on the interaction of hydroclimatology and the biosphere - atmosphere in the Amazon, which we call the Current State. 47. Reduction in water levels and regional warming of the Amazon River from Peru to the Atlantic Ocean in Brazil due to the effects of the 2016 ENSO. 47. Reduction in water levels and regional warming of the Amazon River from Peru to the Atlantic Ocean in Brazil due to the effects of the 2016 ENSO. CLIMATE In this first edition, a field observation involving the socio-climatic parameters in the city of Manaus - Brazil (2021-2022) of the Rio Negro floods and the consequences of the passage of the La Niña phenomenon in 2021 and 2022. 36. Observations of Gravity Wave propagation in the Equatorial Ionosphere after the Hunga Tonga–Hunga Ha'apai eruption with Ionosonde data and HYSPLIT Modeling 36. Observations of Gravity Wave propagation in the Equatorial Ionosphere after the Hunga Tonga–Hunga Ha'apai eruption with Ionosonde data and HYSPLIT Modeling 70. Observations with operational research on the connection of the 11-year Solar Cycle and the El Niño and La Niña phenomena and rainfall in the Amazon (1980-2030). In addition, the scope of the research describes the social and economic view of the Amazon River of the phenomena within the Surface Boundary Layer in the diffusion of localized and non-localized turbulence, wind speed and air temperature, among others. In addition, the scope of the research describes the social and economic view of the Amazon River of the phenomena within the Surface Boundary Layer in the diffusion of localized and non-localized turbulence, wind speed and air temperature, among others. Cover Design and Images Niels N C LIn thisSpecialEdition Newton Silva de Lima (Chief-Editor) September, 2022 September, 2022 CLIMATE 4. Black River in the La Niña Period (2020-2022) and the Climate of the Amazon: Hidrology CLIMATE 15. FRACTUS Experiment African dust scattering, between Sahel and Central Amazon CLIMATE 33. Visit to MUSA (Amazon Natural Museum) SPACE WEATHER 36. Observations of Gravity Wave propagation in the Equatorial Ionosphere after the Hunga Tonga–Hunga Ha'apai eruption with Ionosonde data and HYSPLIT Modeling The Amazon holds several adjectives of grandeur about its rainforest for having the largest river in the world, for having the greatest biodiversity of plant and animal specimens, for being the largest contributor to the capture of atmospheric CO2, also for favoring rainfall throughout the Americas. and influence the global circulation of the atmosphere by balancing the planet's temperature. With all this status quo, it is always a subject of research and, of course, always will be. connections SPACE WEATHER \| CLIMATE 70. Observations with operational research on the connection of the 11-year Solar Cycle and the El Niño and La Niña phenomena and rainfall in the Amazon (1980-2030). Newton Silva de Lima (Chief-Editor) Cover Design and Images Niels N C LIn thisSpecialEdition Cover Design and Images Niels N C LIn thisSpecialEdition amazon Expedition Magazine \| 3 amazon Expedition Magazine \| 3 amazon Expedition Magazine \| 4 Image by Robson Calazães: Negro (Black) River - Manaus Harbour. Consequences of the La Niña phenomenon in Northern Brazil in South America in June 2021 - 2022. Consequences La Niña phenomenon in Manaus City – Brazil 2021-2022, June Consequences of the La Niña phenomenon in Northern Brazil in South America in June 2021 - 2022. Consequences La Niña phenomenon in Manaus City – Brazil 2021-2022, June Image by Robson Calazães: Negro (Black) River - Manaus Harbour. amazon Expedition Magazine \| 4 amazon Expedition Magazine \| 4 View of the Port of São Raimundo from the Bridge over the Black (Negro) River in front of Manaus View of the Port of São Raimundo from the Bridge over the Black (Negro) River in front of Manaus View of the Port of São Raimundo from the Bridge over the Black (Negro) River in front of Manaus amazon Expedition Magazine \| 5 Introduction Its volume is strongly influenced by the seasonal climatological phenomena of El Niño, when it reaches 13.63 m and in the La Niña period, it reaches up to 30.00 m, as the normal elevation oscillates between 17.54 m and 21.77 m, in relation to sea level, which is distant from its mouth, another 1,054 km to the Atlantic Ocean. It is the largest tributary on the left bank of the Amazon River, being the seventh largest river in the world by volume of water, responsible for 7% of the water it discharges into the Atlantic, the Black drains an area corresponding to 10% of the 7 million kilometers squares of the Amazon Basin. In, Zeidemann, 2001, more water flows through its bed than flows in all the rivers in Europe combined. From November 2020 to June 2022, the La Niña phenomenon, and the warming of the South Atlantic in the tropical region, plus the formation of the Intertropical Convergence Zone - ITCZ parking over the region in this period, favored greater rainfall intensities over the Amazon and the entire sector North of South America. From then onwards (second half of June), the Black River (Rio Negro) tends to start its ebb process until it reaches its minimum level. The end of the ebb, in turn, does not have a preferential period, and may occur between October and January of the next year. The present work shows the climatic phenomenology of the Manaus region (Brazil) at the meeting of the waters of the Black River with the Solimões River (Rio Solimões). The Black flows near Manaus over soil from the Mesozoic Geological Era in the Cretaceous Period (144 – 66 million years) after the Jurassic period, while the Solimões River runs over the ground of the Quaternary of the Cenozoic Era (1.8 – 0.15 million years old), in this convergence of two geological formations, the beds are of different depths and ages. At this meeting of the waters, the Black has a depth during the flood period of approximately 90 m and the Solimões 42 m. Introduction Introduction he Black River (Rio Negro) is a river that rises in the Guyanese shield in North South America in Colombian territory (Guainia River) and comes towards the great Amazon River (Brazil), where it has its mouth, where its black waters meet the waters of the Solimões river in front of the city of Manaus (Latitude: - 3.10719 Longitude: - 60.0261). Its volume is strongly influenced by the seasonal climatological phenomena of El Niño, when it reaches 13.63 m and in the La Niña period, it reaches up to 30.00 m, as the normal elevation oscillates between 17.54 m and 21.77 m, in relation to sea level, which is distant from its mouth, another 1,054 km to the Atlantic Ocean. It is the largest tributary on the left bank of the Amazon River, being the seventh largest river in the world by volume of water, responsible for 7% of the water it discharges into the Atlantic, the Black drains an area corresponding to 10% of the 7 million kilometers squares of the Amazon Basin. In, Zeidemann, 2001, more water flows through its bed than flows in all the rivers in Europe combined. In this observation period (June, 2021-2022) indicates the increase in the level in the Black basin, which reached a record level of 30.00 m in Manaus (Brazil), the year of La Niña. At the meteorological stations in sequence to Manaus coming from São Gabriel da Cachoeira, the height ruler is 12.23 m, (average 9 m), in Santa Isabel do Rio Negro it is 7.78 m (average 6.70 m) and in Barcelos it is at 9.39 m (average 6 m), being the next in Manaus. (Brazilian Geological Survay - BGS, 2021). t d d he Black River (Rio Negro) is a river that rises in the Guyanese shield in North South America in Colombian territory (Guainia River) and comes towards the great Amazon River (Brazil), where it has its mouth, where its black waters meet the waters of the Solimões river in front of the city of Manaus (Latitude: - 3.10719 Longitude: - 60.0261). Black River in the La Niña Period (2020-2022) and the Climate of the Amazon: Hydrology Lima(1), N S; Malveira, A O; Façanha(1), E; Ferreira(1), A S; Oliveira(1), L G S; Figueredo(1) R.; Calazães(1), R; Coimbra, A T O; Ramos, N L; Vale, R; Mansur, D V B. Lutheran University Center of Manaus - CEULM \| ULBRA(1) Ave. Carlos Drummond de Andrade, 1460 - Conjunto Atílio Andreazza – Japiim CEP 69077-730 - Manaus/AM – Brazil - Telephone: +55 (92) 3616-9800 · e-mail: acsmao@ulbra.br · e-mail: alfredocoimbra@seduc.net · e-mail: newton.lima@ulbra.br · e-mail: liliam.oliveira@ulbra.br · e-mail: naiana.Lopes.r@gmaim.com (Université de Genève -Faculté Sciences- Switzerland) ·e-mail: rics.fig@gmail.com · e-mail: alan.ferreira@ulbra.br · e- mail: danielvmansur@gmail.com (Martha Falcão College - Wyden – AM \| Brazil) · e-mail: matoscalazaes@gmial.com · e-mail: roseilsondovale@gmail.com (Oeste do Pará Federal University \| Brazil) · e-mail: eriberto.filho@ulbra.br · e-mail: amoliveira@gmai.com (Amazonas Federal University \| Brazil) amazon Expedition Magazine \| 5 amazon Expedition Magazine \| 5 Introduction (3D simulation with Sankey Diagram and didactic view of the Underground Diagram - Subway), to better understand the positions and locations of the cities around the rivers, in addition to the bathymetry at the meeting of the waters shown here in the history of 1978, 1988, 1998 and 2013, illustrating the changes and dynamics of the profiles and turbulence at the site of observations. In this observation period (June, 2021-2022) indicates the increase in the level in the Black basin, which reached a record level of 30.00 m in Manaus (Brazil), the year of La Niña. At the meteorological stations in sequence to Manaus coming from São Gabriel da Cachoeira, the height ruler is 12.23 m, (average 9 m), in Santa Isabel do Rio Negro it is 7.78 m (average 6.70 m) and in Barcelos it is at 9.39 m (average 6 m), being the next in Manaus. (Brazilian Geological Survay - BGS, 2021). Amazon River Basin (Hydrology), with emphasis on Modeling (Underground Diagram - Sankey 3D) for the meeting of the Rio Negro waters with the Rio Solimões. (Manaus-Brazil). Amazon River Basin (Hydrology), with emphasis on Modeling (Underground Diagram - Sankey 3D) for the meeting of the Rio Negro waters with the Rio Solimões. (Manaus-Brazil). 1. Manaus – Novo Airão : 154,8 km (2 h 52 min) (3h)* 2. Novo Airão – Barcelos: 276 km (5 h~ 6 h)* 3. Barcelos – Santa Isabel: 220 km (5 h ~ 6 h)* 4. Santa Isabel – S. Gabriel: 222 km (5 h~ 6 h)* 5. São Gabriel da Cachoeira () in speedboat Barcelos Novo Airão 2 1 3 4 Santa Isabel do Rio Negro S. Gabriel da Cachoeira 5 Route from Rio Negro from Colombia to Manaus-Brazil Manus 5 3 2 Comparative images between El Niño and La Niña periods on the Black (Negro) River in Manaus (Brazil). 4 Route from Rio Negro from Colombia to Manaus-Brazil 5 Comparative images between El Niño and La Niña periods on the Black (Negro) River in Manaus (Brazil). The Black (Negro) River in this period was in a period of low tide, although for the entire route between the cities the level of its level was still high, due to the conditions of heavy rains at its head and also throughout its body of water. Noted for the navigability throughout the route. Introduction (mS/cm) O2 Sat(%) O2 Dissipated (mg/L) Manaus S 03005’32.1” W 60002’22.7” Local Time: 06h 20min 27.6 oC (81.68 oF) 100681 Pa 54.01 m 5.14 53.2 76.6 5.36 Moura\| Remanso S 02004’44.9” W 61009’49.9” Local Time: 00h37min 25 oC (80.6 oF) 100574 Pa 62.21 m 7.3 32.3 78.7 6.20 Barcelos S 00024’38.0” W 63037’47.2” Local Time:18h43min 24,5 oC (76,1 oF) 100504 Pa 67.91 m 6.9 37.5 79.1 6.21 Santa Isabel do Rio Negro S 00018’07.1” W 65056’02.7” Local Time:12h04min 31.4 oC (88.52 oF) 100440 Pa 73.61 m 6.7 35.7 78.0 6.21 São Gabriel da Cachoeira S 00007’44.0” W 67005’25.2” Local Time: 06h 25,4 oC (77.72 oF) 100515 Pa 67.49 m 6.9 31.7 78.1 6.26 (Data obtained between Dec 7 - 8, 2022. Trip started in São Gabriel da Cachoeira to Manaus) Table - Geoindicators \| Black (Negro) River(Dec - 2021) Óbidos (S 01o55'03" and W 55o31'05") is a city on the left bank of the Amazon River in the state of Pará (Brazil), close to the city of Nhamundá in the state of Amazonas. It is the last station to measure the flow of the river, whose distance to the mouth is approximately 1,100 km. This care is taken so that the tides do not influence the flow data. Site Temp (oC - oF) P Atm (pascal) Alt. (m) pH Cond. (mS/cm) O2 Sat(%) O2 Dissipated (mg/L) Manaus S 03005’32.1” W 60002’22.7” Local Time: 06h 20min 27.6 oC (81.68 oF) 100681 Pa 54.01 m 5.14 53.2 76.6 5.36 Moura\| Remanso S 02004’44.9” W 61009’49.9” Local Time: 00h37min 25 oC (80.6 oF) 100574 Pa 62.21 m 7.3 32.3 78.7 6.20 Barcelos S 00024’38.0” W 63037’47.2” Local Time:18h43min 24,5 oC (76,1 oF) 100504 Pa 67.91 m 6.9 37.5 79.1 6.21 Santa Isabel do Rio Negro S 00018’07.1” W 65056’02.7” Local Time:12h04min 31.4 oC (88.52 oF) 100440 Pa 73.61 m 6.7 35.7 78.0 6.21 São Gabriel da Cachoeira S 00007’44.0” W 67005’25.2” Local Time: 06h 25,4 oC (77.72 oF) 100515 Pa 67.49 m 6.9 31.7 78.1 6.26 (Data obtained between Dec 7 - 8, 2022. Trip started in São Gabriel da Cachoeira to Manaus) Table - Geoindicators \| Black (Negro) River(Dec - 2021) Table - Geoindicators \| Black (Negro) River(Dec - 2021) (Data obtained between Dec 7 - 8, 2022. Trip started in São Gabriel da Cachoeira to Manaus) In 2021, June. Introduction “The flow recorded (Óbidos – PA \| Brazil) was 270,392.8 m³/s and is considered the highest in the data series, since the record is made with acoustic measurement equipment. With this flow rate, a city like São Paulo (Brazil) could be supplied with its water needs for one day, in 15 seconds”. (CPRM-ANA). Óbidos (S 01o55'03" and W 55o31'05") is a city on the left bank of the Amazon River in the state of Pará (Brazil), close to the city of Nhamundá in the state of Amazonas. It is the last station to measure the flow of the river, whose distance to the mouth is approximately 1,100 km. This care is taken so that the tides do not influence the flow data. Introduction During El Niño, the headwaters of the river become not very navigable due to rock outcrops along the riverbed. They can also be seen even during the flood season on some sections of the trail. A well- noted feature occurs when approaching the head of the river, is the appearance of mountainous elevations, including in this region the largest mountain in Brazilian territory is observed, which is the Neblina peak in the Imeri mountain range (2,995.30 meters of altitude). hI n the first half of December 2021, a trip up and down the Rio Negro was carried out through its multichannels, from Manaus to São Gabriel da Cachoeira. The purpose was to observe the present state of the river, after a record flood in Manaus, within the period of La Niña installed over the region in early 2018, remaining established until 2022 (June), when the river is already in alert quota. of severe flooding in the city of Manaus (Northern Brazil). I ThI hI During the trip along the river, both ascent and descent, a condition of heavy rain throughout the route between the cities of Manaus-São Gabriel da Cachoeira. The location of the source of the river in the Colombian Amazon in the North of South America, has climate type “Af”, in the Köppen-Geiger classification, until close to Manaus (Brazil), that is, rainy all year round regardless of the seasonality season. . That is, it connotes this river as the highest flow tributary in the Amazon River. The meteorological conditions of the trip and the geochemical analysis of the river water were recorded (Table \| Geoindicators – Black River \| 2021, December). amazon Expedition Magazine \| 7Black (Negro)River In 2021, June. “The flow recorded (Óbidos – PA \| Brazil) was 270,392.8 m³/s and is considered the highest in the data series, since the record is made with acoustic measurement equipment. With this flow rate, a city like São Paulo (Brazil) could be supplied with its water needs for one day, in 15 seconds”. (CPRM-ANA). Site Temp (oC - oF) P Atm (pascal) Alt. (m) pH Cond. River Level and Rainfall River Level and Rainfall River Level and Rainfall Scenario of the Meeting of the Waters showing a Sankey Flow Diagram in 3D (Map, Drawing and Graphic) Scenario of the Meeting of the Waters showing a Sankey Flow Diagram in 3D (Map, Drawing and Graphic) amazon Expedition Magazine \| 11 Bathymetry and dynamic analysis of the geological floor at the meeting of waters in over forty years of observations (Black - Solimões) Turbulence (Meeting of the waters) - Surface View of Horizontal Turbulence Turbulence (Meeting of the waters) - Surface View of Horizontal Turbulence Black River (Rio Negro) in La Niña period in 2021, June. Photo: Natja LimaThe ratio between width and depth of a river is represented for the meeting of the Black (Negro) River and SolimõesRiver (Figure below), showing the specificities of the meeting of two rivers and their main water parameters Black River (Rio Negro) in La Niña period in 2021, June. Photo: Natja LimaThe ratio between width and depth of a river is represented for the meeting of the Black (Negro) River and SolimõesRiver (Figure below), showing the specificities of the meeting of two rivers and their main water parameters Black River (Rio Negro) in La Niña period in 2021, June. Photo: Natja LimaThe ratio between width and depth of a river is represented for the meeting of the Black (Negro) River and SolimõesRiver (Figure below), showing the specificities of the meeting of two rivers and their main water parameters v Cross-section of the channels of the two rivers at the meeting of the waters (Black \|Solimões) Scenarios of El Niño and La Niña Rio Negro (Black River) Manaus Harbour \| Brazil Photos: Robson Calazães and Niels Lima Scenarios of El Niño and La Niña Rio Negro (Black River) Manaus Harbour \| Brazil Photos: Robson Calazães and Niels Lima Black (Negro) River Manaus Solimões River The Figure describes the influence of hydrology on major floods in Manaus-Brazil, resulting from the flooding of the Solimões River in the Amazon Basin. Especially during La Niña periods. Black (Negro) River Manaus Solimões River The Figure describes the influence of hydrology on major floods in Manaus-Brazil, resulting from the flooding of the Solimões River in the Amazon Basin. Especially during La Niña periods. resulting from the flooding of the Solimões River in the Amazon Basin. Especially during La Niña periods. g Solimões River in the Amazon Basin. Especially during La Niña periods. Turbulence (Meeting of the waters) - Surface View of Horizontal Turbulence Configuration criteria: horizontal bursts of N-row at the meeting of the waters Black River (Rio Negro) with the Solimões River. (Lima, et al., 2021) Configuration criteria: horizontal bursts of N-row at the meeting of the waters Black River (Rio Negro) with the Solimões River. (Lima, et al., 2021) Configuration criteria: horizontal bursts of N-row at the meeting of the waters Black River (Rio Negro) with the Solimões River. (Lima, et al., 2021) 1 - Formation of coherent vortices. 2 - horizontal turbulent formation configuration. 3 - Configuration of the set of swirls that carry out shear when approaching. 4 - 5 - 6 - 7- Effects of the formation of eddies on liquid walls with different densities, temperature and speeds at the meeting of the waters. 8 - Artistic view of the Turbulance phenomenon. 4 - 5 - 6 - 7- Effects of the formation of eddies on liquid walls with different densities, temperature and speeds at the meeting of the waters. 8 - Artistic view of the Turbulance phenomenon. 1 - Formation of coherent vortices. 2 - horizontal turbulent formation configuration. 3 - Configuration of the set of swirls that carry out shear when approaching. 1 - Formation of coherent vortices. 2 - horizontal turbulent formation configuration. 3 - Configuration of the set of swirls that carry out shear when approaching. amazon Expedition Magazine \| 12 Artistic View of Turbulence within the Channel (Study based on Turbulence in Fluids - MARCEL LESIEUR, 2008) 1. Continuum liquid body; 2. Horizontally quantized body; 3. n = 1; 4. n = 2; 5. n = 4. 6. n - Flow blades (twist-type turbulence). () n = number of blades . Hydraulic, hydrochemical and sedimentological characteristics of the Solimões and Black rivers at the point where the waters meet. Artistic View of Turbulence within the Channel (Study based on Turbulence in Fluids - MARCEL LESIEUR, 2008) Artistic View of Turbulence within the Channel (Study based on Turbulence in Fluids - MARCEL LESIEUR, 2008) Channel (Study based on Turbulence in Fluids - MARCEL LESIEUR, 2008) ulence in Fluids - MARCEL LESIEUR, 2008) 6. n - Flow blades (twist-type turbulence). () n = number of blades . 1. Continuum liquid body; 2. Horizontally quantized body; 3. n = 1; 4. n = 2; 5. n = 4. 1. Continuum liquid body; 2. Horizontally quantized body; 3. n = 1; 4. n = 2; 5. Turbulence (Meeting of the waters) - Surface View of Horizontal Turbulence n = 4. 1. Continuum liquid body; 2. Horizontally quantized body; 3. n = 1; 4. n = 2; 5. n = 4. 6. n - Flow blades (twist-type turbulence). () n = number of blades . Hydraulic, hydrochemical and sedimentological characteristics of the Solimões and Black rivers at the point where the waters meet. Sternberg, H. O’Reilly. 1975. The Amazon River Of Brazil. GEOGRAPHISCHE ZEITSCHRIFT. BEIHEFTE. p.78 Vivian Karina Zeidemann. Forests of the Black River. The Black Waters River. Chapter 2 – USP – Company of Letters, Paulista University and The New York Botanical Garden, 2001. http://ecologia.ib.usp.br/guiaigapo/florestas.ht ml Acknowledgments Martinelli et al., Discharge of total dissolved solids from the Amazon River and its main tributaries. Geochim. Brazil 3 (2): 141-148, 1989. Martinelli et al., Discharge of total dissolved solids from the Amazon River and its main tributaries. Geochim. Brazil 3 (2): 141-148, 1989. The authors are grateful to the Northwest Navigation Survay - Brazilian Navy (NNS - Vila Buriti \| Manaus- Brazil) for accessing navigation chart 4032, for bathymetry assistance, they are also grateful to the Brazilian Geological Survay (BGS) for the hydrology and climatology database from CPRM and ANA offices, and Amazon Protection System - SIPAM. Likewise, to the photographer Niels Lima, the Engineer Natja Lima and Engineer Robson Calazães for the images of the Black river and the field geochemical analyses. Sternberg, H. O’Reilly. 1975. The Amazon River Of Brazil. GEOGRAPHISCHE ZEITSCHRIFT. BEIHEFTE. p.78 amazon Expedition Magazine \| 14 Yalin, M.S. 1992. River Mechanics. Pergamon Press. p. 220. References Brazilian Geological Survey - BGS (Critical Events Alert System) - CPRM - ANA http://www.cprm.gov.br/sace/ Brazilian (https://www.marinha.mil.br/chm/dados-do- segnav/cartas-raster) Brazilian Geological Survey - BGS (Critical Events Alert System) - CPRM - ANA http://www.cprm.gov.br/sace/ Brazilian (https://www.marinha.mil.br/chm/dados-do- segnav/cartas-raster) Navy. Navy. Yalin, M.S. 1992. River Mechanics. Pergamon Press. p. 220. Franzinelli, E. Morphological characteristics of the confluence of the Black and Solimões rivers (Amazon, Brazil). Brazilian Journal of Geosciences, volume 41 (4), 2011. Geo cities project: integrated urban environmental report: GEO report: Manaus/ Supervision: Ana Lúcia Nadalutti La Rovere, Samyra Crespo; Coordination: Rui Velloso. Rio de Janeiro: Consortium Partnership 21, 2002. 188 p.: 21 cm Horbe, Adriana and Collaborators. Geochemistry of the waters of the middle and lower Wood River and its main tributaries - Amazon – Brazil. ACTA AMAZONICA. VOL. 43(4) 2013: 489 – 504. HAS - Hydrological Alert Service (CPRM - ANA) BULLETIN ON HYDROLOGICAL MONITORING OF THE WOOD RIVER BASIN BULLETIN No. 14/2021 May 7, 2021 – Lesieur, Marcel. Turbulence in Fluids Fourth Revised and Enlarged Edition. Library of Congress Control Number: 200794115. All Rights Reserved © 2008 Springer. amazon Expedition Magazine \| 14 amazon Expedition Magazine \| 14 FRACTUS Experiment (BR) Sahel \| Amazon Amazon, the secret of the rains Amazon, the secret of the rains* (Hypothesis) African aerosols fertilize the trees of the Adolpho Ducke Forest Reserve (Manaus-Brazil), of the Peruvian city of Iquitos, of the Pará city of Santarém (Brazil), and of the forest of the upper Black River (Rio Negro) border between Colombia and Brazil. () The FRACTUS Experiment is in field activity. Its expected completion is for the year 2025. Artistic representation of the FRACTUS Experiment work scenario (Authors, 2022) amazonexpedition.zohosites.com (FRaction Aerosol – Climate – Temperature USable) Artistic representation of the FRACTUS Experiment work scenario (Authors, 2022) Artistic representation of the FRACTUS Experiment work scenario (Authors, 2022) amazon Expedition Magazine \| 15 FRACTUS mosaic of African dust scattering, wind trajectories between Africa and Central Amazon at 15,000 m (red) and 13,500 m (green), intermediate blue, during Amazon wet season, with HYSPLIT modeling for 7 days from January 14 to 22 January 2022. Courtesy: HYSPLIT (NOAA-NASA). References (FRaction Aerosol – Climate – Temperature USable) (FRaction Aerosol – Climate – Temperature USable) FRACTUS mosaic of African dust scattering, wind trajectories between Africa and Central Amazon at 15,000 m (red) and 13,500 m (green), intermediate blue, during Amazon wet season, with HYSPLIT modeling for 7 days from January 14 to 22 January 2022. Courtesy: HYSPLIT (NOAA-NASA). FRACTUS mosaic of African dust scattering, wind trajectories between Africa and Central Amazon at 15,000 m (red) and 13,500 m (green), intermediate blue, during Amazon wet season, with HYSPLIT modeling for 7 days from January 14 to 22 January 2022. Courtesy: HYSPLIT (NOAA-NASA). nuary 2022. Courtesy: HYSPLIT (NOAA NASA). (a) Trajectory of African dust (Dust Optical Thickness) over the “hot spots”, that is, on sites in the Amazon between January 1, 2022 and January 20, 2022 (Courtesy: HYSPLIT\|NOAA\|NASA), (b) Image of the global circulation of the atmosphere (CGA). Courtesy: EOSDIS - NASA) from 22 Jan 2022 at 00 UTC, (c) Image of dispersion and deposition of African dust over the target in Manaus (22 Jan 2022), different colors mean different altitudes being red 15000 m (maximum) and green 12500 m (minimum). Courtesy: HYSPLIT\|NOAA\|NASA. amazonexpedition.zohosites.com amazon Expedition Magazine \| 16 (a) Trajectory of African dust (Dust Optical Thickness) over the “hot spots”, that is, on sites in the Amazon between January 1, 2022 and January 20, 2022 (Courtesy: HYSPLIT\|NOAA\|NASA), (b) Image of the global circulation of the atmosphere (CGA). Courtesy: EOSDIS - NASA) from 22 Jan 2022 at 00 UTC, (c) Image of dispersion and deposition of African dust over the target in Manaus (22 Jan 2022), different colors mean different altitudes being red 15000 m (maximum) and green 12500 m (minimum). Courtesy: HYSPLIT\|NOAA\|NASA. (a) Trajectory of African dust (Dust Optical Thickness) over the “hot spots”, that is, on sites in the Amazon between January 1, 2022 and January 20, 2022 (Courtesy: HYSPLIT\|NOAA\|NASA), (b) Image of the global circulation of the atmosphere (CGA). Courtesy: EOSDIS - NASA) from 22 Jan 2022 at 00 UTC, (c) Image of dispersion and deposition of African dust over the target in Manaus (22 Jan 2022), different colors mean different altitudes being red 15000 m (maximum) and green 12500 m (minimum). Courtesy: HYSPLIT\|NOAA\|NASA. amazon Expedition Magazine \| 16 FRACTUS Modeling Strongest surface dust mass concentration in North Africa with density (dark brown), > 9.0 x10 -8 kg/m3. Courtesy: MERRA-2 (Monthly) \| NASA. lots of rain in the period FRACTUS Experiment x10 -8 kg/m3. Courtesy: MERRA-2 (Monthly) \| NASA. FRACTUS modeling for sites 1 - 2 - 3 – 4 - 5, for the month of January 2022, with data from NOAA HYSPLIT MODEL. amazon Expedition Magazine \| 17 amazon Expedition Magazine \| 17 amazon Expedition Magazine \| 17 amazon Expedition Magazine \| 17 amazon Expedition Magazine \| 18 FRACTUS EXPERIMENT: AMAZON, THE SECRET OF THE RAIN T h i 2 R C N i p V Overview Overview he FRACTUS Experiment began its field activities in December 2021, with the trip to the upper Rio Negro to the São Gabriel da Cachoeira City (Site 4) in the North mosaic of South America in Northwest Brazil with proximity to Colombia and Venezuela. he FRACTUS Experiment began its field activities in December 2021, with the trip to the upper Rio Negro to the São Gabriel da Cachoeira City (Site 4) in the North mosaic of South America in Northwest Brazil with proximity to Colombia and Venezuela. (See:cprm.gov.br/sace/index_bacias_monitoradas.php ?getbacia=bamazonas#), including the entire Black River channel from São Gabriel da Cachoeira to Manaus (Amazonas-Brazil), where the Black River meets the Solimões River from hence it receives the name of Rio Amazonas in Brazil. For the year of January 2022, the level of the Black River at the Black River Station in Manaus, is (54.01 m) with a water quota in relation to sea level, higher than in previous years (January 2020; quota of 20.90 m ), that is, outside the average standard for the period (ANA-CPRM, 2022). The climate observations on the sites under study will start with the Köppen-Geiger classification, a reference in the most widely used global climate classification system worldwide (Hasan Mahmud et al., 2015). Following the Köppen-Geiger climate classification, climatic variables and geochemical analyses, this research work is an attempt to analyze the spatial distribution pattern of climatic elements (African aerosols arriving in the Amazon) and describe the period of seasonality, scientifically quantify to categorize the climatic zones affected by African dust from the Sahel. 1 2 Köppen-Geiger climate type in South America, with emphasis on the Amazon, 1. Manaus City (Amazonas-Brazil); 2. Santarém City; 3. Iquitos City (Loreto-Peru); 4. São Gabriel da Cachoeira City (Amazonas-Brazil). Adapted from M. C. Peel et al.: Updated world Koppen-Geiger climate classification map, 2007. Seasonality and annual mean values of air temperature and precipitation are the parameters for determining the climatic types of the Köppen-Geiger classification (Peel et al., 2007). In this context, it is defined as a megathermal climate of tropical and subtropical regions – Main Group - A, with Af (tropical climate without dry season) and Am (tropical monsoon climate) being the blue color representing the main group A. The year 2021 in the Amazon, the La Niña phenomenon was very present, this event began effectively in July 2020 (NOAA, 2020). The year 2019 was completely neutral on the sea surface temperature (SST) in the Equatorial Pacific T he FRACTUS Experiment began its field activities in December 2021, with the trip to the upper Rio Negro to the São Gabriel da Cachoeira City (Site 4) in the North mosaic of South America in Northwest Brazil with proximity to Colombia and Venezuela. FRACTUS EXPERIMENT: AMAZO Overview amazon Expedition Magazine \| 21 amazon Expedition Magazine \| 19 amazon Expedition Magazine \| 19 1. Departing from the São Raimundo Pier (Manaus-Brasil); 2. Departing from Manaus (Brazil); 3. Arriving in São Gabriel da Cachoeira - CGC (Amazon-Brazil); 4. Santa Isabel Harbour; Journey Overview - FRACTUS São Gabriel da Cachoeira – SGC (Amazon\|Brazil) 5. Port of CAMANAUS in SGC; 6. Fieldwork in SGC; 7. On the way between Santa Isabel do Rio Negro and Barcelos; 8. Barcelos City. Journey Overview - FRACTUS São Gabriel da Cachoeira – SGC (Amazon\|Brazil) 1. Departing from the São Raimundo Pier (Manaus-Brasil); 2. Departing from Manaus (Brazil); 3. Arriving in São Gabriel da Cachoeira - CGC (Amazon-Brazil); 4. Santa Isabel Harbour; 5. Port of CAMANAUS in SGC; 6. Fieldwork in SGC; 7. On the way between Santa Isabel do Rio Negro and Barcelos; 8. Barcelos City. amazon Expedition Magazine \| 20 The climate observations on the sites under study will start with the Köppen-Geiger classification, a reference in the most widely used global climate classification system worldwide (Hasan Mahmud et al., 2015). Following the Köppen-Geiger climate classification, climatic variables and geochemical analyses, this research work is an attempt to analyze the spatial distribution pattern of climatic elements (African aerosols arriving in the Amazon) and describe the period of seasonality, scientifically quantify to categorize the climatic zones affected by African dust from the Sahel. Seasonality and annual mean values of air temperature and precipitation are the parameters for determining the climatic types of the Köppen-Geiger classification (Peel et al., 2007). In this context, it is defined as a megathermal climate of tropical and subtropical regions – Main Group - A, with Af (tropical climate without dry season) and Am (tropical monsoon climate) being the blue color representing the main group A. In the tropical climate (A) there is a climatic zone with two subdivisions, Af (tropical climate with no dry season \| strong blue), Am (tropical monsoon climate \| medium blue) covering the entire Amazon and almost the entire northern part of South America, thus translating into a tropical zone climate in which the average air temperature of the coldest month is greater than or equal to 24oC. Af represents the tropical climate without a dry season and this climate covers the northwest area of Brazil in the locality of the city of São Gabriel da Cachoeira, a region known on the map of Brazil as, “Dog Head”. FRACTUS EXPERIMENT: AMAZON, THE SECRET OF THE RAIN African dust from the Sahara to spread across Europe to the Americas is a well-known theme of global monitoring by satellites and the advent of space travel that began in the 1960s. Furthermore, in BARRY and CHORLEY (2013) they comment that the tanks used in World War II in North Africa churned the surface of the desert in such a way that material transported later was visible in clouds over the Caribbean. Imaging Spectroradiometer\|NASA - Terrestrial Observation System - EOS), provides a product called MODIS Aerossol that performs the global monitoring of the optical thickness of the environmental aerosol in the oceans and on continents, “fine” aerosols (anthropogenic/pollution) and “ongoing” aerosols (natural particles; e.g. dust) in synoptic view of the Optical Depth of Atmospheric Aerosols image, all year round. This work uses information from the MODIS sensor (AQUA). The connection of so-called aerosols (suspended particulate matter, measured in mg.m-3) with rainfall in the Amazon comes from studies of these particles derived from the burning of biomass in central Africa, marine aerosols and dust brought by the wind from North Africa generally reach the central part of the Amazon basin during the rainy season, a theory that gains visibility with the physicist Paulo Artaxo. Artaxo, states that 80% of the rain clouds in the period of greater intensity in the Central Amazon are constituted by African aerosols (2009 a, b). The transport of particulate matter on a micron scale between the African and American continents is done by the General Circulation of the Atmosphere - GCA in the equatorial belt, through the Hardley cell, resulting from the East Trade winds and the equatorial belt of greater heating (thermal equator) , on the border of the northeast and southeast trade winds between the Northern and Southern Hemispheres respectively, results in enormous instability of the convective masses due to the warming of the oceans in the summer hemisphere, giving rise to a semi-stationary region known as the Intertropical Convergence Zone - ITCZ with a predominantly oceanic feature, (Barry and Chorley, 2013). The AQUA platform, started its operation in 2002, through the MODIS sensor (MODerate Resolution Seasonality of the ITCZ on the Central Amazon and the Climatological Normals for the Manaus City and São Gabriel da Cachoeira City– SGC. Source: Barry and Chorley, 2013; ANA- CPRM, 2022. FRACTUS EXPERIMENT: AMAZON, THE SECRET OF THE RAIN In the tropical climate (A) there is a climatic zone with two subdivisions, Af (tropical climate with no dry season \| strong blue), Am (tropical monsoon climate \| medium blue) covering the entire Amazon and almost the entire northern part of South America, thus translating into a tropical zone climate in which the average air temperature of the coldest month is greater than or equal to 24oC. Af represents the tropical climate without a dry season and this climate covers the northwest area of Brazil in the locality of the city of São Gabriel da Cachoeira, a region known on the map of Brazil as, “Dog Head”. Köppen-Geiger climate type in South America, with emphasis on the Amazon, 1. Manaus City (Amazonas-Brazil); 2. Santarém City; 3. Iquitos City (Loreto-Peru); 4. São Gabriel da Cachoeira City (Amazonas-Brazil). Adapted from M. C. Peel et al.: Updated world Koppen-Geiger climate classification map, 2007. Köppen-Geiger climate type in South America, with emphasis on the Amazon, 1. Manaus City (Amazonas-Brazil); 2. Santarém City; 3. Iquitos City (Loreto-Peru); 4. São Gabriel da Cachoeira City (Amazonas-Brazil). Adapted from M. C. Peel et al.: Updated world Koppen-Geiger climate classification map, 2007. Köppen-Geiger climate type in South America, with emphasis on the Amazon, 1. Manaus City (Amazonas-Brazil); 2. Santarém City; 3. Iquitos City (Loreto-Peru); 4. São Gabriel da Cachoeira City (Amazonas-Brazil). Adapted from M. C. Peel et al.: Updated world Koppen-Geiger climate classification map, 2007. The year 2021 in the Amazon, the La Niña phenomenon was very present, this event began effectively in July 2020 (NOAA, 2020). The year 2019 was completely neutral on the sea surface temperature (SST) in the Equatorial Pacific. So, everything indicates that in 2022, following that of 2021, the effects of the event will remain active in this region, as cities throughout the Brazilian Amazon were greatly affected by the record floods. amazon Expedition Magazine \| 21 FRACTUS EXPERIMENT: AMAZON, THE SECRET OF THE RAIN Modeling the scattering of African dust over Europe, the Atlantic Ocean and the Amazon (Adapted from L3 MODIS imagery \| NASA, 2015). Optical Depth of Aerosol in the period of September 2015, for illustration of Sites 1,2,3,4 and 5. (MODIS \| NASA, 2015). Seasonality of the ITCZ on the Central Amazon and the Climatological Normals for the Manaus City and São Gabriel da Cachoeira City– SGC. Source: Barry and Chorley, 2013; ANA- CPRM, 2022. amazon E pedition Maga ine \| 22 Modeling the scattering of African dust over Europe, the Atlantic Ocean and the Amazon (Adapted from L3 MODIS imagery \| NASA, 2015). Optical Depth of Aerosol in the period of September 2015, for illustration of Sites 1,2,3,4 and 5. (MODIS \| NASA, 2015). Seasonality of the ITCZ on the Central Amazon and the Climatological Normals for the Manaus City and São Gabriel da Cachoeira City– SGC. Source: Barry and Chorley, 2013; ANA- CPRM, 2022. Modeling the scattering of African dust over Europe, the Atlantic Ocean and the Amazon (Adapted from L3 MODIS imagery \| NASA, 2015). Optical Depth of Aerosol in the period of September 2015, for illustration of Sites 1,2,3,4 and 5. (MODIS \| NASA, 2015). amazon Expedition Magazine \| 22 Manaus Harbour – Brazil (2016, July) Research Route - FRACTUS In images Tabatinga \| Santa Rosa Tabatinga \| Santa Rosa Tabatinga \| Santa Rosa Crossing point between Tabatinga (Brazil) and Santa Rosa (Peru) Crossing point between Tabatinga (Brazil) and Santa Rosa (Peru) amazon Expedition Magazine \| 24 Crossing point between Tabatinga (Brazil) and Santa Rosa (Peru) Tabatinga \| Santa Rosa 2016, July Crossing point between Tabatinga (Brazil) and Santa Rosa (Peru) Islandia \| Peru Islandia \| Peru Islandia \| Peru Tabatinga \| Santa Rosa 2016, July Crossing point between Tabatinga (Brazil) and Santa Rosa (Peru) Crossing point between Tabatinga (Brazil) and Santa Rosa (Peru) Santa Rosa Harbor – Peru (2016, July) Benjamin Constant Islandia \| Peru Benjamin Constant \| Brazil Islandia \| Peru Neighboring cities (connected by the Amazon River. 2016, July) Benjamin Constant \| Brazil Neighboring cities (connected by the Amazon River. 2016, July) São Gabriel da Cachoeira \| Brazil Black (Negro) River. 2021, December amazon Expedition Magazine \|28 Black (Negro) River. 2021, December amazon Expedition Magazine \|28 amazon Expedition Magazine \|28 Black (Negro) River. 2021, December Santa Isabel do Rio Negro\| Brazil Black (Negro) River. 2021, December Santa Isabel do Rio Negro\| Brazil Santa Isabel do Rio Negro\| Brazil amazon Expedition Magazine \| 29 Choachí \| Colombia Planting in mountains in the Andes, 2022, April. Planting in mountains in the Andes, 2022, April. amazon Expedition Magazine \| 30 amazon Expedition Magazine \| 30 Guatavita Lagoon (Eldorado Ceremony. 2022, April) Guatavita \| Colombia Guatavita \| Colombia Guatavita \| Colombia Guatavita Lagoon (Eldorado Ceremony. 2022, April) Manaus \| Brazil Black (Negro) River Hydrographic Station in Manaus, (July 2022). La Niña period. Manaus \| Brazil Manaus \| Brazil Black (Negro) River Hydrographic Station in Manaus, (July 2022). La Niña period. Black (Negro) River Hydrographic Station in Manaus, (July 2022). La Niña period amazon Expedition Magazine \| 32 The FRACTUS Experiment should start from July 2016 to 2019. Discontinued in 2019 until 2021. Now it continues until 2026. To learn more about the survey, go to: HAL Id: hal-03130887 https://hal.archives-ouvertes.fr/hal-03130887 The FRACTUS Experiment should start from July 2016 to 2019. Discontinued in 2019 until 2021. Now it continues until 2026. To learn more about the survey, go to: HAL Id: hal-03130887 https://hal.archives-ouvertes.fr/hal-03130887 The FRACTUS Experiment should start from July 2016 to 2019. Discontinued in 2019 until 2021. Now it continues until 2026. To learn more about the survey, go to: HAL Id: hal-03130887 https://hal.archives-ouvertes.fr/hal-03130887 Manaus \| Brazil Port region in the La Niña period. (July 2022). Manaus \| Brazil Port region in the La Niña period. (July 2022). Manaus \| Brazil Port region in the La Niña period. (July 2022). amazonexpedition.zohosites.com amazon Expedition Magazine \| 33 amazonexpedition.zohosites.com amazon Expedition Magazine \| 33 amazon Expedition Magazine \| 33 Amazon Research NEWS Amazon Research NEWS amazon Expedition Magazine \| 34 Atmosphere Office 101 The research team from the Atmosphere Office 101 (Yogeshwar Sahai Laboratory) went to Amazon Natural Museum (MUSA) to verify the possibility of developing observations on climate and environment at the MUSA in a dry and wet season in Central Amazon (Brazil), North of South America. The team made a brief observation of the scenery and conditions of collection of atmospheric information at the location, in order to understand the behavior of direction, vertical wind profile, humidity, atmospheric pressure, rainfall, temperature and greenhouse gases. Such investigations will become relevant climate and environment information for the Amazon Natural. Visit to MUSA Site Fieldwork Site Fieldwork Site Fieldwork The climate observations at MUSA will be developed with the support of the Climate and Environment of the Y. Sahai Laboratory team, in the field of Geosciences. Together they intend to build an Orthomosaic GIS for the Museum area with several layers that reflect the climate and environment of the scenario. Acccess: Site Fieldwork Site Climatology Site Climatology Site Climatology Abstract This article reports the propagation of gravity waves in the Ionosphere over the west coast of South America (-11.9523954; -76.8757172) as of 01:03 UTC, after the eruption of the Hunga Tonga-Hunga Ha'apai volcano (-20.8229 ; -175.4376) using Ionosonde (VIPIR) (PEZZOPANE and SCOTTO, 2005) and HYSPLIT Modeling for January 15, 2022 (17:00 UTC). It was observed the real accuracy of this instrumentation for the verification of gravity waves in the Equatorial Ionosphere. Fagundes et al., (2008) proposes to study gravity waves, through ionograms obtained by digital Ionsonde that simulate the same RADAR sounding, so sounding analyzes with high temporal resolution (100 seconds, for example) should be used. In Pillat and Guimarães, (2014), the technology and data acquisition by ionosondes are in the radio frequency range from 1 to 20-30 MHz. The radio frequency signals transmitted by ionosondes are reflected when the transmitted frequency is equal to the natural frequency. of the ionospheric plasma, that is, the echoes return to the Earth's surface, whose graphs resulting from these echoes when performing a frequency sweep are called ionograms. In this work we use a digital Ionosonde composed of several antennas (Vertical Incidence Pulsed Ionospheric Radar – VIPIR), (MENDOZA et al., 2021, TSAI, et al., 2014), and HYSPLIT - (Hybrid Single Particle Lagragian Integrated) modeling. Keywords: Layers F1, F2, F3, Es; Magnetic Equator; ionograms (VIPIR). Introduction In Lima (2009) and Lima et. al., (2011), he says that oscillating ionospheric disturbances such as gravity waves (GW) and traveling ionospheric disturbances (TID - Traveling Ionospheric Disturbances), are treated by some researchers as synonyms (KELLEY, 1989, HARGREAVES, 1992). TIDs, which are oscillations with a period on the order of hours to minutes, are produced in geomagnetic periods disturbed by the Joule effect at high latitudes and travel in the equatorial direction, while GW generally have their origins in the lower atmosphere by tropospheric winds, fronts cold weather or tropical convection (FAGUNDES et al., 2007) and according to Hines (1960), can also be caused by meteors entering the Earth's atmosphere, rocket launches in the F region of the ionosphere or by Solar Eclipse. In addition to nuclear explosion, volcanic eruption (FERREIRA et al., 2018; KOMJATHY et al., 2012, MINSTER, 1996; ROW, 1967). Hegai et al., (2006), investigated disturbances in the F2 region at mid- latitude during nighttime in the ionosphere that were produced by internal gravity waves generated before a strong earthquake through Joule heating due to the electric field of short duration seismogenics, in the Region D, of the ionosphere. Hines (1974) adds that GW originate by transfer of momentum and pressure and gravity gradients in an anisotropic medium. Observations of Gravity Wave propagation in the Equatorial Ionosphere after the Hunga Tonga–Hunga Ha'apai eruption with Ionosonde data and HYSPLIT Modeling Newton Silva de Lima1,2, Alan Ferreira dos Santos1, Eriberto Barroso Façanha Filho1,2, Liliam Gleicy de Souza Oliveira1, Aldemir Malveira de Oliveira2,3, Daniel Barros Valentim Mansur da Silva4. (1) Lutheran University Center of Manaus – CEULM/ULBRA Av. Carlos Drummund de Andrade, 1460. Conj. Atilio Andreazza – Japiim II – CEP 69077-730. Manaus, Amazonas - Brazil. Tel: (+55 92 3616-9800). Email: acsmao@ulbra.br (2) Secretary of State for Education and Sports of the State of Amazonas Street: Waldomiro Lustosa, 250 - Japiim II, Manaus – Amazonas. Brazil. CEP 69076-830 Manaus, Amazonas - Brazil. https://www.seduc.am.gov.br/ (3) University Center for Higher Education of Amazonas - CIESA Rua Pedro Dias, 203 – Flores. CEP 69058-818 Manaus – Amazonas – Brazil. Tel. (+55 92 99401-6229). Email: academico@ciesa.br (4) Martha Falcão College - Wyden Rua Natal, 300 – Adrianópolis - Manaus – Amazonas – Brazil. CEP 69057-090. Tel.: (+55 (92) 2121- 0929). e-mail: newton.lima@ulbra.br; alan.ferreira@ulbra.edu.br; eriberto.filho@ulbra.br; liliam_gso@yahoo.com.br; amoliveira@gmail.com, danielvmansur@gmail.com. e-mail: newton.lima@ulbra.br; alan.ferreira@ulbra.edu.br; eriberto.filho@ulbra.br; liliam_gso@yahoo.com.br; amoliveira@gmail.com, danielvmansur@gmail.com. amazon Expedition Magazine \| 36 The site The site Observations were carried out between coordinates (-20.570; -175.398) and (- 11.9523954; -76.8757172), near the center of the Equatorial Pacific Ocean and the west coast of South America over Peru, respectively. The site Observations were carried out between coordinates (-20.570; -175.398) and (- 11.9523954; -76.8757172), near the center of the Equatorial Pacific Ocean and the west coast of South America over Peru, respectively. Gravity Waves (GW) Gravity Waves (GW) Study the morphological aspects of the GW, that is, seasonal variation, preferential propagation direction, wavelengths, periods and characteristic propagation speed, mainly in the equatorial region of the event (January 15, 2022) with the aid of Trajectory, dispersion, deposition modeling and Ionograms, were performed in this observation. The dynamics of the lower and upper atmosphere, which were excited after the strong volcanic eruption of the Hunga Tonga- Hunga Ha'apai, mainly in the transport of momentum and energy through clouds of ash, steam and gas up to 30 km into the atmosphere at the beginning of the eruption , the plume that initially spread across 260 km in diameter before being distorted by wind and seismic magnitude of M4.8. (NASA; usgs.gov; IRIS – (2022). During propagation of gravity waves in the neutral atmosphere, depending on the type of forcing in the ionospheric plasma, the time in the region of maximum anomaly (in this study, on the magnetic equator) the appearance of stratification for an F3 layer, is sometimes present. , a fact also discussed by Klausner and Fagundes (2005) and shown in Mendoza et al., (2021). amazon Expedition Magazine \| 37 Fig. 1 – (A) The Site: Mosaic of the Observation Site between the Hunga Tonga-Huga Ha'apai volcano and the west coast of South America, wind trajectory modeling for possible dispersion and deposition of ash at tropospheric altitude between days 15 December 2021 to January 18, 2022. Courtesy: HYSPLIT – NASA, (2022). (B) Ionogram of January 15, 2022 at 02:03:04 UTC, in Jicamarca (Peru), using the VIPIR technique. Courtesy: LISN/IGP, (2022). Fig. 1 – (A) The Site: Mosaic of the Observation Site between the Hunga Tonga-Huga Ha'apai volcano and the west coast of South America, wind trajectory modeling for possible dispersion and deposition of ash at tropospheric altitude between days 15 December 2021 to January 18, 2022. Courtesy: HYSPLIT – NASA, (2022). (B) Ionogram of January 15, 2022 at 02:03:04 UTC, in Jicamarca (Peru), using the VIPIR technique. Courtesy: LISN/IGP, (2022). Gravity Waves (GW) Fig.2 - Equipment set (Double-sided): Model – VIPIR: Vertical Incidence Pulsed Ionospheric Radar Very high interference immunity: IP3 > 45 dBm; High Dynamic Range: 115(I) +30(V) dB; Direct RF sampling 14 bits at 80 MHz; Fully digital conversion, receiver and exciter; Waveform Agility: 2 µs to 2 ms pulse/chip width; USB-2 Data and Command/Control Interfaces; 8 coherent receive channels; Frequency: 0.3 – 26 MHz; 4 kW class AB pulse amplifier; 3rd harmonic < - 30 dBc; Precise GPS timing possible for bi-static operation; Radar software Open Source C code; runs under Linux. Cortesy: National Oceanic and Atmospheric Administration – NOAA and National Geophysical Data Center \| Terence.Bullet@colorado.edu (2018). Ionsonde – VIPIR (Vertical Incidence Pulsed Ionospheric Radar) Ionsonde – VIPIR (Vertical Incidence Pulsed Ionospheric Radar) VIPIR is a fully digital frequency radar that operates between 0.3 and 26 MHz. It has 8 digital receivers and a digital transmission exciter. It has extremely high-performance analog receiving electronics and a 4 kW solid-state amplifier provide an interface to the real world. This work shows an application of ionograms recorded by this Ionosonde (Fig. 2). Due to the Earth's magnetic field, the ionosphere is birefringent at radio frequencies - there are two possible propagation modes, called ordinary (O) and extraordinary (X) mode. This means that there is a high frequency (HF) radio band sweep, normally they find two distinct ionospheric returns for each frequency. For automatic scaling of ionograms, it is highly desirable to be able to separate the two modes (HARRIS and PEDERICK, 2017). VIPIR is a fully digital frequency radar that operates between 0.3 and 26 MHz. It has 8 digital receivers and a digital transmission exciter. It has extremely high-performance analog receiving electronics and a 4 kW solid-state amplifier provide an interface to the real world. This work shows an application of ionograms recorded by this Ionosonde (Fig. 2). Due to the Earth's magnetic field, the ionosphere is birefringent at radio frequencies - there are two possible propagation modes, called ordinary (O) and extraordinary (X) mode. This means that there is a high frequency (HF) radio band sweep, normally they find two distinct ionospheric returns for each frequency. For automatic scaling of ionograms, it is highly desirable to be able to separate the two modes (HARRIS and PEDERICK, 2017). Fig.2 - Equipment set (Double-sided): Model – VIPIR: Vertical Incidence Pulsed Ionospheric Radar Very high interference immunity: IP3 > 45 dBm; High Dynamic Range: 115(I) +30(V) dB; Direct RF sampling 14 bits at 80 MHz; Fully digital conversion, receiver and exciter; Waveform Agility: 2 µs to 2 ms pulse/chip width; USB-2 Data and Command/Control Interfaces; 8 coherent receive channels; Frequency: 0.3 – 26 MHz; 4 kW class AB pulse amplifier; 3rd harmonic < - 30 dBc; Precise GPS timing possible for bi-static operation; Radar software Open Source C code; runs under Linux. Cortesy: National Oceanic and Atmospheric Administration – NOAA and National Geophysical Data Center \| Terence.Bullet@colorado.edu (2018). Results and Discussion The event in the Central Pacific began at 17 UTC. At this time on the west coast of South America (Jicamarca – Peru) it was 01 UTC, (Peru is -5 UTC). In order to understand the results of the ionograms and modeling, we will have to start our observations with the records of Geoscience research agencies (NASA, NOAA, INGV, USGS) that justifies the phenomenon recorded on the volcanic eruption event that occurred in the Central Pacific near the island. of Tonga on January 15, 2022. The event in the Central Pacific began at 17 UTC. At this time on the west coast of South America (Jicamarca – Peru) it was 01 UTC, (Peru is -5 UTC). In Fig. 3, at the time of (00:03:04 UTC) it is 19 HL and the Pre-Reverse Peak is taking place in the Electric Field, that is, the inversion of the direction of the Terrestrial Electric Field is taking place, whose effect is a strong elevation altitude of the F layer, at this moment the ionogram registers strong echoes over the F layer, that is, defined close to 300 km of altitude. At, (01:03:04 UTC) there is an echo decrease over the VIPIR signal (transmission) with more definition of the F layer at 350 km altitude. At, (02:03:04 UTC) there is an appearance of Spread-F (Plasma Bubbles) in the signal with altitude 420 km altitude. In, (03:03:04 UTC) F layer wrapped in full Spread-F (Plasma Bubble) at 480 km altitude. In Fig. 3, at the time of (00:03:04 UTC) it is 19 HL and the Pre-Reverse Peak is taking place in the Electric Field, that is, the inversion of the direction of the Terrestrial Electric Field is taking place, whose effect is a strong elevation altitude of the F layer, at this moment the ionogram registers strong echoes over the F layer, that is, defined close to 300 km of altitude. At, (01:03:04 UTC) there is an echo decrease over the VIPIR signal (transmission) with more definition of the F layer at 350 km altitude. At, (02:03:04 UTC) there is an appearance of Spread-F (Plasma Bubbles) in the signal with altitude 420 km altitude. In, (03:03:04 UTC) F layer wrapped in full Spread-F (Plasma Bubble) at 480 km altitude. Ionsonde – VIPIR (Vertical Incidence Pulsed Ionospheric Radar) The acquisition of the ionograms was carried out by the Low-Latitude Ionospheric Sensor Network – Instituto Geofisico del Perú - IGP, at the Jicamarca Radio Observatory (JRO) JM91J, (Lima - Peru), as it has a VIPIR acquisition technique system (Fig. 2), and be ideal for viewing the Magnetic Equator proximity anomaly. The acquisition of the ionograms was carried out by the Low-Latitude Ionospheric Sensor Network – Instituto Geofisico del Perú - IGP, at the Jicamarca Radio Observatory (JRO) JM91J, (Lima - Peru), as it has a VIPIR acquisition technique system (Fig. 2), and be ideal for viewing the Magnetic Equator proximity anomaly. – Instituto Geofisico del Perú - IGP, at the Jicamarca Radio Observatory (JRO) JM91J, (Lima - Peru), as it has a VIPIR acquisition technique system (Fig. 2), and be ideal for viewing the Magnetic Equator proximity anomaly. amazon Expedition Magazine \| 38 Fig. 3 - Panel of Ionograms of the times (00 UTC; 01 UTC; 02 UTC and 03 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. Fig. 3 - Panel of Ionograms of the times (00 UTC; 01 UTC; 02 UTC and 03 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. Results and Discussion Comparing the energy of the volcanic explosion with that released by another type of explosion, such as the Hiroshima bomb (Mw = 4.8), the magnitude Mw = 5.7 calculated for the Tonga eruption corresponds to a load of 355,000 tons of TNT (equivalent to TNT), therefore, at least 25 times larger than the Hiroshima explosion (equivalent to 15,000 tons of TNT), (INVG, 2022). For this observation, Ionosonda Digital - VIPIR in Jicamarca records the day of January 15, 2022, (Fig. 3), in the first four ionograms of a total of 24 ionograms in the 1 h time sequence between them, for this work. amazon Expedition Magazine \| 39 Fig. 4 - Ionogram Panel of the times (04 UTC; 05 UTC; 06 UTC and 07 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. Fig. 4 - Ionogram Panel of the times (04 UTC; 05 UTC; 06 UTC and 07 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. At, Fig. 6, (12:03:04 UTC), the GW remain in the high atmosphere environment scattering the ionospheric plasma and begin to appear at an altitude of 100 km approximately in the form of a sporadic E layer, with the radar signal producing strong echoes and the definition of the heights of layers F1 and F2 confused at this time. For the times of (13:03:04 UTC), (14:03:04 UTC) and (15:03:04 UTC) in the range of 4 h to 6 h from the beginning of the volcano eruption, a strong connection is noted of the reflected signal from the plasma between 100 km to 600 km with definition of F1 and F2 and the acquisition signals of O and X, well defined. It is known that when the ionosphere has regions with very rarefied plasma density, the acquisitions (O and X) are more difficult to define the heights of the F1 and F2 layers. In Fig. 4, we have at (04:03:04 UTC) a fully diffused signal in Spread-F, close to 400 km of altitude. In, (05:03:04 UTC) Plasma bubbles involved in the F layer with strong echo in the Radar signal - VIPIR. At, (06:03:04 UTC) very strong echoes for all sweep frequencies of the Ionsonde Plasma Bubble formation. (07:03:04) decreases the Plasma Bubbles and the ordinary and extraordinary signals now appear to be more defined for F1 and F2 and signal reflection for F2 with ionospheric plasma scattering. Results and Discussion For Fig. 5, from (08:03:04 UTC) the Radar signal (VIPIR) tends to disappear, remaining thus at (09:03:04 UTC) and (10:03:04 UTC), returning slowly appearing at (11:03:04 UTC), but for the low frequencies of Ionosonde at 300 km altitude. This effect on the ionograms is strongly linked to Gravity Waves (GW) brought to the Ionosphere by tropospheric winds from the eruption of the Hunga Tonga-Hunga Ha'apai volcano at 08 UTC, in the Central Pacific. In Fig. 7, at (16:03:04 UTC), (17:03:04 UTC), (18:03:04 UTC) and (19:03:04 UTC) we observe the influence of GW on the ionospheric plasma, with Es formation for all Ionosonde scanning frequencies and F3 layer formation after 17 UTC. amazon Expedition Magazine \| 40 Fig. 4 - Ionogram Panel of the times (04 UTC; 05 UTC; 06 UTC and 07 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. Fig. 4 - Ionogram Panel of the times (04 UTC; 05 UTC; 06 UTC and 07 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. Fig. 5 – Timetable Ionogram Panel (08 UTC; 09 UTC; 10 UTC and 11 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. amazon Expedition Maga Fig. 5 – Timetable Ionogram Panel (08 UTC; 09 UTC; 10 UTC and 11 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. amazon Expedition Mag ble Ionogram Panel (08 UTC; 09 UTC; 10 UTC and 11 UTC) in Jicamarca for January 15, LISN \| IGP 2022 amazon Expedition Magazine \| 41 Fig. 6 - Timetable Ionogram Panel (12 UTC; 13 UTC; 14 UTC and 15 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. Fig. 6 - Timetable Ionogram Panel (12 UTC; 13 UTC; 14 UTC and 15 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. amazon Expedition Magazine Fig.7 - Timetable Ionogram Panel (16 UTC; 17 UTC; 18 UTC and 19 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. Fig.7 - Timetable Ionogram Panel (16 UTC; 17 UTC; 18 UTC and 19 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. amazon Expedition Magazine \| 42 Fig. 8 - Timetable Ionogram Panel (20 UTC; 21 UTC; 22 UTC and 23 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. Fig. Results and Discussion 8 - Timetable Ionogram Panel (20 UTC; 21 UTC; 22 UTC and 23 UTC) in Jicamarca for January 15, 2022. Courtesy: LISN \| IGP, 2022. The modeling of the dispersion and deposition of volcanic ash shows the global dimension of the phenomenon. Comparing the energy of the volcanic explosion with that released by another type of explosion, such as the Hiroshima bomb (Mw = 4.8), the magnitude Mw = 5.7 calculated for the Tonga eruption corresponds to a load of 355,000 tons of TNT (equivalent to TNT), therefore at least 25 times greater than the Hiroshima explosion (equivalent to 15,000 tons of TNT) and P and S seismic waves, traveling at speeds between > 340 m/s (in air) at 1450 m/s (in water). (INGV, 2022). The consequence of the shock wave at the ocean-atmosphere interface is devastating and produces a large difference in pressure, rumble and ejection of particulate matter that are projected in the first moment at an altitude greater than 15 km (NASA Earth Observatory System, 2022). Conclusion Observations in the Equatorial Ionosphere using Digital Ionosonde – VIPIR in Jicamarca (- 11.9523954; -76.8757172) and HYSPLIT Modeling of trajectory and ash dispersion, showed efficient and strong accuracy. They also showed the propagation of Gravity Waves, shortly after the eruption of the Hunga Tonga- Hunga Ha'apai volcano (17 HL Tonga, 08 HL London and 03 HL Jicamarca). This work begins by announcing a series of 24 Ionograms with a 1 h interval, which described the behavior of the Equatorial Ionosphere very close to (or over) the Magnetic Equator on the west coast of South America. Confirming the point location anomaly (-11.9523954); - 76.8757172), by enabling the visualization by Ionograms at the time of (00:03:04 UTC to 07:03:04 UTC) the development of Ionospheric Plasma Bubbles throughout the dawn of January 15, 2022. It also identifies the propagation of Gravity Waves in the equatorial position during (08:03:04 UTC and 10:03:04 UTC) the visual identification by Ionograms, through the disappearance or removal (profile of the F layer) in the target of the echoes that should return to the Radar Digital, ie the scanning of the VIPIR Fig. 9 shows the modeling in the volcano scenario and the ash dispersion to 15 km of altitude in the first 12 h of the event, if we compare the aerial of the spot, with the city of São Paulo in Brazil, which is 1,521 km2, the largest city in South America, the spot is more than 36 times bigger, even in the first 12 hours. amazon Expedition Magazine \| 43 frequency range (0.3 – 26 MHz) was compromised by GW. The profiles very slowly return to appear on the VIPIR screen at (11:03:04 UTC). Tropospheric winds of > 340 m/s in the atmosphere (NASA, USGS, INGV) (with a certain inclination, shear with stratospheric winds, also with a certain inclination within the atmospheric envelope of the Earth Magnetic Field, inducing an altitude of approximately 100 km (on Screen of VIPIR) the formation of a Sporadic layer – Es. A fact already discussed by Hines, 1964, confirming Lorentz's theory for particles or charges immersed with great speed in a magnetic field, the effect of rotation and displacement. volcano expels particulate material rich in metallic ions (HERRMANN, et al., 1978), which rise and favor the construction of Es throughout 15 January 2022. amazon Expedition Magazine \| 44 Conclusion Also, during the daytime phase (18:03:04 UTC – 14 HL), formation of a third layer F (F3) appears and remains until (20:03:04 UTC). of the Hunga Tonga- Hunga Ha'apai, in the range of (07:03:04 UTC to 23:03:04). During the day phase, the acquisition of information by the O/X signal (ordinary and extraordinary) are well defined in the ionograms, since, during the night, the Spread-F formation is constant, confusing the visualization of a given signal. The trajectory HYSPLIT Modeling (Fig. 1-A) and ash dispersion (Fig. 9) confirm the efficiency and accuracy of the VIPIR in Jicamarca. The authors, however, indicate that more information can be extracted with these two instruments used in this observation. Fig. 9 – Modeling of the dispersion and deposition of ash from the Hunga Tonga-Huga Ha'apai volcano (-20.8229; -175.4376) for the first 12 h of the beginning of the event, at an altitude of 15 km. Courtesy: HYSPLIT – Vulcan Ash. (NASA). (Authors' Note: Although on January 14, 2022, magnetometer (forecasting and monitoring) data from the WDC (World Data Center - Kyoto \| Japan) indicated a geomagnetic storm (-88 nT at 24 GMT) in comparison with of the same time of the Auroral Electrojet (WDC data), for the equatorial latitude it is not possible to inform a strong influence of this storm. Therefore, the disturbances in the equatorial ionosphere east of South America (January 15, 2022) are strongly affected by Gravity Waves resulting from the explosion of the Hunga Tonga-Huga Ha'apai volcano as shown in Fig. 1A and Fig. 9 of this paper viewed by HYSPLIT modeling). amazon Expedition Magazine \| 44 Acknowledgments HARGREAVES, J. K. (1992). The solar- terrestrial environment, Cambridge: Cambridge University Press, v.5, 420p. The authors are immensely grateful for the courtesy of Low-Latitude Ionospheric Sensor Network – LISN, Instituto Geofisico del Perú – IGP and the Ministry of the Environment of Peru, for making the information available by Ionograms (VIPIR \| O/X) from Station JM91J: Jicamarca Radio Observatory – JRO. We also thank the Air Resources Laboratory (NOAA-NASA) for making available the HYSPLIT - models that simulate the dispersion and trajectory of substances transported and dispersed through our atmosphere, on local to global scales. We thank the support of the researchers' institution: CEULM/ULBRA – SEDUC-AM – CIESA – FMF- Wyden, Dr. Evandro Barbosa Brandão (CEULM/ULBRA-Manaus-Brasil) to Dr. Troy Beldini (UFOPA-Santarém-Brazil), the agencies NASA, NOAA, INGV, USGS for relevant information about the research object of the article. HARRIS, T. J., PEDERICK, L. H. (2017). A Robust Automatic Ionospheric O/X Mode Separation Technique for Vertical Incidence Sounders. Radio Science. https://doi.org/10.1002/2017RS006279. HEGAI, V. V.; LIU, J. Y.; KIM, V.P. (2006) The ionospheric effect of atmospheric gravity waves excited prior to strong earthquake. Advances in Space Research 37-653-659. Available at: https://www.researchgate.Net/publication/2 22556220. HERRMANN, U., EBERHARDT, P., HIDALGO, M. A., KOPP, E., SMITH, L. G. (1978). Metal ions and isotopes in sporadic E-layers during the Perseid meteor shower. Space Res. 18, 249. References HINES, C. O. et al. (1974). The upper atmosphere in motion, Canadian Journal of Geophysical, American Geophysical Union, Washington, D.C, v.75, 2563-2586. HINES, C. O., (1964). The formation of mid- latitude sporatic-E layers. Journal Geophysical Research. Volume 69, Issue 5. 1 Mar 1964. Pages 1018-1019. https://doi.org/10.1029/JZ069i005p01018. BULLETT, T., MALAGNINI, A., PEZZOPANE M., SCOTTO C (2010). Application of Autoscala to ionograms recorded by the VIPIR ionosonde. Advances in Space Research. Volume 45, Issue 9, 3 May 2010, Pages 1156-1172. https://doi.org/10.1029/JZ069i005p01018. FAGUNDES, P.R., KLAUSNER, V., SAHAI, Y., PILLAT, V. G., BECKER-GUEDES, F., BERTONI, F.C., BOLZAN, M. J. A., ABALDE, J. R. (2007). Observations of daytime F2-layer stratification under the southern crest of the equatorial ionization anomaly region, Journal of Geophysical Research, vol 112, doi:10.1029/2006JA011888. HINES, C.O. (1960). Internal atmospheric gravity waves at ionospheric heights. Canadian Journal of Physics, 38, 1441-1481. HYSPLIT. Hybrid Single-Particle Lagrangian Integrated Trajectory (2015). Models simulate the dispersion and trajectory of substances transported and dispersed through our atmosphere, over local to global scales. https://doi.org/10.1175/BAMS-D-14- 00110.1. FAGUNDES, P.R., MUELLE, M. T. A. H., BITTENCOURT, J. A., SAHAI, Y., LIMA, W. L. C., GUARNIERI, F. L., PILLAT, V. G., BECKER-GUEDES, FERREIRA, A. S., LIMA, N. S. (2008) - Nighttime ionosphere–thermosphere coupling observed during an intense geomagnetic storm, Advances in Space Research 41, 539–547. https://www.ready.noaa.gov/HYSPLIT.php. FERREIRA, A., LIMA, N., SILVA, R., GÓES, K., FARIAS, T. (2018). Investigation of the propagation of gravity waves in the earthquake in PERU on 26 November 2015 at 05:45:18 (UTC) observed at GPS stations in the Amazon. VII Brazilian Geophysics Symposium – (Ouro Preto – MG – Brazil). INGV - ISTITUTO NAZIONALE DI GEOFISICA E VULCANOLOGIA, (2022). Boletim INGV \| nº 01 \| 2022 \| ano XVI (/it/ingv-newsletter-n- 01-2022-anno-xvi) - Un redação do presidente INGV - ISTITUTO NAZIONALE DI GEOFISICA E VULCANOLOGIA, (2022). Boletim INGV \| nº 01 \| 2022 \| ano XVI (/it/ingv-newsletter-n- 01-2022-anno-xvi) - Un redação do presidente amazon Expedition Magazine \| 45 Recovery of Ionospheric Signals Using Fully Convolutional DenseNet and Its Challenges. Recovery of Ionospheric Signals Using Fully Convolutional DenseNet and Its Challenges. Sensors 2021, 21, 6482. https://doi.org/10.3390/s21196482. IRIS - Incorporated Research Institutions for Seismology, (2022). https://www.iris.edu/hq/. Accessed on January 22, 2022. KELLEY, M. C. (1989). The Earth´s Ionosphere Plasma Physics and Eletrodynamics, Academic Press, San Diego – USA – California, 487p. MINSTER, J. BERNARD. (1995) GPS detection of ionospheric perturbations following the January 17, 1994, Northridge Earthquake. DOI: 10.1029/95GL00168. Geophysical Research Letters. References Vol. 22. Pages 1045– 1048, May 1995. KLAUSNER, V., FAGUNDES, P. R. (2005). Observations of Gravity Waves in Region F during the day period. X Latin American Meeting of Scientific Initiation and VI Latin American Postgraduate Meeting – University of Vale do Paraíba. (São José dos Campos, SP, Brazil). NOAA - National Oceanic and Atmospheric Administration - Air Resources Laboratory’s (ARL); https://www.ready.noaa.gov. Accessed on January (15 – 31), 2022. KOMJATHY, J.A.; GALVAN, V.D.A.; STEPHENS, P.; BUTALA, M.D.; AKOPIAN, V.; WILSON, B.; VERKHOGLYADOVA, O.; MANNUCCI, A.J.; HICKEY, M. (2012). Detecting ionospheric TEC perturbations caused by natural hazards using a global network of GPS receivers: The Tohoku case study. Earth Planets Space. 64, 1287–1294. PEZZOPANE, M. and SCOTT, C. (2005). The INGV software for the automatic scaling of foF2 and MUF (3000) F2 from ionograms: A performance comparison with ARTIST 4.01 from Rome data. Journal of Atmospheric and Solar-Terrestrial Physics 67, 1063–1073. LIMA, N. S., FERREIRA, A. S., ARAÚJO, R. C., YAMAMOTO, K. C. (2011). Observations of MSTIDs/GWs at the F2 layer heights in the near equatorial region. Geophysical Research Abstracts Vol. 13, EGU2011-122, 2011 EGU General Assembly. PILLAT, V.G. and GUIMARÃES, L.N.F. (2014). Identification of the Ionosphere Profile Using Fuzzy Logic: Part I. Trends in Applied and Computational Mathematics, 15, N.1, 47-57. doi: 10.5540/tema.2014.015.01.0047. LIMA, N. S., (2009). Study of the Propagation of Gravity Waves in the Equatorial Ionosphere, using Observations in Manaus (2.9OS, 60OW), Master's Dissertation in Geosciences, Federal University of Amazonas, UFAM/Brazil. (Available in: https://tede.ufam.edu.br/handle/tede/3275). ROW, R. V. (1967). Acoustic gravity waves in upper atmosphere due to a nuclear and an earthquake. Journal of Geophysical Research, 72 (5); 1599. STEIN, A. F., DRAXLER, R. R., ROLPH, G. D., STUNDER, B. J. B., COHEN, M. D., and NGAN, F. (2015). NOAA’S HYSPLIT Atmospheric Transport and Dispersion Modeling System. AMERICAN METEOROLOGICAL SOCIETY. December – BAMS \| 2059. LISN - Low Latitude Ionospheric Sensor Network. In collaboration of Instituto Geofísico del Peru (IGP) and Jicamarca Radio Observatory (JRO) (2022). http://lisn.igp.gob.pe/ Accessed on January (15-31), 2022. TSAI, L.C.; TIEN, M.H.; CHEN, G.H.; ZHANG, Y., (2014). HF radio angle-of-arrival measurements and ionosonde positioning. Terr. Atmos. Ocean. Sci. 25, 401–413. NASA - Earth System Observatory, (2022). Dramatic Changes at Hunga Tonga-Hunga Ha‘apai. https://science.nasa.gov/earth- science/earth-system-observatory Accessed on January 22, 2022. USGS - United States Geological Survey - https://earthquake.usgs.gov/earthquakes/ma/. Accessed on January 22, 2022. MENDOZA, M. M., CHANG, Y., DMITRIEV, A. References V., LIN, C., TSAI, L., LI, Y., HSIEH, M., HSU, H., HUANG, G., Lin, Y. and TSOGTBAATAR, E. (2021). amazon Expedition Magazine \| 46 Almeirim border of the state of Pará with Amapá (Brazil, in the North of South America), on the Amazon River (December, 2016). Almeirim border of the state of Pará with Amapá (Brazil, in the North of South America), on the Amazon River (December, 2016). Almeirim border of the state of Pará with Amapá (Brazil, in the North of South America), on the Amazon River (December, 2016). Abstract Therefore, hydroclimatic variability in South America is strongly coupled, on a large scale, to oceanic and atmospheric phenomena. Specifically, the El Niño Southern Oscillation (ENSO) that affects climatological and hydrological conditions has a “terrestrial – atmospheric” mechanism that forms a bridge between these two domains and connects the anomalies of SST of the Pacific and Atlantic Oceans (Paveda and Mesa, 1997). According to Jiménez-Muñoz et al (2016), this event was associated with warming that was without precedent and an extreme drought in the Amazon, compared to other strong ENSO events in 1982/83 and 1997/98. The typical conditions of drought caused by the ENSO were observed and described by Jiménez-Muñuz et al, (2016), as occurring only in the eastern Amazon, while in the western region of the Amazon there prevailed an uncommon level of humidity. For researchers this situation can be attributed to the humid-dry dipole at the location of maximum warming of the surface of the equatorial central Pacific Ocean. This humid-dry dipole was also confirmed in the current study through a time series of temperature readings at 4 distinct points (edge, middle of the canal, 1 m deep below the surface, and ambient air temperature) along the Amazon River from the west in Iquitos, Peru, to the east in Macapá, Brazil by the research team of the Amazon River Expedition from Peru to Brazil in 2016. According to Erfanian, Wang and Fomenko (2016), the empirical relationships between rainfall and sea surface temperatures (SST) in the Pacific and Atlantic Oceans represent the factors of tropical ocean variability responsible for the observed precipitation anomalies. These results indicate that the warmer than normal SST for the tropical Pacific and Atlantic Oceans (including El Niño events) were the principal causes of extreme droughts in South America, however, researchers are still unable to explain the severity of the precipitation deficits observed in 2016 in a substantial portion of the Amazon region. Therefore, hydroclimatic variability in South America is strongly coupled, on a large scale, to oceanic and atmospheric phenomena. Specifically, the El Niño Southern Oscillation (ENSO) that affects climatological and hydrological conditions has a “terrestrial – atmospheric” mechanism that forms a bridge between these two domains and connects the anomalies of SST of the Pacific and Atlantic Oceans (Paveda and Mesa, 1997). Reduction in water levels and regional warming of the Amazon River from Peru to the Atlantic Ocean in Brazil due to the effects of the 2016 ENSO. Lima, NS.; Malveira, AO.; Façanha, EF.; Braga, OS.; Pietzsch, MR.; Figueiredo, RS.; Calazães, RM.; Quispe, WD; Ferreira, AS. Lima, NS.; Malveira, AO.; Façanha, EF.; Braga, OS.; Pietzsch, MR.; Figueiredo, RS.; Calazães, RM.; Quispe, WD; Ferreira, AS. amazon Expedition Magazine \| 47 Abstract In situ observations of the tendencies of the prolonged ENSO (El Niño Southern Oscillation) phenomenon combined with a trend of regional warming in both western and eastern Amazonia were registered by the Amazon River Peru-Brazil Expedition on the Amazon River. Temperatures were taken at four positions on the river (edge, middle of the canal, 1 m deep below the surface, and ambient air temperature), air pressure and humidity, and the velocity and direction of the wind were the parameters that were sampled from the Peruvian city of Iquitos beginning in July 2016, to the Brazilian city of Macapá at the mouth of the Amazon River in December 2016. The results suggest that there was a decline in water levels along the river during the entire observation period due to the prolonged El Niño event that occurred in 2014, 15 and 16. According to Jiménez-Muñoz et al (2016), this event was associated with warming that was without precedent and an extreme drought in the Amazon, compared to other strong ENSO events in 1982/83 and 1997/98. The typical conditions of drought caused by the ENSO were observed and described by Jiménez-Muñuz et al, (2016), as occurring only in the eastern Amazon, while in the western region of the Amazon there prevailed an uncommon level of humidity. For researchers this situation can be attributed to the humid-dry dipole at the location of maximum warming of the surface of the equatorial central Pacific Ocean. This humid-dry dipole was also confirmed in the current study through a time series of temperature readings at 4 distinct points (edge, middle of the canal, 1 m deep below the surface, and ambient air temperature) along the Amazon River from the west in Iquitos, Peru, to the east in Macapá, Brazil by the research team of the Amazon River Expedition from Peru to Brazil in 2016. According to Erfanian, Wang and Fomenko (2016), the empirical relationships between rainfall and sea surface temperatures (SST) in the Pacific and Atlantic Oceans represent the factors of tropical ocean variability responsible for the observed precipitation anomalies. These results indicate that the warmer than normal SST for the tropical Pacific and Atlantic Oceans (including El Niño events) were the principal causes of extreme droughts in South America, however, researchers are still unable to explain the severity of the precipitation deficits observed in 2016 in a substantial portion of the Amazon region. Sampling and chronogram For monitoring of weather and climate during the period of the research, a I - FLIR-E60 thermal imager, (Tab. 1), and II –Mira digital thermometer– LASER, Minipa MT-360 sensors were used. Measurement of ambient air temperature, and the temperature at the edge of the river, middle of the canal, and at 1.0 m below the river´s surface a Davis meteorological station with uninterrupted recording (15 days + 15 days) with data collection (ambient air temperature, humidity, pressure, wind speed and direction, dew point) were measured every 5 minutes, and in situ two liter water samples were taken at each sampling point along the entire river. 1-Alkalinity (APHA, 2003; GOLTERMAN, 1970); 2- Calcium (Ca +), hardness, and Magnesium (Mg +) APHA, 1985; GOLTERMAN & CLYMO, 1978); 3 – Chlorides ((FENANTHROLINE – FIA) (MACKERETH, et. al., 1978: GOLTERMAN & CLYMO, 1971); 4- DQO with potassium dichromate (APHA, 2003; MACKERETH, et. al., 1978); 5 – Total and Dissolved iron (FIA) (APHA, 2003); 6 - Phosphate (PO4) (APHA, 2003; GOLTERMAN, 1970; 7 - Total phosphorus and nitrogen (N and P-TOTAL) (VALDERRAMA, 1980); 8 – Total phosphorus (FIASTAR) (APHA, 2003, ISO 5861, s/d); 9 – Nitrate (FIA) (GOLTERMAN, 1971); 10 – Ammoniacal nitrogen (NH3) (FIA - Flow Injection Analysis) Nessler reagent method; – 11 –Silicates (Silica – Molybdenum blue method) (GOLTERMAN, 1980, MACKERETH, et. al., 1978); 12 – pH (hydrogen ion concentration) (APHA, 2003), 13 – Potassium and sodium by flame emission spectroscopy (MACKERETH, et. al., 1978), 14 – Total suspended solids (STS) (APHA, 2003), 15 – Sulfate (APHA, 2003), 16 – Temperature (FLIR-E60); 17 – Turbidity (turbidity meter); 18 - Color (Spectrophotometer). 9 – Nitrate (FIA) (GOLTERMAN, 1971); 10 – Ammoniacal nitrogen (NH3) (FIA - Flow Injection Analysis) Nessler reagent method; – 11 –Silicates (Silica – Molybdenum blue method) (GOLTERMAN, 1980, MACKERETH, et. al., 1978); 12 – pH (hydrogen ion concentration) (APHA, 2003), 13 – Potassium and sodium by flame emission spectroscopy (MACKERETH, et. al., 1978), 14 – Total suspended solids (STS) (APHA, 2003), 15 – Sulfate (APHA, 2003), 16 – Temperature (FLIR-E60); 17 – Turbidity (turbidity meter); 18 - Color (Spectrophotometer). Temperature values are a composite of 10 in situ readings taken at each sampling point. Introduction Changes in atmospheric circulation in the tropical zone (Walker cell) induce change in rainfall patterns, devastating floods, and severe droughts that can drastically affect the lives of millions of people (MOHTADI et al, 2017). In the mosaic of landscapes that is tropical South America the tendencies for rainfall, in the Amazon in eastern Brazil, to the northwest of Peru are well-defined by long-term hydrological data for the Amazon basin that were recorded during the 20th century. During this period the tendency for rainfall during the three most humid months and for the subsequent superficial runoff rate during the three months with the greatest runoff for the northeastern region of Brazil demonstrated a slow increase over long periods (MAREGO, TOMASELLA, UVO, 1998). In 2016 the Amazon River Expedition from Peru to Brazil observed tendencies in which a prolonged ENSO event combined with a trend of regional warming increased the demand for water from the reservoirs of Brazilian hydroelectric plants in the Northeast, Central-West, and Southeastern regions of Brazil (CCEE, 2017), and caused strong rains in the Southern region of Brazil (CPTEC, 2016). amazon Expedition Magazine \| 48 amazon Expedition Magazine \| 48 Material and Methods Fig. 1: Image of the mosaic of regions of tropical South America and the 2016 route of the Amazon River Expedition from Peru to Brazil (solid line), and the 2017/18 route (dotted line) (3rd phase, modified route). Map of the Expedition (Arequipa-Peru; Iquitos-Peru; Macapá-Brazil) Map of the Expedition (Arequipa-Peru; Iquitos-Peru; Macapá-Brazil) Map of the Expedition (Arequipa-Peru; Iquitos-Peru; Macapá-Brazil) Fig. 1: Image of the mosaic of regions of tropical South America and the 2016 route of the Amazon River Expedition from Peru to Brazil (solid line), and the 2017/18 route (dotted line) (3rd phase, modified route). Fig. 2: Ships used in the Amazon River Expedition (A) Voyager III (Manaus-Tabatinga). (B) M. Monteiro II (Tabatinga-Manaus). (C) Flipper (Santa Rosa-Iquitos- Santa Rosa ). (D) S. Bartolomeu III and IV, (Manaus– Santarém– Santana—Manuas) July through December, 2016. An automatic meteorological station was installed on roof of the five passenger transport ships (Fig. 2) used in this research expedition (Fig. 1). The station was free from obstacles that would impede accurate measurement of the variables of interest (temperature, humidity, pressure, wind speed and direction, dew point, and rainfall). amazon Expedition Magazine \| 49 Fig. Introduction 3 - Footprint of the boat-mounted meteorological station, Lagragian Footprint Model, Weather Station - DAVIS - Vantage – Vue, 300 m ≤R ≤500 m, on the ship M. Monteiro II, July 2016. Fig. 3 - Footprint of the boat-mounted meteorological station, Lagragian Footprint Model, Weather Station - DAVIS - Vantage – Vue, 300 m ≤R ≤500 m, on the ship M. Monteiro II, July 2016. Statistical modeling and georeferencing of data. Statistical modeling and georeferencing of data. The time series of temperature reading along the Amazon River were processed and analyzed using the Marine Modeling and Analysis Branch Oper. H. R. (Verification ensemble) of NOAA/NWS/NCEP/EMC, Sampling and chronogram The geographic coordinates of the sampling points were taken along with a description of the weather (climate) and the time at the moment of collection, and samples were labeled accordingly (Steps 1 and 2 of the Amazon River Expedition protocol). () Done only between Manaus (Brazil) and Macapá (Brazil). Additionally, in situ analyses of pH, O2, conductivity, and O2 saturation were conducted on all water samples. amazon Expedition Magazine \| 50 Table 1. Physical characteristics of the FLIR-E60 thermal imager. Table 1. Physical characteristics of the FLIR-E60 thermal imager. map for sampling points was done using ArqGis®. Flux measurements (temperature, humidity, pressure, wind speed and direction) were taken using a meteorological station (Vantage-Vue/DAVIS Instruments Corporation, WeatherLink 6.0.3), using the static method (Lima, 2017), for covariances (Eddy Covariance). Results and Discussion Figure 4 below shows systems that were responsible for the atmosphere dynamics on July 26, 2016 between 05:00 LT and 17:0 LT, the first day of the Amazon River Expedition when it left Iquitos, Peru, for Manaus, Brazil. The Intertropical Convergence Zone (ITCZ) is in the north of South America and reaches the Amazon region, and there is a cold front that is developing in the southern Atlantic Ocean and is penetrating the southern region of Brazil. (ftp://ftpprd.ncep.noaa.gov/pub/data/nccf/co m/gfs/prod). For the characterization of the composition of the El Niño event during this period the temperature gradients of the SST of the equatorial Atlantic and the eastern equatorial Pacific were constructed. All sampling points were georeferenced using a GPS (GARMIN – E60 and the software TrackMaker®), and the creation of a thematic Fig.4: Meteorological conditions on July 26, 2016, between 05:00 LT and 17:00 LT for South America. (Source: CPTEC/INPE, 2016). Fig.4: Meteorological conditions on July 26, 2016, between 05:00 LT and 17:00 LT for South America. (Source: CPTEC/INPE, 2016). amazon Expedition Magazine \| 51 Figure 5 shows a graphical rhythmic analysis of weather types between Iquitos (Loreto-Peru), Tabatinga and São Paulo de Oliveira (State of Amazonas-Brazil) on July 26, 2016, between 05:00 LT and 18:00 LT in Peru, and July 27 and 28, 2016 (12:00 h to 03:00 h LT, in Brazil), in which the climatic elements involved in this analysis of atmosphere dynamics are evident (CPTEC/INPE). Figure 5 shows a graphical rhythmic analysis of weather types between Iquitos (Loreto-Peru), Tabatinga and São Paulo de Oliveira (State of Amazonas-Brazil) on July 26, 2016, between 05:00 LT and 18:00 LT in Peru, and July 27 and 28, 2016 (12:00 h to 03:00 h LT, in Brazil), in which the climatic elements involved in this analysis of atmosphere dynamics are evident (CPTEC/INPE). The observations from this study suggest regional warming of temperature gradients in the stretch between Iquitos-Peru to Manaus- Brazil) in July, 2016, (dry season), with average ambient temperature at the ship (in the shade) of 30.410C, at the river´s surface (100 m from the edge) of 27.340C, and at the middle of the canal of 24.730C, (Fig.6). Results and Discussion 7: Time series of temperature along the Amazon River during the second stage of the Expedition (Manaus/Brasil – Macapá/Brasil), and compared to data from the Marine Modeling and Analysis Branch Oper. H. R. (Verification Ensembles) of NOAA/NWS/NCEP/EMC. Source: Amazon River Expedition and NOAA, 2016. Manaus, Brazil) was 5.54 m (at the end of the rainy season it was 11.62 m at this sampling point in May 2016), and the highest level recorded here was in May 1999 when the depth was 13.38 m with respect to sea level (ANA/CPRM/SIPAM, 2017). During this period of the expedition the El Niño climate phenomenon was already firmly established in the region since it had begun in 2014, lasted for all of 2015, and was still strong in 2016. The effects of this drought were clearly visible during the entire voyage along the river from Peru to the Atlantic, principally due to the marks left on trees in the várzea areas at the river’s edge by the previous high-water season. Manaus, Brazil) was 5.54 m (at the end of the rainy season it was 11.62 m at this sampling point in May 2016), and the highest level recorded here was in May 1999 when the depth was 13.38 m with respect to sea level (ANA/CPRM/SIPAM, 2017). During this period of the expedition the El Niño climate phenomenon was already firmly established in the region since it had begun in 2014, lasted for all of 2015, and was still strong in 2016. The effects of this drought were clearly visible during the entire voyage along the river from Peru to the Atlantic, principally due to the marks left on trees in the várzea areas at the river’s edge by the previous high-water season. Fig. 7: Time series of temperature along the Amazon River during the second stage of the Expedition (Manaus/Brasil – Macapá/Brasil), and compared to data from the Marine Modeling and Analysis Branch Oper. H. R. (Verification Ensembles) of NOAA/NWS/NCEP/EMC. Source: Amazon River Expedition and NOAA, 2016. Fig. 7: Time series of temperature along the Amazon River during the second stage of the Expedition (Manaus/Brasil – Macapá/Brasil), and compared to data from the Marine Modeling and Analysis Branch Oper. H. R. (Verification Ensembles) of NOAA/NWS/NCEP/EMC. Source: Amazon River Expedition and NOAA, 2016. Results and Discussion During the 2nd stage of the Expedition (Manaus-Brazil to Macapá-Brazil) in December 2016, the rainy season had already begun and average temperatures were slightly reduced, with average ambient temperature at the ship of 28.970C, at the river´s surface (100 m from the edge) of 26.060C, and at the middle of the canal of 24.040C. The interval between the first and second stages was taken in order to be able to verify the effect of drought on the river due to the time necessary for water to flow across the large distance from Iquitos- Peru to Macapá-Brazil (Fig.7). The margin of error is shown in Figures 6 and 7 and in Table Figure 6 shows the time series of temperature that was taken at three positions (ambient temperature at the ship – 100 m from the edge of the canal – middle of the canal) during the first stage of the expedition (Iquitos/Peru – Manaus/Brazil), using the FLIR E60 thermal imager. The image next to the time series shows SST in Real Time Global (RTG), High Resolution (HR) and was obtained by NOAA/NCEP/NWS by analyzing satellite images, ocean floats, sea ice cover, salinity, and conducting mathematical modeling in a second degree polynomial series (Branch analysis method), ftp://ftpprd.ncep.noaa.gov/pub/data/nccf/com/gfs/ prod), and indicates correlation with the results obtained by the Amazon River Expedition. 1. Fig.5: Meteorological conditions on July 27, 2016, between 10:30 and 14:30 GMT, for South America. (Source: CPTEC/INPE, 2016). Fig.5: Meteorological conditions on July 27, 2016, between 10:30 and 14:30 GMT, for South America. (Source: CPTEC/INPE, 2016). amazon Expedition Magazine \| 52 Fig. 6: Time series of temperature along the Amazon River during the first stage of the Expedition (Iquitos/Peru – Manaus/Brazil), and compared to data from the Marine Modeling and Analysis Branch Oper. H. R. (Verification Ensembles) of NOAA/NWS/NCEP/EMC. Source: Amazon River Expedition and NOAA, 2016. Fig. 6: Time series of temperature along the Amazon River during the first stage of the Expedition (Iquitos/Peru – Manaus/Brazil), and compared to data from the Marine Modeling and Analysis Branch Oper. H. R. (Verification Ensembles) of NOAA/NWS/NCEP/EMC. Source: Amazon River Expedition and NOAA, 2016. Fig. 7: Time series of temperature along the Amazon River during the second stage of the Expedition (Manaus/Brasil – Macapá/Brasil), and compared to data from the Marine Modeling and Analysis Branch Oper. H. R. (Verification Ensembles) of NOAA/NWS/NCEP/EMC. Source: Amazon River Expedition and NOAA, 2016. Fig. Conclusion At the end of December 2016, the Tapajós River in Santarém, Pará, Brazil, located in the lower Amazon region, was more than 6 m below the base of the contention dike that serves as a waterfront walkway for urban dwellers along the Tapajós’ edge in front of the city. A vertical line near the pier in front of the church of Nossa Senhora da Conceição approximately 5m above the base of this dike represented the maximum extent of the previous high-water mark, and this mark extended for more than 60 m horizontally to near the municipal fish market. Rainfall is still sporadic during this period of the year in Santarém and almost always occurs early in the first hours of the morning before sunrise or at the end of the afternoon, but is always brief in duration. The Amazon River, during the dry season of 2016, was influenced by a prolonged El Niño climatic tendency (2014, 2015 and 2016). The results show that there was a reduction in water levels along the entire sampling trip on the river, from the city of Iquitos in Peru to the Brazilian city of Macapá near the interface of Brazil and the Atlantic Ocean. The sea surface temperature stimulated the establishment of an increasing temperature gradient in the equatorial region along the river, up to its mouth at the Atlantic Ocean, where the river accompanied the same temperature regime as the ocean during this period. This gradient established the climatic phenomenon called the wet-dry dipole, combined with a tendency for regional warming during the El Niño event of 2016. Nearing the mouth of the Amazon River, the weather was constantly cloudy with grey and dark, low nimbostratus (Ns) clouds at about 2,000 to 3,000 m, with a constant fine rain near Prainha (Pará/Brazil) and Almeirim (Pará/Brazil), (08:20 LT), and the air temperature and the dew point temperature at the level of the river’s surface were very similar, indicating a condition of saturation. There was fog on the horizon, and this fog goes by the name of hot fog because the drops are well above the freezing temperature. Conclusion It was most likely an advective fog in function of the horizontal dynamic of atmospheric migration that was in a situation that was more adequate for saturation, since, being nearer to the Atlantic Ocean (approximately 300 km), the ocean breeze that penetrates the coast of Amapá at Macapá (Brazilian Atlantic coast), in this period of the year has favorable conditions for the trade winds, including for the ITCZ, that can stimulate the development of climatic ariation in this region of the Amazon River. Results and Discussion The analyses of the water samples from both stages of the expedition are list in Tables 2 (1st stage) and 3 (2nd stage), and these data describe the “actual state” of the Amazon River in 2016 during the dry season in the Amazon. In Tabatinga at the entry of the Amazon River into Brazil, during the month of July 2016, the level of the Solimões River (the name given to the river from this point t amazon Expedition Magazine \| 53 Recommendation However, the quality of the water from the Amazon River at the 39 georeferenced sample points was satisfactory and within the standard for potable water for human consumption by communities adjacent to the river’s edge from the western portion of the basin to the Atlantic, although basic sanitation services are a preoccupation for all the communities located at these 39 sampling points, including for Iquitos (Peru), Manaus, Santarém and Macapá (Brazil). However, the quality of the water from the Amazon River at the 39 georeferenced sample points was satisfactory and within the standard for potable water for human consumption by communities adjacent to the river’s edge from the western portion of the basin to the Atlantic, although basic sanitation services are a preoccupation for all the communities located at these 39 sampling points, including for Iquitos (Peru), Manaus, Santarém and Macapá (Brazil). At the site (https://amazonexpedition.zohosites.com) there is more information about the “actual state” of the Amazon River in 2016, not only with respect to climatology, but also with respect to the life of people in the communities in this region. Acknowledgements The authors are grateful to the Lutheran University Center of Manaus (Centro Universitário Luterano, Manaus, (CEULM/ULBRA)) for the help with setting up this bi-national research trip, the Brazilian Navy in the Amazon (western and eastern regiments) for information that helped with navigation, the Foundation for the Support of Research of the State of Amazonas (Fundação de Amparo e Pesquisa do Amazonas (FAPEAM)) that provided a scholarship student to conduct the water analyses, the Max Planck Chemistry Institute (Mainz- Germany) for support with the chemical analyses, and the Mauá group at INPA in Manaus/Brazil. Furthermore, we thank the Secretary of Education and Quality of Teaching of the State of Amazonas, that through the DEPPE, amazon Expedition Magazine \| 54 ERFANIAN, A.; WANG, G.; FOMENKO, L. Unprecedented drought over tropical South América in 2016: significantly under-predicted by tropical SST. Scientific Reports. 5811(2017) doi: 10.1038/s415998-017- 05373-2. provided logistical support in sampling areas in the State of Amazonas, Brazil, and the Environmental Engineering sector of Honda of the Amazon, the engineers Feijão, Mirian, Mário, and Murakami for physical support with material used in the field, and also the Consul of Peru in Manaus, Dr. Javier Arteta Valencia for his orientation, and the Consul of Brazil Dr. Salvador R. Vecchio in Iquitos for providing access to sites in Peru. Additionally, the authors thank the Institute of Investigation of the Peruvian Amazon (IIAP) in Iquitos, Peru, specificaly the Director Dr. Luis Campo Baca and his team, for the logistical support and the dissemination and promotion of this reasearch expedition, the Superintendency of the Federal Police –Tabatinga-Amazonas/Brazil, the Migration and Foreign Visitor Service of Peru (Manaus, Santa Rosa and Iquitos), and the collaborators Eliomar Oliveira, Maurício Benzecry, Abrahão Barros, Gilberto Carvalho, and Francisco Santana. GOLTERMAN, H.L., CLYNO, R.S. & OHNSTAD, M.A.M. Methods for physical and chemical analysis of freshwaters. 2nd ed. Blackwell, Oxford, 1978. GOLTERMAN, H. L. (Edit., with the assistance of R. S. Clymo): Methods for Chemical Analysis of Fresh Waters—IBP Handbook No. 8—Oxford & Edinburgh (Blackwell Scientific Publications), 1971. JIMÉNEZ-MUÑOZ, J.; MATTAR, C.; BARICHIVICH, J.; SANTAMARIA-ARTIGAS, A.; TAKAHASHI, K.; MALHI, Y.; SOBRINO, J. A.; SCHRIER G. Record-breaking warming and extreme drought in the Amazon rainforest during course of El Niño 2015-2016. Scientific Reports. 33130 (2016) doi: 10.1038/srep33130. References APHA - American Public Health Association. Standard Methods for the examination of water and wastewater. 19th ed, Washington DC, USA. 1985. LIMA N.S., TÓTA J., BOLZAN, M.J.A., FERREIRA, A.S. and PIETZSCH, M.R. A brief observation of the formation of coherent structures and turbulence over a rain forest área in central Amazonia: THE ATTO-CLAIRE/IOP – 1/2012 EXPERIMENT. Revista Brasileira de Geofísica (2017) 35(3): 187-199. (2017) Sociedade Brasileira de Geofísica. ISSN 0102- 261X, www.scielo.br/rbg. DOI: http://dx.doi.org/10.22564/rbgf.v35i3.882. APHA - American Public Health Association. Standard Methods for the examination of water and wastewater. 19th ed, Washington DC, USA. 2003. ANA – CPRM - SIPAM: Monitoramento Hidrológico. Agência Nacional de Água – Serviço Geológico do Brasil - Sistema de Proteção da Amazônia. Boletim no. 18 (2016). Disponível em; https://www.cprm.gov.br/sace/boletins/Amazo nas/20160513_19-20160513%20- %20191650.pdf EE ANA – CPRM - SIPAM: Monitoramento Hidrológico. Agência Nacional de Água – Serviço Geológico do Brasil - Sistema de Proteção da Amazônia. Boletim no. 18 (2016). Disponível em; htt // b / /b l ti /A ANA – CPRM - SIPAM: Monitoramento Hidrológico. Agência Nacional de Água – Serviço Geológico do Brasil - Sistema de Proteção da Amazônia. Boletim no. 18 (2016). Disponível em; https://www.cprm.gov.br/sace/boletins/Amazo nas/20160513_19-20160513%20- %20191650.pdf EE Câmara de Comercialização de Energia Elétrica. Em https:://economia.uol.com.br/noticias/Reuters/ 2017/09/20. CPTEC – INPE: Centro de Previsão de Tempo e Estudos Climáticos – INSTITUTO NACIONAL DE PESQUISAS ESPACIAIS: Em, http://satelite.cptec.inpe.br/home/index.jsp, (acesso em julho e dezembro 2016). MACKERETH, F.J.H., Heron, J. & Talling, J.F. Water analysis: some revised methods for limnologists. Freshwater Biological Association, London. (Scientific Publications, 36), 1978. MAREGO J.A.; TOMASELLA J.; UVO, C.R. Trends in streamflow and rainfall in tropical South America, eastern Brazil, and northwestern. Journal of Geophysical Research Atmospheres. 103: (D2) 1775-1783 (1998). Câmara de Comercialização de Energia Elétrica. Em https:://economia.uol.com.br/noticias/Reuters/ 2017/09/20. MOHTADI, M.; PRANGE, M.; SCFUB, E.; JENNERJAHN, T. Circulation in Southeastern South America and it’s influence from El Niño events. Journal of the Meteorological Society of Japan, 80, 21-22. Article number: 1015 (2017) doi: 10.1038/s41467-017-00855-3n, Nature Communications. CPTEC – INPE: Centro de Previsão de Tempo e Estudos Climáticos – INSTITUTO NACIONAL DE PESQUISAS ESPACIAIS: Em, http://satelite.cptec.inpe.br/home/index.jsp, (acesso em julho e dezembro 2016). CPTEC – INPE: Centro de Previsão de Tempo e Estudos Climáticos – INSTITUTO NACIONAL DE PESQUISAS ESPACIAIS: Em, http://satelite.cptec.inpe.br/home/index.jsp, (acesso em julho e dezembro 2016). amazon Expedition Magazine \| 55 In, NOAA/NCEP/NWS/EMC: In, ftp://ftpprd.ncep.noaa.gov/pub/data/nccf/co m/gfs/prod, (acess: July/December, 2016/July, 2017). NOAA/NCEP/NWS/EMC: In, ftp://ftpprd.ncep.noaa.gov/pub/data/nccf/co m/gfs/prod, (acess: July/December, 2016/July, 2017). References NOAA/NCEP/NWS/EMC: In, ftp://ftpprd.ncep.noaa.gov/pub/data/nccf/co m/gfs/prod, (acess: July/December, 2016/July, 2017). POVEDA G.; MESA, O. J. Feedbacks between hydrological processes in tropical South America and large-scale ocean-atmospheric phenomena. Journal of Climate. 2690-2702 (1997). VALDERRAMA, J. G. The similaritons analysis of total nitrogen and total phosphorus in natural waters. Mar. Chem, v.10, 1981 VALDERRAMA, J. G. The similaritons analysis of total nitrogen and total phosphorus in natural waters. Mar. Chem, v.10, 1981 VALDERRAMA, J. G. The similaritons analysis of total nitrogen and total phosphorus in natural waters. Mar. Chem, v.10, 1981 amazon Expedition Magazine \| 56 Transport of Soy on the Madeira River between Porto Velho and Manaus (Brazil) Transport of Soy on the Madeira River between Porto Velho and Manaus (Brazil) ABSTRACT work for more specific analysis of gills, liver, and kidneys of specimens, collection in other habitats in the floodplain environment, and inclusion of mercury (Hg) in the aquatic field sampling. The presence of toxic metals in the gold mining process conducted in the region of the Madeira River and its effect on fish metabolism and contamination of the várzea floodplain environment and the vegetables that are grown there is a relevant factor for the need for geoindicators to monitor gold mining activities in this watershed, especially between Novo Aripuanã and Manicoré. The objective of this study was to identify geoindicators and concentrations of the metals Cu, Cd, Cr, Co, Pb,, Zn, Mn and Ni in the fish called pacu (Mylossoma spp), plankton, and vegetables and fruits such as squash, watermelon, and banana in the adjacent floodplain communities of Jenipapo, Vencedor, and Providência. The risk of contamination was analyzed, and a brief evaluation of the current state of the water taken from the Madeira River by these communities was made. Samples of fish, plankton, and vegetables and fruits were taken in these floodplain communities along the Madeira River between Novo Aripuanã and Manicoré, along with water samples and depth at each community. Atomic absorption was used to analyze the metal concentrations, and geochemical analysis of water samples was done, including pH, conductivity, associated O2 and oxygen concentration for in situ determination of water quality. The results show that the geoindicators require further fieldwork to better describe the parameters necessary for monitoring of water, vegetable, and fruit consumption by these communities. The necessary adjustments include improving the use of taxonomy of larger fish during field collection work for more specific analysis of gills, liver, and kidneys of specimens, collection Keywords: toxic metals, contamination, várzea floodplain, atomic absorption Keywords: toxic metals, contamination, várzea floodplain, atomic absorption Introduction This mining activity makes the elaboration of geoindicators of contamination of the elements that compose this ubiquitous aquatic ecosystem an important priority. The establishment of geoindicators, according to Berger and Iams (1996), and VALLADARES SOARES, et al., (2006), involves estimating a temporal range of effects and disturbances in an ecosystem. Investigations of the behavior of the Madeira River, which flows carrying great quantities of alluvium, is a highly challenging undertaking because the natural conditions of this river are far from simple, with the flow varying in time and space, and the granular structure and cohesive properties of the alluvium are rarely homogeneous along its course (YALIN, 1992). Mining activities that exist along the Madeira River are intense and require monitoring, a theme that is discussed in detail by BASTOS and LACERDA (2004). This mining activity makes the elaboration of geoindicators of contamination of the elements that compose this ubiquitous aquatic ecosystem an important priority. Figure 2 shows the delimitation of the research area along the Madeira River from Porto Velho to Manaus (Fig. 02). The stretch of river between the cities of Novo Aripuanã and Manicoré is where samples were collected of fish, fruit, vegetables, and plankton. Water samples were collected from Manicoré to Manaus. The Madeira is an alluvial river is with meanders, floodplains, and erosion along its banks, known as terras caídas in Portuguese (Fig. 03). This geofluvial scenario has allowed for the development of gold mining along the river (Figs. 04 A - B). The choice of the sample collection regions (Tab. 01) is a function of the strategic position of Novo Aripuanã, which is on the river with the same name. This river is a tributary of the Madeira, and the confluence of these two rivers is in front of Novo Aripuanã. The Aripuanã River has the Roosevelt River as a tributary, and the city of Manicoré (S 5058’32” W61018’00”) (Fig. 05) is the nearest city upriver from Novo Aripuanã (S 5008’00” W 60022’30”). p p y According to Stumm and Morgan (1981), since the industrial revolution, the burning of fossil fuels, production of paper and cement, mining, among other human activities have caused an increase in the levels of metals in aquatic ecosystems around the world. Introduction McDonald and Wood (1993) related that the potentially toxic metals in aquatic ecosystems that affect fish are, in decreasing order of severity, Hg, Pb, Cd, Cu, Zn, Al, Mn and Cr. In the current study, all these metals were investigated, except for mercury (Hg). The motive for omitting Hg was to focus on other metals since Hg receives an inordinate amount of attention in studies of environmental contamination in this region, especially because it is essential to gold mining activities in the region and would therefore be relevant as a geoindicator. The effects of exposition to potentially toxic metals in aquatic ecosystems affects fish morphology as well as physiology, according to Mazon et al. (1999), and Val and Almeida-Val (1999). Fig. 03 – Artistic representation of the profiles of the geoindicators for terrestrial and aquatic ecosystems along the Madeira River, with emphasis on the effects of erosion caused by vortices as observed in the 2018 field campaign, (not to scale). Introduction “Egypt is a gift of the Nile”, an observation that is identical in relation to a habitat in the western world in South America, specifically communities in the north of Brazil such as Porto Velho, Humaitá, Manicoré, Novo Aripuanã, Borba and Nova Olinda do Norte, which can be thought of as gifts from the Madeira River. The Madeira River is the most inhabited and commercially exploited of the tributaries of the right (southern) bank of the Solimões-Amazonas River system (RAPP PY- DANIEL, 2007). The river received this name because of the enormous quantity of tree trunks that its waters carry throughout the entire year (STERNBERG, 1975). The true grandeur of this river lies in its richness in fish species (DORIA et al., 2012) along it margins, vast alluvial plains have developed through deposition of sediments carried in the water (HORBE et al., 2013), which also contain gold. Its geographic location allows for a shipping route for soybeans produced in the Center- West region of Brazil to the Atlantic Ocean (Correa and Ramos, 2010), besides providing ample hydroelectrical energy through the dams at Jirau (Fig. 01) and Santo Antônio, which do not use conventional reservoirs (ANEEL). Fig. 01 – Control of the natural flow rate at the Jirau hydroelectric dam (1967-2020). Source: ANA – National Water and Sanitation Agency, 2020. amazon Expedition Magazine Fig. 01 – Control of the natural flow rate at the Jirau hydroelectric dam (1967-2020). Source: ANA – National Water and Sanitation Agency, 2020. Fig. 01 – Control of the natural flow rate at the Jirau hydroelectric dam (1967-2020). Source: ANA – National Water and Sanitation Agency, 2020. amazon Expedition Magazine \| 58 amazon Expedition Magazine \| 58 The establishment of geoindicators, according to Berger and Iams (1996), and VALLADARES SOARES, et al., (2006), involves estimating a temporal range of effects and disturbances in an ecosystem. Investigations of the behavior of the Madeira River, which flows carrying great quantities of alluvium, is a highly challenging undertaking because the natural conditions of this river are far from simple, with the flow varying in time and space, and the granular structure and cohesive properties of the alluvium are rarely homogeneous along its course (YALIN, 1992). Mining activities that exist along the Madeira River are intense and require monitoring, a theme that is discussed in detail by BASTOS and LACERDA (2004). Material and Methods amazon Expedition Magazine \| 59 Sites along the Madeira River Fig. 02 – Map of the Amazon River watershed, with the Madeira River in detail. Source: ANA – National Water and Sanitation Agency, 2018. Fig. 03 – Artistic representation of the profiles of the geoindicators for terrestrial and aquatic ecosystems along the Madeira River, with emphasis on the effects of erosion caused by vortices as observed in the 2018 field campaign, (not to scale). Sites along the Madeira River Fig. 02 – Map of the Amazon River watershed, with the Madeira River in detail. Source: ANA – National Water and Sanitation Agency, 2018. Fig. 03 – Artistic repr geoindicators for ter along the Madeira effects of erosion ca the 2018 field campa Sites along the Madeira River Fig. 02 – Map of the Amazon River watershed, with the Madeira River in detail. Source: ANA – National Water and Sanitation Agency, 2018. Fig. 03 – Artistic re geoindicators for t along the Madei effects of erosion the 2018 field cam Sites along the Madeira River Sample collection community of Vencedor (S 05° 20.130' W060° 44.776') before Novo Aripuanã, and included watermelon (Citrullus lanatus), squash from the family Cucurbitaceae – order Cucurbitales, which are native to várzea floodplain areas in the Amazon and are abundant in the month of July. Along the banks of the Madeira River in the community of Jenipapo (S 050 34.598’ W 0600 55.803’) plankton (limnoplâncton) were collected based on Raunkiaer et al. (1934), in according to Gonçalves and Lorenzi (2011). The banana called pacovã (Musa paradisíaca) is a pseudoberry or an epigynous berry from the community of Providência (S 050 34.797’ W 0600 55.801’) along the stretch of river from Manicoré - Novo Aripuanã (Tab. 01). This banana sample was collected at Borba, where it was being sold in the market. Water sample collection occurred during the entire trajectory between the cities along the Madeira River to Manaus. Sample collection occurred between the cities of Manicoré and Novo Aripuanã (Fig. 06), with sampling trips in different communities. In the public market of Manicoré ten specimens of Characiformes - Mylossoma spp that are commonly marketed in the community of Rosarinho (upriver from Manicoré, on the Manicoré River, 20 min by motorboat). An additional ten specimens (Mylossoma spp) were collected at Fazenda Providência which were captured in Xadá Lake, between Manicoré and Novo Aripuanã. Although there were species of the order Siluriformes available during the sample collection, the choice of species of the order Characiforme was made due to the abundance of these fish during the study period, and also because these species are greatly appreciated by the local communities and therefore economically support fishing activities. Collection of fruits and vegetables occurred near the Fig. 06 – Panel of sample collection in the field: (1) squash and watermelon in the community Vencedor. (2) water sample collection in Providência. (3) mining plant in Providência. (4) cataloging fish (Mylossoma spp) in Manicoré. (5) bananas being shipped in Providência. (6) weighing of fish in the public market of Manicoré. (7) public fish market (species of Characiformes and Siluriformes) in Manicoré. (8) Plankton collection in Jenipapo. (9) water collection in Novo Aripuanã. (10) Bananas in Borba. All the products that were being shipped on the Madeira River were destined for the Municipal Market of Manaus. Fig. 06 – Panel of sample collection in the field: (1) squash and watermelon in the community Vencedor. (2) water sample collection in Providência. Sites along the Madeira River Sites along the Madeira River Fig. 03 – Artistic representation of the profiles of the geoindicators for terrestrial and aquatic ecosystems along the Madeira River, with emphasis on the effects of erosion caused by vortices as observed in the 2018 field campaign, (not to scale). Fig. 02 – Map of the Amazon River watershed, with the Madeira River in detail. Source: ANA – National Water and Sanitation Agency, 2018. Fig. 02 – Map of the Amazon River watershed, with the Madeira River in detail. Source: ANA – National Water and Sanitation Agency, 2018. amazon Expedition Magazine \| 59 Fig. 05 – Aerial view in the direction Porto Velho to Manaus of the city of Manicoré on the Madeira River in July 2018. Fig. 05 – Aerial view in the direction Porto Velho to Manaus of the city of Manicoré on the Madeira River in July 2018. Figs. 04 A - B – Madeira River Alluvial Gold Mining (A). In detail, 15 active mining plants, cross-section cut and bathymetry of the georeferenced location on the map of the region - S 050 17' 52.0'' W 0600 41' 14.3‘’ (B). (2017DEC24 – 11:32:24 LT). (Source: SHNN-09/Map 4726.tif – Madeira River; Google Maps, 2018) Figs. 04 A - B – Madeira River Alluvial Gold Mining (A). In detail, 15 active mining plants, cross-section cut and bathymetry of the georeferenced location on the map of the region - S 050 17' 52.0'' W 0600 41' 14.3‘’ (B). (2017DEC24 – 11:32:24 LT). (Source: SHNN-09/Map 4726.tif – Madeira River; Google Maps, 2018) amazon Expedition Magazine \| 60 Sample collection (3) mining plant in Providência. (4) cataloging fish (Mylossoma spp) in Manicoré. (5) bananas being shipped in Providência. (6) weighing of fish in the public market of Manicoré. (7) public fish market (species of Characiformes and Siluriformes) in Manicoré. (8) Plankton collection in Jenipapo. (9) water collection in Novo Aripuanã. (10) Bananas in Borba. All the products that were being shipped on the Madeira River were destined for the Municipal Market of Manaus. amazon Expedition Magazine \| 61 amazon Expedition Magazine \| 61 Geochemical analysis method for potentially toxic metals After collection, the samples were sent to the where they were washed with tap water then with distilled water and prepared for analysis. Samples of fish gills and pieces of fruits and vegetables were used for the atomic absorption analysis, while plankton were analyzed in their whole form, and all samples were dried at 65°C for 48 h. From each sample a subsample of 0.5000 g was taken in duplicate and digested in 10 mL of (Synth) on a heating plate at 90 °C for 1 hour. The digested sample were filtered, quantitatively transferred to a 50 mL volumetric flask and the volume was completed with distilled water. Subsequently, the samples were stored in flasks and refrigerated until the atomic absorption analysis. The same procedures were conducted for the control and blank samples. The standard concentrations used were (mg/mL): Cu (0.5, 1.0, 2.0, and 3.0), Pb (1.0, 3.0, and 5.0), Zn (0.1, 1.0, 3.0) and 6.0, Cd (0.5, 2.0, and 3.0), Ni (1.0, 3.0, and 6.0), Mn (0.1, 0.5, 1.0 and 2.0), Cr (1.0, 3.0, and 5.0), Co (1.0, 5.0, 10, and 20). All biogeochemical analyses followed the procedures described by Bruno (2014). Table 01 – Sample points during the on the Madeira River between Novo Aripuanã and Manicoré in 2018 (Ner. of samples). mple points during the on the Madeira River between Novo Aripuanã and Manicoré in 2018 Madeira River from Manicoré to Nova Olinda do Norte, which is the last city before the mouth of the river, between the cities of Autazes (right bank) and Itacoatiara (left bank), each coming after Manaus, on the Amazon River. amazon Expedition Magazine \| 62 Sample collection Water quality analysis at the collection points points All the parameters for geoindicators of water quality were measured in situ, thus allowing for a sampling scheme that was able to accurately capture the characteristics of the Madeira river- atmosphere ecosystem for the data collection period of December 2017, and January and July of 2018. The sampled parameters are shown in (Tab. 03), with their respective geochemical analyses (pH, conductivity, associated oxygen, and oxygen concentration) and water temperature, for the Madeira River to the Negro River in Manaus. Additionally, bathymetric profiles were conducted with the aid of maps in raster and tiff formats from Northwest Hydrology and Navigation Service – (SHNN-09) for the sample collection points. This sampling will serve to describe the current state of the bed of the Fig. 07 –Concentration toxic metal ions in aquatic and terrestrial environments on the Madeira River between the cities of Manicoré and Novo Aripuanã in July 2018. amazon Expedition Magazine \| 62 Fig. 07 –Concentration toxic metal ions in aquatic and terrestrial environments on the Madeira River between the cities of Manicoré and Novo Aripuanã in July 2018. Fig. 07 –Concentration toxic metal ions in aquatic and terrestrial environments on the Madeira River between the cities of Manicoré and Novo Aripuanã in July 2018. amazon Expedition Magazine \| 62 amazon Expedition Magazine \| 63 Results and Discussion this may be a result of the operations of the port which involve manipulation of contaminating fluids and fuels that contain oil and gasoline (Tab. 03). Observations made of the mining plants on the Madeira River from Porto Velho to Borba revealed that all these plants use a diesel generator to generate electricity, and very few of them use gasoline powered generators, and then only to power lights and small electronic equipment. The filling of storage tanks with fuel and equipment maintenance is done at the mining site and spilling of contaminants into the river commonly occurs. There are frequently from 3 up to 20 mining plants operating in close proximity, and in this way water, plankton, and fish are constantly in contact with contaminating material discarded into the river during mining activities. The plankton had higher values for the concentrations of the elements Cu, Pb, Zn and Mn (Fig. 09). The concentrations of Mn (manganese) were high in all the species sampled in this study, and it is known that there is an antagonistic interaction between Mn and iron (Fe) wherein an excess of Mn in the diet will reduce the absorption of Fe, causing anemia, and affects the central nervous system as well as the reproductive and respiratory systems (SOARES, 2009). The Mn geoindicator is strong for concentration in fish species in Xadá Lake, as well as for most species sampled in this study, except for fish and watermelon from Manicoré. Ni (nickel) had a significant peak only for squash. According to the Environmental Agency of the State of São Paulo (CETESB; Appendix E-2016), conductivity is the numerical expression of the capacity of water to conduct an electrical current. This capacity depends on ionic concentration and temperature, and indicates the quantity of salts present in the water column and is therefore an indirect measure of pollutant concentration. In general, levels above 100 µS/cm (Fig. 10) indicate impacted environments (metals). The data taken in front of the city of Manicoré (104 µS/cm), show that this aquatic ecosystem has been impacted, and Temperature is influenced by variables such as latitude, altitude, seasonality, time of day, water flow rate, and depth (Fig. 11). The conditions of the Madeira River drastically change with depth and flow rate every six months. Results and Discussion Health Organization (WHO) is 0.07 mg/L; however, the Ministerial Order 2914/2011 does not establish a recommended concentration for nickel. The description of geoindicators is shown in (Tab. 02) for the eight (8) analyzed chemical elements (Fig. 07). Zn and Mn have the largest values, but only for the fish (Mylossoma spp), and Cd, Cr, Co and Ni had the lowest values. The Ministerial Order 2914/2011 of the Brazilian Ministry of Health determines that the values of the peaks shown in (Fig. 08) for Zn and Mn are above the acceptable limits for human consumption. For zinc the recommended concentration is 5 mg/L and acceptable levels are in the range of 10 – 40 mg/L. For manganese the recommended concentration is 0.1 mg/L and acceptable levels are within 0.2 mg/L. Recommended concentrations for human consumption the recommended acceptable levels are in the range of 1 – 10 mg/L, and for nickel the recommended concentration limit established by the World There is a total absence of Cd (cadmium) for all sampled species, which, according to the WHO (2010) and the Food and Agriculture Organization (FAO; 1997) the Provisional Tolerable Weekly Intake (PTWI) is 400-500 mg, or 7 mg / kg of body mass (assuming a body mass of 60 kg). The presence of cadmium in foods is a result of soil and water contamination from natural as well as anthropogenic sources. Is known that cadmium accumulates in tissues, particularly the liver and kidney, in terrestrial as well as aquatic animals, especially those that feed on detritus, such as mollusks and Siluriforme species like catfish. Table 02 – Concentration of toxic metal ions in aquatic and terrestrial environments on the Madeira River between the cities of Manicoré and Novo Aripuanã during the dry season in July 2018. Table 02 – Concentration of toxic metal ions in aquatic and terrestrial environments on the Madeira River between the cities of Manicoré and Novo Aripuanã during the dry season in July 2018. amazon Expedition Magazine \| 63 Fig. 08 – Indices of potentially toxic metals in the fish pacu - Mylossoma spp, in the Rosarinho community (Manicoré River) in blue, and the Xadá Lagoon in red, July 2018. Fig. 08 – Indices of potentially toxic metals in the fish pacu - Mylossoma spp, in the Rosarinho community (Manicoré River) in blue, and the Xadá Lagoon in red, July 2018. amazon Expedition Magazine \| 65 Results and Discussion The pH geoindicator for water from the Madeira River showed that it is alkaline (6.4 to 7.7) up to its mouth at the Amazon River, and is much more acid at Manaus (5.45). The water-associated oxygen geoindicator had a large decrease in Autazes (1.08 mg/L) at the mouth of the Madeira, and the oxygen saturation (40.8%) peaked at the mouth of the river. amazon Expedition Magazine \| 64 Fig. 09 – Panel showing the geoindicators for the potentially toxic metals Cu, Pb, Zn, Cd, Ni, Mn, Cr and Co, for fish, vegetables, fruits, and plankton, between Manicoré and Novo Aripuanã on the Madeira River, Brazil, in July 2018. Fig. 09 – Panel showing the geoindicators for the potentially toxic metals Cu, Pb, Zn, Cd, Ni, Mn, Cr and Co, for fish, vegetables, fruits, and plankton, between Manicoré and Novo Aripuanã on the Madeira River, Brazil, in July 2018. Table 03 – Water quality of the Madeira River in July 2018. Partly Partly Partly Partly Partly Clear Clear amazon Expedition Mag Table 03 – Water quality of the Madeira River in July 2018. Table 03 – Water quality of the Madeira River in July 2018. Partly Partly Partly Partly Partly Clear Clear amazon Expedition Magazine \| 65 Fig. 10 – Water quality of the Madeira River between Manicoré and Manaus – Brazil, in July 2018. Fig. 10 – Water quality of the Madeira River between Manicoré and Manaus – Brazil, in July 2018 Fig. 11 – Panel showing transversal cross sections and bathymetry profiles of the Madeira River and the sampling points at indicated cities between Manicoré and Nova Olinda do Norte. (Source: Northwest Hydrology and Navigation Service – 09, Brazilian Navy, 2018). Fig. 11 – Panel showing transversal cross sections and bathymetry profiles of the Madeira River and the sampling points at indicated cities between Manicoré and Nova Olinda do Norte. (Source: Northwest Hydrology and Navigation Service – 09, Brazilian Navy, 2018). amazon Expedition Magazine \| 66 Conclusion The authors are immensely grateful to the chemistry technician Eila B. Vasconcelos (CRQ-AM: 023790) for conducting the laboratory analyses, and This study of geoindicators of potentially toxic metals conducted on the Madeira River between Manicoré and Novo Aripuanã followed the guidelines established by the FAO (Report FSF/FOS 97.5) / the WHO (Technical Report – Series 960), National Health Foundation (FUNASA; Water Analysis Manual – 4ª. Ed.), CETESB (Appendices D and E – Environmental and Sanitary Importance of Water Quality Variables and of Sediments, and Analytic and Sampling Methods), the Minsterial Order 2914/2011 – of the Brazilian Ministry of Health, besides the contributions of Brazilian governmental agencies such as ANA, ANEEL, IBAMA, SHNN-09. 023790) for conducting the laboratory analyses, and to the Northwest Hydrology and Navigation Service – (SHNN-09) of the Brazilian Navy (Vila Buriti/Manaus) for the bathymetry maps (raster and tiff) of the Madeira River. We also thank the Biodiversity Authorization and Information System - IBAMA/ICMBio (CR-02 - km 3 of the BR-174/Manaus highway) for authorization No. 64309 to conduct scientific activities at the National Center for Research and Conservation of Continental Fish - CEPTA, and the National Civil Aviation Agency (ANAC) for registering No. PP- 201800724 in the Unmanned Aircraft Register System (SISANT) for non-recreational use to fly over the Madeira River. Additionally, we thank the Social Assistant and MSc. Isaque Dantas da Unidade Hospitalar of Manicoré for logistical help in Manicoré during the Period of Intense Observation (POI). We extend a warm thanks to the captain Matheus and his team for the use of the boat N/M – Lindo Amanhecer III and the small motorboat, who contributed to our work through their skills and experience in piloting on the Madeira River and its streams, bays, and flooded forests, all having many meanders and canals which can make navigation difficult. to the Northwest Hydrology and Navigation Service to the Northwest Hydrology and Navigation Service to the Northwest Hydrology and Navigation Service – (SHNN-09) of the Brazilian Navy (Vila Buriti/Manaus) for the bathymetry maps (raster and tiff) of the Madeira River. We also thank the Biodiversity Authorization and Information System - IBAMA/ICMBio (CR-02 - km 3 of the BR-174/Manaus highway) for authorization No. 64309 to conduct scientific activities at the National Center for Research and Conservation of Continental Fish - CEPTA, and the National Civil Aviation Agency (ANAC) for registering No. amazon Expedition Magazine \| 67 Conclusion PP- 201800724 in the Unmanned Aircraft Register System (SISANT) for non-recreational use to fly over the Madeira River. Additionally, we thank the Social Assistant and MSc. Isaque Dantas da Unidade Hospitalar of Manicoré for logistical help in Manicoré during the Period of Intense Observation (POI). We extend a warm thanks to the captain Matheus and his team for the use of the boat N/M – Lindo Amanhecer III and the small motorboat, who contributed to our work through their skills and experience in piloting on the Madeira River and its streams, bays, and flooded forests, all having many meanders and canals which can make navigation difficult. However, the use of geoindicators to collect data in the field should be frequently conducted in order to maintain robust, updated scientific information that can help to improve the lives of the populations that live along the Madeira River. It is also important to state that the omission of the metal Hg in this study should be corrected so it is included in future studies since gold mining activities in the region are in continuous operation and depend on the use of Hg due to its ability to rapidly amalgamate gold, although it contaminates the user as well as the surrounding aquatic environment and also enters the atmosphere to be subsequently deposited into waters and land at the sites visited in this study. Future studies could include Siluriforme species such as (catfish, and surubim (Pseudoplatystoma species)) for gill, liver, and kidney analyses, since these fish are bottom feeders and feed on detritus and smaller fish. These results reveal the current state of the Madeira River between Manicoré and Novo Aripuanã, and the analyzed material provides an initial slate of parameters that should be investigated using the geoindicators developed in this study in order to support decision-making with respect to use and conservation of aquatic and terrestrial ecosystems in the study region. Since this study is of a preliminary character, it is nevertheless important, and it should be emphasized that future studies should complement the current one in order to reinforce the conclusions or correct possible errors made in conducting this research. amazon Expedition Magazine \| 67 References ANA – Agência Nacional de Água e Saneamento Básico: https://www.ana.gov.br/as-12-regioes-hidrograficas- brasileiras/amazonica. (Acesso em agosto, 2020). ANA – Agência Nacional de Água e Saneamento Básico: https://www.ana.gov.br/as-12-regioes-hidrograficas- brasileiras/amazonica. (Acesso em agosto, 2020). CORREA, Vivian Helena Capacle; RAMOS, Pedro. A precariedade do transporte rodoviário brasileiro para o escoamento da produção de soja do Centro-Oeste: situação e perspectivas. Rev. Econ. Sociol. Rural- - vol.48 no.2 Brasília abr./jun. 2010. https://doi.org/10.1590/S0103- 20032010000200009. ANEEL - Agência Nacional de Energia Elétrica: https://www.aneel.gov.br. (Acesso em agosto, 2020). vol.48 no.2 Brasília abr./jun. 2010. https://doi.org/10.1590/S0103- 20032010000200009. BASTOS, W. R. & LACERDA, L. D. A contaminação por mercúrio na bacia do Madeira: Uma breve revisão. Geochim. Brasil. 18(2)099- 114. 2004. DORIA, Carolina Rodrigues da Costa [et al.]. A pesca comercial na bacia do rio Madeira no estado de Rondônia, Amazônia brasileira. ACTA AMAZONICA. vol. 42(1) 2012: 29 – 40. DORIA, Carolina Rodrigues da Costa [et al.]. A pesca comercial na bacia do rio Madeira no estado de Rondônia, Amazônia brasileira. ACTA AMAZONICA. vol. 42(1) 2012: 29 – 40. DOWN, Randy D., LEHR , Jay H., Environmental Instrumentation and Analysis Handbook, John Wiley & Sons, 2005 ISBN 0-471-47332-4. BERGER, A. R. and IAMS, W. J., eds, 1996, Geoindicators—assessing rapid environmental changes in earth systems: Rotterdam, A.A. Balkema, 466p. DOWN, Randy D., LEHR , Jay H., Environmental Instrumentation and Analysis Handbook, John Wiley & Sons, 2005 ISBN 0-471-47332-4. BRASIL. Fundação Nacional de Saúde. Manual prático de análise de água / Fundação Nacional de Saúde – 4. ed. – Brasília: Funasa, 2013. 150 p. FAO/WHO – Food consumption and exposure the assessment chimicals. WHO/FSF/FOS/97.5. (Consultations and Workshops. Geneva, Switzerland, 10-17 February, 1997). BRASIL. Ministério da Saúde – MS. Portaria No. 2914 de 12 de Dezembro de 2011. https://bvsms.saude.gov.br/bvs/saudelegis/gm/2011/p rt2914_12_12_2011.html. FARMER, Franklin H., JARRETT, Olin, BROWN, Clarence A., United States. National Aeronautics and Space Administration. Scientific and Technical Information Branch, Visible absorbance spectra: a basis for In Situ and passive remote sensing of phytoplankton concentration and community composition, National Aeronautics and Space Administration, Scientific and Technical Information Branch, 1983 OCLC 9839377 - Biblioteca Digital HathiTrust’s,NASA. BRASIL. Serviço de Hidrografia e Navegação do Noroeste – No. 09 - (SHNN-09) (Manaus-Amazonas). Marinha do Brasil. https://www.marinha.mil.br/chm/dados-do- segnav/cartas-raster.(2018) BRUNO, Alessandra Nejar. Biotecnologia I: Princípios e Métodos, Artmed Editora, 2014 ISBN 8-582-71101-8. BRUNO, Alessandra Nejar. Biotecnologia I: Princípios e Métodos, Artmed Editora, 2014 ISBN 8-582-71101-8. CETESB - Companhia Ambiental do Estado de São Paulo. References Apêndice E Significado Ambiental e Sanitário das Variáveis de Qualidade das Águas e dos Sedimentos e Metodologias Analíticas e de Amostragem (Apêndice - E). https://www..gcetesb.spov.br/ - E. pdf, (2016). CTESB - Companhia Ambiental do Estado de São Paulo. Apêndice D Significado Ambiental e Sanitário das VariáveisE de Qualidade. https://www.cetesb.sp.gov.br/Apêndice - D. pdf , (2014). CHANG, Raymond. Físico-Química - 3.ed.: Para as Ciências Químicas e Biológicas, McGraw Hill Brasil, 2010 ISBN 8-563-30830-0. CETESB - Companhia Ambiental do Estado de São Paulo. Apêndice E Significado Ambiental e Sanitário das Variáveis de Qualidade das Águas e dos Sedimentos e Metodologias Analíticas e de Amostragem (Apêndice - E). https://www..gcetesb.spov.br/ - E. pdf, (2016). GONÇALVES, Eduardo Gomes e LORENZI, Harri. Morfologia Vegetal: organografia e dicionário ilustrado de morfologia das plantas vasculares. 2ª. Edição – São Paulo: Instituto Plantarum de Estudo da Flora, 2011. CTESB - Companhia Ambiental do Estado de São Paulo. Apêndice D Significado Ambiental e Sanitário das VariáveisE de Qualidade. https://www.cetesb.sp.gov.br/Apêndice - D. pdf , (2014). HORBE, A. M. C., QUEIROZ, M. M. A., MOURA C. A. V., TORO, M. A. G. Geoquímica das águas do médio e baixo rio Madeira e seus principais tributários - Amazonas – Brasil. ACTA AMAZONICA, VOL. 43(4) 2013: 489 – 504. CHANG, Raymond. Físico-Química - 3.ed.: Para as Ciências Químicas e Biológicas, McGraw Hill Brasil, 2010 ISBN 8-563-30830-0. amazon Expedition Magazine \| 68 amazon Expedition Magazine \| 68 IUPAC – International Union of Pure and Applied Chemistry (2020) - https://iupac.org/ KAMAT, Prashant and SCHATZ, George C. How to Make Your Next Paper Scientifically Effective". J. Phys. Chem. Lett. (4): 1578−1581. 2013. doi:10.1021/jz4006916. 2013. IUPAC – International Union of Pure and Applied Chemistry (2020) - https://iupac.org/ KAMAT, Prashant and SCHATZ, George C. How to Make Your Next Paper Scientifically Effective". J. Phys. Chem. Lett. (4): 1578−1581. 2013. doi:10.1021/jz4006916. 2013. IUPAC – International Union of Pure and Applied Chemistry (2020) - https://iupac.org/ STUMM, W. and MORGAN, J. J. (1981). Aquatic Chemistry. An Introduction Emphasizing Chemical equilibria in Natural Water. New York: John Wiley & Sons. KAMAT, Prashant and SCHATZ, George C. How to Make Your Next Paper Scientifically Effective". J. Phys. Chem. Lett. (4): 1578−1581. 2013. doi:10.1021/jz4006916. 2013. THOMÉ-SOUZA, Mário J. F. Estatística Pesqueira do Amazonas e Pará - 2004 / Mario J. F. Thomé- Souza... [et al.]. – Manaus: Ibam/ProVárzea, 2007. 74 p. : il. color.; 20,8x29,7 cm. Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis. References LAKOWICZ, Joseph R, Principles of fluorescence spectroscopy, 3ª edição, Springer, 2009. ISBN978- 0-387-46312-4 doi:10.1007/s00216-007-1822- x ISSN 1618-2642. Livro, ISSN 1618-2650 e-Book MAZON, A. F. et al. (1999). Acute copper exposure in freshwater fish: Morphological and physiological efffects. (In Biology of Tropical Fishes. Edited by A. L. Val and V. M. F. Almeida- Val. Chapter 21, pp. 263 – 275, INPA, Manaus, 1999). VAL, Adalberto Luís e ALMEIDA-VAL, Vera Maria Fonseca. Biology of Tropical Fishes, INPA, Manaus, 1999. VALLADARES SOARES, P., YOSHINAGA PEREIRA S., SIMÕES S J C., BERNARDES G P., Aplicação do Conceito de Geoindicadores na Avaliação da Disponibilidade Hídrica em Bacias Hidrográficas – Uma Abordagem Introdutória. RBRH – Revista Brasileira de Recursos Hídricos Volume 11 n.1 Jan/Mar 2006, 111-117. MORAES, Érica Siani – Histórias (Heródoto e o Egito. Tradução e Comentários do Livro II) Dissertação de Mestrado - UNICAMP, 1999. RAPP PY-DANIEL, Lucia [et al.]. Biodiversidade do médio Madeira: bases científicas para propostas de conservação – Manaus; INPA; [Brasília]: MMA: MCT, 2007. VOET, Donald, G. VOET, Judith, PRATT, Charlotte W. Fundamentos de Bioquímica - 4.ed.: A Vida em Nível Molecular; Artmed Editora, 2014, ISBN 8- 582-71066-6. RAUNKIAER, Christen Christensen; FAUSBØL, Annie Ingeborg; GILVERT-CARTER, Humphrey; TANSLEY Sir– The life forms of plants and statistical plant geography. Clarendon Presss, Oxford, 1934. 623 pp. WHITE, Robert. Chromatography/Fourier Transform Infrared Spectroscopy and its Applications, CRC Press, 1989 ISBN 0-824-78191- 0. SKOOG, WEST, HOLLER, CROUCH, Fundamentos de Química Analítica, Tradução da 8ª Edição norte-americana, Editora Thomson, São Paulo-SP, 2006. WHO - World Health Organizatio: technical report series; no. 960. Evaluation of certain food additives and contaminants: seventy-third report of the Joint FAO/WHO, 2010. SOARES, Cristina Pondé Prisco Paraíso Sarno. Avaliação do teor de manganês em alimentos procedentes do município de Simões Filho - BA / Cristina Ponde Prisco Paraíso Sarno Soares - 2009. (Dissertação de Mestrado – UFBA). YALIN, Mehmet Selim. River Mechanics. Permanon Press. 219 p. YALIN, Mehmet Selim. River Mechanics. Permanon Press. 219 p. SRINIVASAN, Damodaran; PARKIN, Kirk L.; FENNEMA, Owen R. Quimica de Alimentos de Fennema, Artmed, 2010 ISBN 8-536-32334-5 STEPHENSON, Frank H., Calculations for Molecular Biology and Biotechnology: A Guide to Mathematics in the Laboratory 2thEd., Academic Press, 2010 ISBN 0-123-75691-X STERNBERG, Hilgard O’Reilly. The Amazon River of Brazil (GEOGRAPHISCHE ZEITSCHRIFT). Franz Steiner Verlag Gmbh – Wiesbaden, 1975. References amazon Expedition Magazine \| 69 amazon Expedition Magazine \| 69 Community on the bank of the Madeira River in the El Niño period, when the river is at an elevation of approximately 12 m with the upper edge of Terra Firme (solid ground) (July, 2017). Observations with operational research on the connection of the 11-year Solar Cycle and the El Niño and La Niña phenomena and rainfall in the Amazon (1980-2030). Newton Silva de Lima††, Eriberto Façanha††, Alan dos Santos Ferreira, Aldemir Malveira† ††, José Cavalcante††, Roseilson Souza do Vale‡, #Daniel Valentim Mansur Lutheran University Center of Manaus (Geosciences - Mathematics) / †Federal University of Amazonas (Mathematics)/ ‡Federal University of West of Pará (Geosciences), †† (Secretary of Education and Quality of Teaching of the State of Amazonas), # Martha Falcão College - Wyden. †† * †Manaus/ ‡Santarém (Brazil) amazon Expedition Magazine \| 70 amazon Expedition Magazine \| 70 Abstract- The stochastic bias was used to describe the correlation between the 11-year solar cycle compared to the El Niño Southern Oscillation (ENSO) time series and rainfall over the city of Manaus (Brazil), looking for a connection between these events. For this, the time series of Solar Spots and El Niño and La Niña events and rainfall over Manaus were studied by means of the time series tools for the investigated periods. This operational research work shows that from 1980 until the 2030 projection, there is a strong correlation of connection of these data to the observed phenomena. caused strong rains in the Southern region of Brazil [4]. According to [5] [6] [7], this event was associated with warming that was without precedent and an extreme drought in the Amazon, compared to other strong ENSO events in 1982/83 and 1997/98. The typical conditions of drought caused by the ENSO were observed and described by [5], as occurring only in the eastern Amazon, while in the western region of the Amazon there prevailed an uncommon level of humidity. For researchers this situation can be attributed to the humid-dry dipole at the location of maximum warming of the surface of the equatorial central Pacific Ocean. About the causes of these changes are analyzed over the last two decades the average SST (Sea Surface Temperature), anomalies are weakened towards the west, in direction of the central Pacific, and this represents an indicator that needs more observation [8]. Keywords - Connection, Solar Cycle 11 years, El Niño, La Niña, Amazon. The sun emits radiation along the entire electromagnetic spectrum. The solar structure can be understood from the point of view of two regions; the interior, which goes from its center to the surface and divided into layers, while the second is the external atmosphere of constant activity in the emission of radiation and mass in the interplanetary environment. Observations with operational research on the connection of the 11-year Solar Cycle and the El Niño and La Niña phenomena and rainfall in the Amazon (1980-2030). When there is an increase in the speed and concentration of the solar wind and upon reaching the earth, it causes sudden ionospheric disturbances (SID), which constitute true ionospheric storms or magnetic storms. The electromagnetic waves, UVE and X-R, do not interact with the magnetosphere and pass through it normally, but the solar wind consisting of particles (electrons and He+) collides with the magnetosphere causing a compression in the field lines, [9]. Keywords - Connection, Solar Cycle 11 years, El Niño, La Niña, Amazon. INTRODUCTION Changes in atmospheric circulation in the tropical zone (Walker cell) induce change in rainfall patterns, devastating floods, and severe droughts that can drastically affect the lives of millions of people [1]. In the mosaic of landscapes that is tropical South America the tendencies for rainfall, in the Amazon in eastern Brazil, to the northwest of Peru are well-defined by long-term hydrological data for the Amazon basin that were recorded during the 20th century. During this period the tendency for rainfall during the three most humid months and for the subsequent superficial runoff rate during the three months with the greatest runoff for the northeastern region of Brazil demonstrated a slow increase over long periods [2]. In 2016 the Amazon River Expedition from Peru to Brazil observed tendencies in which a prolonged ENSO (El Niño Southern Oscillation), event combined with a trend of regional warming increased the demand for water from the reservoirs of Brazilian hydroelectric plants in the Northeast, Central- West, and Southeastern regions of Brazil [3], and The variation between the phases of solar activity is 11 years, in energetic and transient phenomena since the number of explosions as well as coronal mass ejections that will constitute solar wind and the number of sunspots that is related to the polarity inversion. However, the number of dark spots on the surface, the brightness of X-rays, is more intense during half of the cycle and the other half of the cycle is less intense, [9]. amazon Expedition Magazine \| 71 amazon Expedition Magazine \| 71 Figure. 3: Climate Statistics between 11-year Solar Cycle and ENSO (El Niño). Recommendation CCEE – Electric Energy Trading Chamber. https://economia.uol.com.br/noticias/Reute rs/2017/09/20 At the site [13], there is more information about the “actual state” of the Amazon River in 2016, not only with respect to climatology, but also with respect to the life of people in the communities in this region. CPTEC – INPE: Center for Weather Forecasting and Climate Studies - NATIONAL INSTITUTE OF SPACIAL RESEARCH: (Access in: http://satelite.cptec.inpe.br/home/index.jsp , (Access in: 2016, july and december). CONCLUSION J. Jiménez-Muñoz; C. Mattar; J. Barichivich; A.Santamaria-Artigas; K. Takahashi; Y. Malhi; J. A. Sobrino; G. Schrier. Record-breaking warming and extreme drought in the Amazon rainforest during course of El Niño 2015-2016. Scientific Reports. 33130 (2016) doi: 10.1038/srep33130. Although the statistical methods lead us to a great operational solution, we must always understand the method uncertainties, because we work with approximations and averages. However, the method shows a strong correlation between the phenomena. A. Erfanian; G. Wang; L. Fomenko. Unprecedented drought over tropical South América in 2016:significantly under- predicted by tropical SST. Scientific Reports. 5811(2017) doi: 10.1038/s415998-017- 05373-2. REFERENCES Figures 2 and 3 show the interrelationships between the precipitation time series over the city of Manaus (Amazonia-Brazil), the 11-year sunspot index from 1980 to 2019, and also the ENSO Multivariate index from the same period. In the comparison through investigation by operational research, it is found the strong correlation of the observed phenomena within the considered range, configuring to a tendency that the rains in the Amazon suffer from the solar cycle of 11 years of the Sun. M. Mohtadi; M. Prange; E. Scfub; T. Jennerjahn. Circulation in Southeastern South America and it’s influence from El Niño events. Journal of the Meteorological Society of Japan, 80, 21-22. Article number: 1015 (2017) doi: 10.1038/s41467-017- 00855-3n, Nature Communications. J. A. Marego; J. Tomasella; C. R. Uvo. Trends in streamflow and rainfall in tropical South America, eastern Brazil, and northwestern. Journal of Geophysical Research Atmospheres. 103: (D2) 1775-1783 (1998). MATERIAL AND METHODS Figure. 1: Time series of temperature along the Amazon River during the first stage of the Expedition (Iquitos/Peru – Manaus/Brazil), and compared to data from the Marine Modeling and Analysis Branch Oper. H. R. (Verification Ensembles) of NOAA/NWS/NCEP/EMC. Source: Amazon River Expedition and NOAA, [11] [12]. 2016. The variables involved are the number of sunspots observed from 1980 to the 2013 projection, sea surface temperature (TSM) Figs.1 and 2, variations that influence ENSO and precipitation over the city of Manaus. To show the connection between the upper and lower atmosphere, linear regression is used to obtain the interaction between the phenomena, showing through Operational Research (OR) the points of optimal connection solution within the period from 1980 to 2030. The coincident points on the Optimal Solution Line validate the solution that is on the resonance of phenomena over the observed period. Using statistical moments and linear regression, [10]. Figure. 1: Time series of temperature along the Amazon River during the first stage of the Expedition (Iquitos/Peru – Manaus/Brazil), and compared to data from the Marine Modeling and Analysis Branch Oper. H. R. (Verification Ensembles) of NOAA/NWS/NCEP/EMC. Source: Amazon River Expedition and NOAA, [11] [12]. 2016. amazon Expedition Magazine \| 72 amazon Expedition Magazine \| 72 RESULTS AND DISCUSSION Figure. 2: Precipitation Time Series Panel, 11 Year Solar Cycle and ENSO. Figure. 2: Precipitation Time Series Panel, 11 Year Solar Cycle and ENSO. Figure. 3: Climate Statistics between 11-year Solar Cycle and ENSO (El Niño). amazon Expedition Magazine \| 73 amazon Expedition Magazine \| 73 ACKNOWLEDGEMENTS The authors are grateful to the Lutheran University Center of Manaus, the Foundation for the Support of Research of the State of Amazonas (FAPEAM) that provided a scholarship student to conduct the water analyses, the Max Planck Chemistry Institute (Mainz-Germany) for support with the chemical analyses, and the Mauá group at INPA in Manaus/Brazil. Furthermore, we thank the Secretary of Education and Quality of Teaching of the State of Amazonas. G. Poveda; O. J. Mesa. Feedbacks between hydrological processes in tropical South America and large-scale ocean-atmospheric phenomena. Journal of Climate. 2690-2702 (1997). S. Hu; A. V. Fedorov. Cross-equatorial winds control El Niño diversity and change, Nature Climate Change volume 8, pages798– 802 (2018). S. Hu; A. V. Fedorov. Cross-equatorial winds control El Niño diversity and change, Nature Climate Change volume 8, pages798– 802 (2018). amazon Expedition Magazine \| 74 ANA – CPRM - SIPAM: Hydrological Monitoring. National Water Agency - Geological Survey of Brazil - Protection System of the Amazon. Bulletin no. 18 (2016). (Access in; https://www.cprm.gov.br/sace/boletins/Am azonas/20160513_19-20160513%20- %20191650.pdf. A. Roque da Silva. Nossa Estrela O SOL. Ed. Livraria da Física. Sociedade Brasileira de Física – SBF. São Paulo, 1a. Edição. (2006). Von Zuben. EA932 - DCA/FEEC/Unicamp. Tópico 8 - Fundamentos para Processos Estocásticos. (2016). Access : AMAZON Manifest COP26 \| Amazon Messages (https://www.youtube.com/watch?v=YIDZ0jCQ_Xo) VV V V V VVVVV GLOBAL WARMING IN RELATION TO THE PRE-INDUSTRIAL PERIOD COP-21 (2015) PARIS AGREEMENT Observed increase Simulated from the influence of natural and human factors Simulated only by natural factors Source: Observed increase Simulated from the influence of natural and human factors Simulated only by natural factors Access : AMAZON Manifest COP26 \| Amazon Messages (https://www.youtube.com/watch?v=YIDZ0jCQ_Xo) amazon Expedition Magazine \| 75 SCIENTIFIC COMMITTEE PRESIDENT  Aldemir Malveira de Oliveira Final considerations n its epistemology, this journal could be characterized as an investigation effort into the possibility of presenting a collection of articles on the consequences of climatic phenomena observed in the Amazon. However, starting from a reflection on each article that composes this work, it is concluded that this collection becomes a contribution to those who research on the subject around the planet. Even knowing all the difficulties that emerged in the course of each investigation, the authors of the research did not bend towards decline, but transformed each casualty into an observatory filled with energies and concerns that can only be explained by the researchers of “Climatology”. However, in essence, this magazine and its collection of articles will help those who seek to better understand atmospheric conditions, not only in the Amazon, but their interference in the planet's climate. Also, it is noteworthy that none of the works presented here do not constitute closed investigations, but seek to open a range of options for researchers who wish to embark on confronting and/or refuting the phenomena described throughout the journal. I I SCIENTIFIC COMMITTEE PRESIDENT  Aldemir Malveira de Oliveira amazon Expedition Magazine \| 76 G Climate uatavita Lagoon National Park (Colombia), in this place we find the famous Frailejon (Big Monk) that captures water from the clouds in its plumes that condenses and converts into water for the Guatavita Chief Lagoon (3,683 m), located in the Eastern Cordillera of the Colombian Andes, in the municipality of Sesquilé, 57 km northeast of Bogotá, capital of Colombia (2022, April). G amazon Expedition Magazine Bogotá, capital of Colombia (2022, April). Collaborator Julio Escobar (Colombia-Bogotá) amazon Expedition Magazine
https://openalex.org/W2756609766	https://europepmc.org/articles/pmc5622455?pdf=render	English	null	Cross-sectional and prospective mediating effects of dietary intake on the relationship between sedentary behaviour and body mass index in adolescents	BMC public health	2,017	cc-by	7,952	* Correspondence: elly.fletcher@deakin.edu.au 1Deakin University, Geelong, Australia, Institute for Physical Activity and Nutrition (IPAN), School of Exercise and Nutrition Sciences, Geelong, Australia Full list of author information is available at the end of the article © The Author(s). 2017 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. Cross-sectional and prospective mediating effects of dietary intake on the relationship between sedentary behaviour and body mass index in adolescents Elly A. Fletcher1* , Karen E. Lamb1, Sarah A. McNaughton1, Sarah P. Garnett2, David W. Dunstan1,3,4,5,6,7,8, Louise A. Baur2 and Jo Salmon1 Elly A. Fletcher1* , Karen E. Lamb1, Sarah A. McNaughton1, Sarah P. Garnett2, David W. Dunstan1,3,4,5,6,7,8, Louise A. Baur2 and Jo Salmon1 Fletcher et al. BMC Public Health (2017) 17:751 DOI 10.1186/s12889-017-4771-0 Fletcher et al. BMC Public Health (2017) 17:751 DOI 10.1186/s12889-017-4771-0 Background [15, 16]. However, a major limitation of both of these studies was their cross-sectional design which limits causal inference. Whereas, a longitudinal design would allow both the temporal order of associations to be ex- amined and many other aspects of a mediation model to be explored. g Adolescent obesity is a major public health concern. The combined rates of overweight and obesity among adoles- cents have increased over the last two decades world- wide [1]. In the United States, the proportion of obese adolescents has risen from 10.5% in 1988–1994 to 20.6% in 2013–2014 [2]. Australia has experienced similar in- creases with almost one in three adolescents currently overweight or obese [3]. Given that obesity tracks from adolescence to adulthood [4], it is imperative to under- stand the lifestyle risk factors associated with adolescent obesity, particularly prospectively, in order to inform effective interventions. Against this background, the primary aim of the study was to explore both the cross-sectional and prospective mediating effects of the consumption of discretionary foods, SSB and takeaway foods on the association be- tween TV viewing and BMI z-score (zBMI) in a cohort of Australian adolescents, and to examine whether these findings differ when total sedentary time and sedentary bout duration are examined. The secondary aims of the study were to explore the individual associations between the sedentary behaviour variables (TV viewing, sedentary time and sedentary bout duration) and dietary intake vari- ables (discretionary foods, SSB and takeaway foods), as well as their associations with zBMI. Based on the existing evidence, we hypothesised that consumption of discretion- ary foods, SSB and takeaway foods would partially mediate the cross-sectional and prospective association between TV viewing and zBMI, but would not mediate the cross- sectional or prospective association between total seden- tary time and sedentary bouts with zBMI. Sedentary behaviour – defined as any waking behav- iours characterised by low energy expenditure (< 1.5 METS) while in a sitting or reclining posture – has emerged as a new research focus for obesity prevention [5]. High amounts of television (TV) viewing, a common leisure-time sedentary behaviour, during adolescence have both immediate and long-term health conse- quences, including a higher risk of obesity [6]. However, there are inconsistent associations between total time spent in sedentary time [7, 8], or time spent in periods, or ‘bouts’, of sedentary time [9], and indicators of adipos- ity (e.g. BMI, waist circumference) in adolescents. Abstract Background: Cross-sectional evidence suggests TV viewing, but not objectively-measured sedentary time or bouts of sedentary time, is consistently associated with body mass index (BMI) in adolescents. However, it is unclear whether dietary intake is a potential mediator of these relationships. The aim of this study was to explore the cross-sectional and prospective mediating effects of dietary intake on the association of sedentary behaviour with BMI z-score (zBMI) in a cohort of Australian adolescents. Methods: Cross-sectional and prospective analyses were conducted in adolescents aged 12–15 years participating in the 2002/03 (baseline) and 2004/05 (follow-up) Nepean Growing Up Study. The independent variables were television (TV) viewing, an objective measure of total sedentary time and average sedentary bout duration, and the outcome variable zBMI. Using the Sobel-Goodman method with bootstrapping, mediation analyses were conducted examining three dietary components (discretionary foods, sugar- sweetened beverages [SSB] and takeaway foods) as mediators of associations between TV viewing and zBMI (n = 259) and between total sedentary time and average sedentary bout duration with zBMI (n = 140). Results: No significant cross-sectional or prospective total or direct associations were observed for TV viewing, total sedentary time and average sedentary bout duration with zBMI. However, TV viewing was positively associated with consumption of takeaway foods cross-sectionally (β = 0.06; 95% CI 0.01 to 0.12), prospectively at baseline (β = 0.07; 95% CI 0.01 to 0.12) and prospectively at follow-up (β = 0.10; 95% CI 0.04, 0.16), and average sedentary bout duration was inversely associated with SSB consumption both cross-sectionally (β = −0.36; 95% CI -0.69 to −0.02) and prospectively at baseline (β = −0.36; 95% CI -0.70 to −0.02). No mediation effects were identified. Conclusions: TV viewing, total sedentary time and bouts of sedentary time were not associated cross- sectionally or prospectively with adolescents’ zBMI, and three elements of dietary intake (e.g. intake of discretionary foods, SSB and takeaway foods) did not mediate this relationship. The role of dietary intake and sedentary behaviour in relation to adolescent health requires further clarification. Keywords: Television viewing, Sedentary behaviour, Dietary intake, BMI, Adolescents Fletcher et al. BMC Public Health (2017) 17:751 Page 2 of 10 Page 2 of 10 Page 2 of 10 Study design y g In 2002/03 (baseline), 348 adolescents aged 12–13 years participated in the Nepean Kids Growing-Up Study. The adolescents were originally part of a birth cohort study (the “Nepean Study”) which involved 2314 infants born between 1989 and 1990 at the Nepean Hospital (western Sydney, Australia). Written consent was obtained from the parent or guardian and assent from the adolescent. The Ethics Committees of The Children’s Hospital at Westmead and Wentworth Area Health Service gave ethical approval. Full details about the original study and eligibility criteria have been previously published [17]. Briefly, the study involved adolescents attending the clinic at Nepean Hospital, where they had their height and weight measured and completed a questionnaire on their demographics and physical activity levels, and a semi-quantitative food frequency questionnaire (FFQ). Afterwards, adolescents wore an accelerometer for 7 days during all waking hours. In 2004/05, the adoles- cents were recontacted and invited to participate in the follow-up study. In total, 63 adolescents were unable to be contacted or withdrew from the study, leaving 285 adolescents participating at both time points. A systematic review examining whether associations between sedentary behaviour and health outcomes in adolescents were independent of dietary intake found TV viewing, screen time and overall sedentary time were positively related to BMI, independent of dietary intake [14]. The systematic review also identified very few studies had specifically examined the mediating role of dietary intake in the TV viewing and BMI rela- tion; with only two studies (out of the 21 studies identi- fied) exploring this and reporting no mediation effects Background y g One potential behavioural mechanism that could explain why TV viewing has more consistent associa- tions with body mass index (BMI) when compared to total or bouts of sedentary time in adolescents, is an in- crease in energy-dense, nutrient poor foods and sugar- sweetened beverages (SSB). For example, TV viewing has consistently been reported to be associated with a higher energy intake, and an increased consumption of dis- cretionary foods, SSB, and fast food/takeaway foods in adolescents [10], whereas few studies have reported as- sociations between objectively-measures of sedentary time with dietary intake [11, 12]. In addition, no study to date has explored whether prolonged bouts of sedentary time are related to dietary intake among adolescents. The latter is important as studies with adults have shown that, independent of how much total sedentary time is accumulated, those with fewer interruptions in sedentary time (i.e., prolonged bouts) have poorer car- diometabolic health profiles [13]. Independent variables (sedentary behaviour) Adolescents completed a self-report questionnaire on their time spent watching TV (hours/day) on a usual school day (Monday to Friday) and a usual weekend day (Saturday and Sunday). The questionnaire has previously been shown to have good to excellent reliability (per- centage agreement = 70%–99%) [20]. To calculate ave- rage daily hours spent watching TV over a usual week, daily weekday TV hours was multiplied by five and daily weekend TV hours was multiplied by 2, then summed together and divided by seven. Sedentary time was measured objectively by an ActiGraph AM-7164 accelerometer (ActiGraph Inc., Florida). At both time points, adolescents were asked to wear the monitor on their right hip during all waking hours for 7 days, except when bathing, swimming and sleeping. Data were downloaded in 1-min epochs and non-wear time was defined as at least 20 min of zero counts. Sedentary time was defined as all wear-time mi- nutes with an average activity count of ≤100 counts per minute (cpm), and was standardised for wear time using the residual method [21]. Average sedentary bout duration was calculated by summing all uninterrupted minutes ≤100 cpm, and then taking the midpoint of all sedentary bouts that lie on the accumulation curve for each individual [22]. Analyses were limited to partici- pants who had ≥8 h of wear time on ≥3 week days and ≥7 h of wear time on ≥1 weekend day [23]. Covariates The covariates considered for the analyses included age at baseline, sex, maternal education (an indicator for family socioeconomic status), pubertal status and accelerometry-measured moderate-to-vigorous physical activity (MVPA) collected at baseline. Maternal educa- tion was collapsed into three categories: “low” (some secondary education or less); “medium” (completing sec- ondary school, an apprenticeship or technical certifi- cate); and “high” (university or tertiary qualification). Pubertal status was self-assessed using the ‘Tanner Stages’ of breast development and commencement of menses (girls) and pubic hair and genitalia (boys) [25]. For analyses, participants were categorised as early pu- berty, mid-pubertal, late-pubertal and post-pubertal. Accelerometry-measured MVPA at baseline was calcu- lated based on the Freedson accelerometer age-cut points [26] and considered as a covariate for the analyses involving sedentary time and sedentary bouts with zBMI. Mediating variables (dietary intake) Usual dietary intake was measured using a 56-item semi-quantitative FFQ, which was developed based on data from the 1995 Australian National Nutrition Survey [24]. Adolescents were asked to report how often they ate certain foods and beverages (either in times per week or per day) over the previous 7 days. The 8-item frequency response scale was converted to times per week as follows: 1) “not consumed last week” = 0; 2) “consumed once last week” = 0.143; 3) “consumed 2–3 times last week” (average number used) = 0.357; 4) “con- sumed 4–6 times last week” (average number used) = 0.714; 5) “consumed once a day” = 1; 6) “con- sumed 2 times a day” = 2; 7) “consumed 3 times a Outcome variable (zBMI) Height and weight were measured at both time points by a trained research assistant and study dietitian at the Fletcher et al. BMC Public Health (2017) 17:751 Page 3 of 10 Page 3 of 10 day” = 3; 8) “consumed 4–6 times a day” (average number used) = 5. For the analyses, a combination of food and beverage items were summed together to create three dietary mediators at both time points: 1) frequency of consuming discretionary foods, which in- cluded any savoury or plain biscuits, sweet pastries, cakes, doughnuts, chocolate, confectionary, and potato chips; 2) frequency of consuming SSB, which included non-diet soft drink, non-diet cordial and fruit juice and not sweetened milk drinks or energy drinks; and 3) fre- quency of consuming takeaway foods, which included savoury pastries (e.g. meat pies and sausage rolls), ham- burgers, pizza, hot chips and spring rolls/dim sims. The FFQ was tested for reproducibility and overall showed fair to excellent reliability for sweet snacks (ICC = 0.61), savoury snacks (ICC = 0.63), SSBs (ICC = 0.77), and fast food (ICC = 0.44). Frequency of consuming discretionary foods and frequency of consuming SSB were multiplied by 7 to convert to times per day. Participants with miss- ing data for any of the dietary items listed were excluded (n = 2). In all analyses, the dietary mediators were treated as continuous variables. clinic. Height was measured to the nearest 0.1 cm and weight was measured without shoes and in light clothing to the nearest 0.1 kg with electronic scales (Wedderburn, Summer Hill, NSW, Australia). Height and weight were used to calculate each participants’ BMI and zBMI was determined using the age- and sex-specific CDC 2000 reference data [18]. Overweight and obesity was deter- mined using the International Obesity Task Force (IOTF) criteria [19]. In all analyses, zBMI was treated as a continuous variable. Statistical analyses As shown in Fig. 1, to be included in the analyses involv- ing TV viewing and zBMI, participants were required to have complete data for age, sex, maternal education, pu- bertal status, dietary intake and TV viewing at baseline, and complete data for zBMI at baseline and follow-up (n = 259). To examine the association of total and bouts of sedentary time, dietary intake and zBMI, separate ana- lyses were undertaken from a subsample of participants who met the previous inclusion criteria as well as Page 4 of 10 Fletcher et al. BMC Public Health (2017) 17:751 Fig. 1 Flow diagram of participants for analyses Fig. 1 Flow diagram of participants for analyses meeting the accelerometry inclusion criteria at baseline (n = 140). Prior to the main analyses, all variables were checked for normality. Discretionary foods, SSB and takeaway food intake at baseline were not normally dis- tributed and were log-transformed. Fig. 2 Theoretical diagram of the cross-sectional (Model 1) and prospective (Model 2) mediation pathway g Analyses were conducted using Stata/SE v14.0 (StataCorp LP, College Station, Texas, 2015). Figure 2 illustrates two theoretical models of the cross-sectional and prospective mediation pathways examined [27]. For all mediation analyses, the Sobel-Goodman mediation method with bootstrapping with 5000 replications was used to estimate standard errors and 95% confidence intervals [28]. For the cross-sectional analyses, only baseline variables were used to test the following associ- ations: 1) association between the independent variable and the mediator (a-coefficient pathway); 2) association between the mediator and the outcome variable, adjust- ing for the independent variable (b-coefficient pathway); 3) total association between the independent variable and outcome variable (c-coefficient pathway); 4) direct association between the independent variable and out- come variable, accounting for each mediator (c′-coeffi- cient pathway); and 5) indirect association (e.g. mediating effect) of the mediator on the independent variable and outcome variable. For the prospective ana- lyses, similar pathways were tested. However, dietary in- take at baseline and dietary intake at follow-up were examined separately as potential mediators in the associ- ations between the independent variable at baseline and the outcome variable at follow-up. All analyses were adjusted for age, sex, pubertal status and maternal edu- cation, with objectively-measured MVPA adjusted for in the analyses involving total and bouts of sedentary time Fig. 2 Theoretical diagram of the cross-sectional (Model 1) and prospective (Model 2) mediation pathway Fig. Associations between TV viewing, dietary intake and zBMI Cross-sectionally Overall, 259 and 140 adolescents were included in the TV viewing and zBMI, and the sedentary time and zBMI analytic samples, respectively (Table 1). Those excluded in the TV viewing and zBMI analyses had mothers with a lower maternal education and those excluded in the y There was no evidence of total or direct cross-sectional as- sociations of TV viewing with zBMI (Table 2). A small, positive association was observed between hours spent watching TV per day and frequency of consuming take- away foods each week (a-coefficient pathway); with each additional hour of TV viewing, adolescents consumed an additional 0.06 serves (95% CI 0.01 to 0.12; p < 0.05) of takeaway foods each week. An inverse association was also observed for consuming discretionary foods each day and zBMI (b-coefficient pathway); with each additional serving of discretionary foods consumed each day, zBMI was lower by −0.39 units (95% CI -0.65 to −0.13; p < 0.01). None of the dietary variables were significant mediators of the cross-sectional association of TV viewing with zBMI. Prospectively h There was no evidence of total or direct prospective associations between TV viewing at baseline and zBMI at follow-up. Similar to the cross-sectional associations, a positive prospective association was observed between hours spent watching TV per day and frequency of con- suming takeaway foods each week at baseline and at follow-up. For example, for each additional hour of TV viewing, adolescents consumed an additional 0.07 (95% CI 0.01 to 0.12; p < 0.05) serves of take-away foods each week at baseline, and an additional 0.10 (95% CI 0.04 to 0.16; p < 0.05) serves of take-away foods each week at follow-up. However, no significant association remained for any of the dietary variables consumed at baseline and at follow-up with zBMI at follow-up (b-coefficient pathway). None of the dietary variables were significant mediators of the pro- spective association between TV viewing and zBMI. Associations between TV viewing, dietary intake and zBMI Cross-sectionally Table 1 Baseline characteristics of participants included in analyses Variables TV and zBMI (n = 259) Sedentary time and zBMI (n = 140) Age, years 12.9 (12.9, 13.0) 12.9 (12.9, 13.0) Sex, % Male 47.5 (41.4, 53.6) 50.0 (41.7, 58.3) Female 52.5 (46.4, 58.4) 50.0 (41.7, 58.3) Maternal education, % Low 6.9 (4.4, 10.8) 5.0 (2.3, 10.2) Medium 65.3 (59.2, 70.8) 62.8 (54.4, 70.5) High 27.8 (22.7, 33.6) 32.1 (24.8, 40.4) Pubertal status, % Early puberty 19.3 (8.0, 4.6) 14.3 (35.1, 56.2) Mid-pubertal 35.5 (30.0, 41.6) 41.4 (33.5, 50.0) Late-pubertal 35.5 (30.0, 41.6) 35.0 (27.5, 43.4) Post-pubertal 27.0 (21.9, 32.8) 22.1 (16.0, 30.0) BMI kg/m2 20.7 (20.2, 21.3) 20.3 (19.7, 21.0) z-score 0.4 (0.3, 0.5) 0.3 (0.1, 0.5) Overweight, % 18.9 (14.6, 24.2) 13.6 (8.8, 20.4) Obese, % 9.2 (6.3, 13.5) 9.2 (5.4, 15.4) Sedentary behaviour TV viewing (hours/day) 3.2 (3.0, 3.4) Total sedentary time (hours/day) 6.1 (6.0, 6.2) Average sedentary bout (minutes/day) 6.6 (6.5, 6.6) MVPA (minutes/day) 48.2 (35.0, 61.4) Dietary intake Discretionary foods (freq/day) 2.4 (2.2, 2.6) 2.4 (2.1, 2.7) Sugar-sweetened beverages (freq/day) 2.1 (1.8, 2.3) 1.9 (1.6, 2.2) Takeaway food (freq/week) 3.4 (3.1, 3.6) 3.2 (2.8, 3.6) BMI body mass index, zBMI BMI z-score, TV television, MVPA moderate-to- vigorous physical activity; freq: frequency Footnote: Maternal education: low = some secondary education or less; medium = completing secondary school, an apprenticeship or technical certificate; high = university or tertiary qualification. Pubertal status self-assessed using the ‘Tanner Stages’ [25]. BMI z-score determined using the age- and sex-specific CDC 2000 reference data [18]. Overweight and obesity determined using the International Obesity Task Force (IOTF) criteria [19] Table 1 Baseline characteristics of participants included in Statistical analyses 2 Theoretical diagram of the cross-sectional (Model 1) and prospective (Model 2) mediation pathway Page 5 of 10 Fletcher et al. BMC Public Health (2017) 17:751 with zBMI. The prospective analyses were additionally adjusted for zBMI at baseline. The significance level was set at p < 0.05 for all statistical tests. sedentary time and zBMI analyses were older, had a lower maternal education and had a higher proportion of overweight participants (Additional file 1: Tables S1 and S2 in the online supplement file). Cross-sectionally There was no evidence of total or direct cross-sectional associations for total sedentary time (hours/day) and zBMI, or between total sedentary time and any of the diet- ary mediators (a-coefficient pathway) (Table 3). An inverse association was observed between frequency of consum- ing discretionary foods per day and zBMI; with each additional discretionary food consumed each week, zBMI was lower by −0.42 units (95% CI -0.77 to −0.07; p < 0.05) (b-coefficient). There were no significant mediating effects for any of the dietary variables in the cross-sectional asso- ciation of total sedentary time with zBMI. Fletcher et al. BMC Public Health (2017) 17:751 Page 6 of 10 Table 2 Cross-sectionala and prospectiveb associations of dietary intake (mediator), TV viewing (independent) and zBMI (outcome) (n = 259) Outcome: zBMI c`-coefficient (direct) β (95% CI) a-coefficient β (95% CI) b-coefficient β (95% CI) ab/indirect (mediated/indirect)c Independent: TV viewing Uncorrected β (95% CI) Bias-corrected β (95% CI) Cross-sectional mediators Discretionary food (freq/day) 0.05 (−0.02, 0.13) 0.01 (−0.03, 0.05) −0.39 (−0.65, −0.13)** −0.04 (−0.18, 0.10) −0.04 (−0.23, 0.12) SSB (freq/day) 0.05 (−0.03, 0.13) 0.02 (−0.03, 0.07) 0.03 (−0.15, 0.22) 0.01 (−0.04, 0.05) 0.01 (−0.07, 0.09) Takeaway food (freq/week) 0.05 (−0.02, 0.13) 0.06 (0.01, 0.12)* −0.07 (−0.24, 0.09) −0.05 (−0.16, 0.07) −0.05 (−0.22, 0.06) Prospective mediators At baseline Discretionary foods (freq/day) −0.01 (−0.04, 0.02) 0.01 (−0.02, 0.05) 0.02 (−0.09, 0.13) 0.01 (−0.01, 0.02) 0.01 (−0.02, 0.04) SSB (freq/day) −0.01 (−0.04, 0.02) 0.02 (−0.03, 0.07) 0.03 (−0.04, 0.11) 0.01 (−0.02, 0.03) 0.01 (−0.02, 0.04) Takeaway food (freq/week) −0.01 (−0.04, 0.02) 0.07 (0.01, 0.12)* −0.01 (−0.07, 0.06) −0.01 (−0.04, 0.05) −0.01 (−0.06, 0.05) At follow-up Discretionary foods (freq/day) −0.01 (−0.04, 0.02) 0.02 (−0.02, 0.07) −0.04 (−0.13, 0.06) −0.01 (−0.04, 0.02) −0.01 (−0.06, 0.02) SSB (freq/day) −0.01 (−0.04, 0.02) −0.01 (−0.05, 0.04) 0.03 (−0.06, 0.11) −0.001 (−0.01, 0.01) −0.001 (−0.03, 0.02) Takeaway food (freq/week) −0.01 (−0.04, 0.02) 0.10 (0.04, 0.16)* 0.02 (−0.05, 0.08) 0.02 (−0.05, 0.08) 0.02 (−0.04, 0.09) aCross-sectional total effects (c-pathway) of TV viewing and zBMI β = 0.05 (95% CI -0.03 to 0.13), adjusting for age, sex, mother’s education and pubertal status. bProspective total effects (c-pathway) of TV viewing and zBMI β = −0.01 (95% CI -0.04 to 0.02), adjusting for age, sex, mother’s education, pubertal status and zBMI at baseline. cDue to the small units of measure, the indirect effects have been multiplied by 10 Significant *p < 0.01, p < 0.05. Cross-sectionally TV: television; SSB: sugar-sweetened beverages; zBMI: body mass index z-score; freq: frequency Prospectively Total sedentary time at baseline was not significantly associated with zBMI at follow-up, accounting for mediation by dietary intake at baseline and at follow-up. No significant associations were also observed for any of the total, direct and indirect pathways, nor with the a- coefficient and b-coefficient pathways. No significant total or direct cross-sectional associations were observed for average sedentary bout duration (minutes/day) with zBMI (Table 4). Average sedentary bout duration was inversely related to frequency of Table 3 Cross-sectionala and prospectiveb associations of dietary intake (mediator), sedentary time (independent) and zBMI (outcome) (n = 140) Outcome: zBMI c`-coefficient (direct) β (95% CI) a-coefficient β (95% CI) b-coefficient β (95% CI) ab/indirect (mediated/indirect)c Independent: total sedentary time Uncorrected β (95% CI) Bias-corrected β (SE) Cross-sectional mediators Discretionary food (freq/day) 0.27 (−0.03, 0.58) −0.01 (−0.15, 0.14) −0.42 (−0.77, −0.07)* 0.01 (−0.61, 0.62) 0.01 (−0.62, 0.69) SSB (freq/day) 0.27 (−0.05, 0.58) −0.16 (−0.38, 0.06) −0.04 (−0.29, 0.20) 0.07 (−0.33, 0.46) 0.07 (−0.31, 0.61) Takeaway food (freq/week) 0.27 (−0.05, 0.58) −0.06 (−0.30, 0.18) −0.04 (−0.26, 0.18) 0.02 (−0.13, 0.18) 0.02 (−0.36, 0.35) Prospective mediators At baseline Discretionary food (freq/day) 0.05 (−0.07, 0.17) 0.03 (−0.12, 0.17) 0.03 (−0.10, 0.17) 0.01 (−0.05, 0.07) 0.01 (−0.08, 0.15) SSB (freq/day) 0.05 (−0.06, 0.17) −0.15 (−0.37, 0.07) 0.02 (−0.07, 0.11) −0.03 (−0.17, 0.11) −0.03 (−0.22, 0.13) Takeaway food (freq/week) 0.05 (−0.07, 0.17) −0.05 (−0.30, 0.19) −0.03 (−0.12, 0.05) 0.02 (−0.08, 0.11) 0.02 (−0.12, 0.16) At follow-up Discretionary food (freq/day) 0.05 (−0.07, 0.17) 0.08 (−0.09, 0.24) 0.01 (−0.11, 0.13) 0.01 (−0.05, 0.10) 0.01 (−0.16, 0.17) SSB (freq/day) 0.05 (−0.06, 0.17) −0.04 (−0.23, 0.15) 0.01 (−0.10, 0.12) −0.01 (−0.05, 0.04) −0.01 (−0.11, 0.09) Takeaway food (freq/week) 0.05 (−0.06, 0.17) 0.21 (−0.57, 0.47) −0.01 (−0.08, 0.07) −0.01 (−0.16, 0.15) −0.01 (−0.19, 0.17) aCross-sectional total effects (c-pathway) of total sedentary time and zBMI β = 0.27 (95% CI -0.04 to 0.58), adjusting for age, sex, mother’s education and pubertal status. bProspective total effects (c-pathway) of total sedentary time and zBMI β = 0.05 (95% CI -0.06 to 0.17), adjusting for age, sex, mother’s education, pubertal status and zBMI at baseline. cDue to the small units of measure, the indirect effects have been multiplied by 10. Significant p < 0.05. aCross-sectional total effects (c-pathway) of total sedentary time and zBMI β = 0.27 (95% CI -0.04 to 0.58), adjusting for age, sex, mother’s education and pubertal status. bProspective total effects (c-pathway) of total sedentary time and zBMI β = 0.05 (95% CI -0.06 to 0.17), adjusting for age, sex, mother’s education, pubertal status and zBMI at baseline. cDue to the small units of measure, the indirect effects have been multiplied by 10. Significant p < 0.05. SSB: sugar-sweetened bever- ages; zBMI: body mass index z-score; freq: frequency Prospectively SSB: sugar-sweetened bever- ages; zBMI: body mass index z-score; freq: frequency nala and prospectiveb associations of dietary intake (mediator), sedentary time (independent) and zBMI Table 3 Cross-sectionala and prospectiveb associations of dietary intake (mediator), sedentary time (indepen (outcome) (n = 140) aCross-sectional total effects (c-pathway) of total sedentary time and zBMI β = 0.27 (95% CI -0.04 to 0.58), adjusting for age, sex, mother’s education and pubertal status. bProspective total effects (c-pathway) of total sedentary time and zBMI β = 0.05 (95% CI -0.06 to 0.17), adjusting for age, sex, mother’s education, pubertal status and zBMI at baseline. cDue to the small units of measure, the indirect effects have been multiplied by 10. Significant p < 0.05. SSB: sugar-sweetened bever- ages; zBMI: body mass index z-score; freq: frequency Fletcher et al. BMC Public Health (2017) 17:751 Page 7 of 10 Table 4 Cross-sectionala and prospectiveb associations of dietary intake (mediator), sedentary bouts (independent) and zBMI (outcome) (n = 140) Outcome: zBMI c`-coefficient (direct) β (95% CI) a-coefficient β (95% CI) b-coefficient β (95% CI) ab/indirect (mediated/indirect)c Independent: sedentary bouts Uncorrected β (95% CI) Bias-corrected β (SE) Cross-sectional mediators Discretionary food (freq/day) 0.12 (−0.36, 0.61) −0.08 (−0.31, 0.15) −0.42 (−0.77, −0.06) 0.32 (−0.67, 1.32) 0.32 (−0.55, 1.37) SSB (freq/day) 0.14 (−0.36, 0.64) −0.36 (−0.69, −0.02)* −0.06 (−0.30, 0.19) 0.20 (−0.71, 1.11) 0.20 (−0.68, 0.12) Takeaway food (freq/week) 0.16 (−0.33, 0.65) 0.03 (−0.34, 0.41) −0.05 (−0.28, 0.18) −0.02 (−0.21, 0.18) −0.02 (−0.62, 0.34) Prospective mediators At baseline Discretionary food (freq/day) 0.08 (−0.10, 0.26) −0.06 (−0.29, 0.17) 0.04 (−0.10, 0.17) −0.02 (−0.14, 0.10) −0.02 (−0.24, 0.14) SSB (freq/day) 0.09 (−0.09, 0.27) −0.36 (−0.70, −0.02)* 0.02 (−0.07, 0.11) −0.09 (−0.42, 0.24) −0.09 (−0.52, 0.24) Takeaway food (freq/week) 0.08 (−0.10, 0.26) 0.04 (−0.34, 0.41) −0.04 (−0.12, 0.04) −0.01 (−0.15, 0.13) −0.01 (−0.27, 0.16) At follow-up Discretionary food (freq/day) 0.08 (−0.10, 0.26) −0.08 (−0.34, 0.17) 0.02 (−0.10, 0.14) −0.02 (−0.12, 0.09) −0.02 (−0.28, 0.16) SSB (freq/day) 0.08 (−0.10, 0.26) 0.11 (−0.18, 0.40) 0.01 (−0.010, 0.11) 0.01 (−0.11, 0.12) 0.01 (−0.19, 0.20) Takeaway food (freq/week) 0.08 (−0.10, 0.26) 0.06 (−0.35, 0.48) −0.01 (−0.08, 0.07) −0.01 (−0.05, 0.04) −0.01 (0.18, 0.17) aCross-sectional total effects (c-pathway) of average sedentary bout and zBMI β = 0.16 (95% CI -0.33 to 0.65), adjusting for age, sex, mother’s education and pubertal status. Prospectively b Prospective total effects (c-pathway) of average sedentary bout and zBMI β = 0.08 (95% CI -0.10 to 0.26), adjusting for age, sex, mother’s education, pubertal status and zBMI at baseline. cDue to the small units of measure, the indirect effects have been multiplied by 10. Significant p < 0.05. SSB: sugar-sweetened beverages; zBMI: body mass index z-score; freq: frequency aCross-sectional total effects (c-pathway) of average sedentary bout and zBMI β = 0.16 (95% CI -0.33 to 0.65), adjusting for age, sex, mother’s education and pubertal status. b Prospective total effects (c-pathway) of average sedentary bout and zBMI β = 0.08 (95% CI -0.10 to 0.26), adjusting for age, sex, mother’s education, pubertal status and zBMI at baseline. cDue to the small units of measure, the indirect effects have been multiplied by 10. Significant p < 0.05. SSB: sugar-sweetened beverages; zBMI: body mass index z-score; freq: frequency with TV viewing, average sedentary bout duration and zBMI, none of the dietary variables significantly medi- ated the relationships between the sedentary variables and zBMI cross-sectionally or prospectively. consuming SSB each day (a-coefficient pathway); with each additional minute spent in a sedentary bout, the frequency of consuming SSB was lower by nearly half a serve each day (β = −0.36, 95% CI -0.69 to −0.02; p < 0.05). An inverse association was also observed for frequency of consuming discretionary foods each week and zBMI (b-coefficient pathway); with each additional discretionary food consumed each week, zBMI was lower by −0.42 units (95% CI -0.77 to −0.06; p < 0.05). None of the dietary variables significantly mediated cross-sectional associations of sedentary bout duration with zBMI. consuming SSB each day (a-coefficient pathway); with each additional minute spent in a sedentary bout, the frequency of consuming SSB was lower by nearly half a serve each day (β = −0.36, 95% CI -0.69 to −0.02; p < 0.05). An inverse association was also observed for frequency of consuming discretionary foods each week and zBMI (b-coefficient pathway); with each additional discretionary food consumed each week, zBMI was lower by −0.42 units (95% CI -0.77 to −0.06; p < 0.05). None of the dietary variables significantly mediated cross-sectional associations of sedentary bout duration with zBMI. Prospectively The null finding for the association of TV viewing with zBMI in the current study is in contrast to previous studies in youth that have consistently shown significant and positive associations both cross-sectionally [6] and prospectively [29, 30]. The differences in findings could be attributed to the homogeneity of the current sample being examined, with higher than average number of hours spent watching TV [31] and a lower zBMI com- pared to the population average [32]. The null associ- ation for total sedentary time and average sedentary bout duration with zBMI is supported by some previous studies [8, 33–35], but not others [7]. Research examin- ing accelerometer-measured sedentary time with health indictors among children and youth appears to be mixed; it is unclear whether an association exists in only some populations or if there are inconsistencies in meas- uring sedentary time and/or the analytical approaches undertaken. Prospectively When examining the prospective associations between average sedentary bout duration at baseline and zBMI at follow-up, no significant total or direct associations were observed. However, a significant inverse association remained for average sedentary bout duration with f- requency of consuming SSB at baseline (β = −0.36; 95% CI -0.70 to −0.02; p < 0.05), but not at follow-up for the a-coefficient pathways. No significant associations were observed for the b-coefficient pathways or mediating effects. The positive association observed for TV viewing with the consumption of takeaway foods, both cross-sectionally and prospectively is consistent with previous research [36, 37]. This link could be partially explained by the large extent of TV advertising of foods high in fat and energy during peak times when children and adolescents are likely to be watching TV [38]. In contrast, no evidence of an association was observed for total sedentary time with any of the dietary Discussion This study found no evidence of direct or indirect associations for TV viewing, total sedentary time and average sedentary bout duration with adolescents’ zBMI, either cross-sectionally or prospectively. Although some of the dietary variables were independently associated Fletcher et al. BMC Public Health (2017) 17:751 Page 8 of 10 Page 8 of 10 (both subjective and objectively measured) and health indicators (e.g. BMI, metabolic syndrome). variables. The null finding is consistent with previous lit- erature in youth [11, 12] where, compared to TV viewing, fewer significant associations are observed for total seden- tary time with elements of a less healthy diet. The null finding could be due to the measure used to capture total sedentary time. For example, accelerometers measure all time spent being sedentary, and thus may capture times where adolescents may not be eating/drinking (e.g. sitting in school, sitting in the car). In addition, due to accelerom- eters being unable to determine posture (e.g. standing still versus sitting), time spent standing may have been included in total sedentary time and thus may diminish the oppor- tunity to engage in an eating occasion. Unexpectedly, the study found higher sedentary bout duration was inversely associated with a lower consumption of SSB, both cross- sectionally and prospectively. One possible reason for this could be due to the adolescents not breaking up their sed- entary time in order to retrieve a SSB from another room (e.g. the kitchen, school canteen). However, given the current study is one of the first to examine individual asso- ciations between sedentary bout duration and dietary in- take, further research is urgently needed in this area. To our knowledge, the current study is the first to examine both the cross-sectional and prospective medi- ating effects of dietary intake on the association between sedentary behaviour and zBMI in an adolescent popula- tion, and the first to examine this using objective measures of sedentary time. Other strengths include ad- justment for a variety of confounders, including mater- nal education and adolescent pubertal status, and examining the dietary mediators (discretionary foods, SSB, takeaway foods) separately in all models. Limita- tions of the study include participants self-reporting their dietary intake using an FFQ and hours spent watching TV, and the low sample size for mediation ana- lyses. In addition, the semi-quantitative FFQ used in the current study was limited with the number of healthy food items included (e.g. Conclusion In conclusion, despite identifying some significant associations between TV viewing and average sedentary bout duration with frequency of consuming takeaway foods and SSB, and between frequency of consuming discretionary foods and zBMI, no significant associations were observed for any of the sedentary behaviour variables with zBMI, either cross-sectionally or prospect- ively. In addition, none of the dietary variables were found to be significant mediators of the associations between sedentary behaviour and zBMI. Given the unacceptably high levels of adolescent overweight and obesity, further studies are warranted to elucidate the complex relationships between TV viewing, sedentary time, dietary intake and health indicators. gy g g Only one other study has explored the prospective as- sociations between TV viewing, dietary intake and BMI; however, that study examined a younger population of pre-school children aged 0–5 years over a 2-year period [42]. In contrast to the current study, Fuller-Tyszkiewicz et al. reported a significant positive association between TV viewing and BMI that was bi-directional, with those children characterised with high amounts of TV viewing having higher BMI, and children with higher BMI watching a greater amount of TV. In addition, that study reported the prospective associations between TV viewing and BMI among 4 year olds were mediated by discretionary foods and soft drink consumption [42]. The differences in findings between the current study and previous study could be contributed to the different study populations and dietary mediators being examined, and that the previous study only examined TV view- ing and BMI. Thus, further prospective studies are needed to explore whether dietary intake mediates the relationship between various sedentary behaviours Discussion fruit and vegetables) and did not have information on portion sizes. There were also differences in those that were included and excluded in the analysis of this study and thus may limit the repre- sentative of the findings. Further, the data presented in the current study was collected more than a decade ago and thus the behaviours reported in the study may not reflect the contemporary sedentary and dietary behav- iours adolescents are engaging in today. In contrast, the cross-sectional, inverse association found for discretionary foods with zBMI, and the null finding for SSB and takeaway foods is in contrast with previous studies that have found positive associations for unhealthy dietary intake with BMI [39, 40]. Our findings could be a consequence of under-reporting, where overweight or obese children and adolescents have been found to under-report their energy intake by 20–40% [41]. Alternatively, it is possible that some participants in the current study with a higher zBMI may have changed their behaviour by decreasing their discretionary food intake over time as a strategy to manage their weight. Additional file Additional file 1: Supplement file. Comparison of baseline characteristics of participants included in the TV viewing and zBMI analyses compared to those excluded in the analyses. (PDF 29 kb) Additional file 1: Supplement file. Comparison of baseline characteristics of participants included in the TV viewing and zBMI analyses compared to those excluded in the analyses. (PDF 29 kb) Competing interests Competing interests The authors declare that they have no competing interests. Abbreviations BMI: Body mass index; Cpm: Counts per minute; FFQ: Food frequency questionnaire; MVPA: Moderate-to-vigorous physical activity; SSB: Sugar- sweetened beverages; TV: Television; zBMI: Body mass index z-score Page 9 of 10 Page 9 of 10 Fletcher et al. BMC Public Health (2017) 17:751 Funding h 6. Tremblay MS, LeBlanc AG, Kho ME, Saunders TJ, Larouche R, Colley RC, Goldfield G, Connor GS. Systematic review of sedentary behaviour and health indicators in school-aged children and youth. Int J Behav Nutr Phys Act. 2011;8(98):98. The Nepean longitudinal study was funded by The Children’s Hospital at Westmead Grant Research Scheme, a National Health and Medical Research Council Project Grant #206501 and Meat and Livestock Australia. SAM is funded by an NHMRC Career Development Fellowship Level 2, ID1104636 and was previously funded by an ARC Future Fellowship (2011-2015, FT100100581). DWD is funded by NHMRC Senior Research Fellowship (APP1078360). JS is funded by a NHMRC Principal Research Fellowship (APP1026216). This work was also partially funded by an OIS grant from the Victorian State Government and a National Health & Medical Research Council, Centre of Research Excellence grant (APP1057608). 7. Martínez-Gómez D, Eisenmann JC, Gómez-Martínez S, Veses A, Marcos A, Veiga OL. Sedentary behavior, adiposity, and cardiovascular risk factors in adolescents: the AFINOS study. Rev Esp Cardiol. 2010;63(3):277–85. 8. Chaput JP, Lambert M, Mathieu ME, Tremblay MS, O’ Loughlin J, Tremblay A. Physical activity vs. sedentary time: independent associations with adiposity in children. Pediatr Obes. 2012;7(3):251–8. 9. Colley R, Garriguet D, Janssen I, Wong S, Saunders T, Carson V, Tremblay M. The association between accelerometer-measured patterns of sedentary time and health risk in children and youth: results from the Canadian Health Measures Survey. BMC Public Health. 2013;13(1):1–9. Publisher’s Note 17. Garnett SP, Cowell CT, Baur LA, Fay RA, Lee J, Coakley J, Peat JK, Boulton TJ. Abdominal fat and birth size in healthy prepubertal children. Int J Obes Relat Metab Disord. 2001;25(11):1667–73. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 18. Kuczmarski RJ, Ogden CL, Guo SS, et al. 2000 CDC growth charts for the United States: Methods and development. National Center for Health Statistics Vital Health Stat 11. 2002;11(246):1–190. Authors’ contributions JS, LB, and SG were involved in the conception and design of the study. EF carried out the data analysis with statistical guidance from KL. EF led the writing of the article, with contributions from authors KL, SM, JS, LB, SG, and DD. All authors read and approved the final manuscript. 12. Borghese MM, Tremblay MS, Leduc G, Boyer C, Bélanger P, LeBlanc AG, Francis C, Chaput J-P. Independent and combined associations of total sedentary time and television viewing time with food intake patterns of 9- 11 year-old Canadian children. Appl Physiol Nutr Metab. 2014;39(8):937–43. 13. Healy GN, Dunstan DW, Salmon J, Cerin E, Shaw JE, Zimmet PZ, Owen N. Breaks in sedentary time: beneficial associations with metabolic risk. Diabetes Care. 2008;31(4):661–6. Availability of data and materials The dataset supporting the conclusions of this article has not been approved to be made publicly available by the ethics committees. To require information about the content and analyses of the dataset, contact Sarah Garnett: sarah.garnett@health.nsw.gov.au 10. Pearson N, Biddle S. Sedentary behavior and dietary intake in children, adolescents, and adults: a systematic review. Am J Prev Med. 2011;41(2):178–88. 11. Vissers PAJ, Jones AP, van Sluijs EMF, Jennings A, Welch A, Cassidy A, Griffin SJ. Association between diet and physical activity and sedentary behaviours in 9–10-year-old British White children. Public Health. 2013;127(3):231–40. Received: 29 January 2017 Accepted: 18 September 2017 24. Australian Bureau of Statistics: National Nutrition Survey 1997: Nutrient intakes and physical measurements (Report No. 4805.0). Canberra, Australia: Australian Bureau of Statistics; 1998. Acknowledgements h h ld l k 4. Singh AS, Mulder C, Twisk JW, van Mechelen W, Chinapaw MJ. Tracking of childhood overweight into adulthood: a systematic review of the literature. Obes Rev. 2008;9(5):474–88. The authors would like to thank Dr. Vanessa Shrewsbury for collecting the data and her work on the project. We would like to thank all the families that generously donated their time to participate in this study. 5. Sedentary Behaviour Research Network. Letter to the Editor: standardized use of the terms “sedentary” and “sedentary behaviours”. Appl Physiol Nutr Metab. 2012;37(3):540–2. Author details 1 1Deakin University, Geelong, Australia, Institute for Physical Activity and Nutrition (IPAN), School of Exercise and Nutrition Sciences, Geelong, Australia. 2The Children’s Hospital at Westmead Clinical School, University of Sydney, Westmead, Australia. 3Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia. 4Department of Medicine, Monash University, Melbourne, Australia. 5The University of Queensland, School of Public Health, Brisbane, Australia. 6Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia. 7School of Sport Science, Exercise and Health, The University of Western Australia, Perth, Australia. 8Mary MacKillop Institute for Health Research, Australian Catholic University, Melbourne, Australia. 19. Cole TJ, Bellizzi MC, Flegal KM, Dietz WH. Establishing a standard definition for child overweight and obesity worldwide: international survey. BMJ. 2000;320(7244):1240. 20. 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Appetite. 2012;59(3):945–8. • 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: • 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
https://openalex.org/W4377821781	https://zenodo.org/records/7638207/files/Credit%20Card%20Fraud%20Detection%20-Formatted%20Paper.pdf	English	null	Credit Card Fraud Detection Using Machine Learning	International journal for research in applied science and engineering technology	2,023	cc-by	2,398	ABSTRACT ABSTRACT The number of customers for the credit cards (CC) has grown of the last decade. These cards have aggravated the cashless systems of payments as well as alleviated the use of cash credit, which is termed as a short-term continuous loan. The credit cards are known to increase the purchasing power of citizens, and let them meet their daily needs, gadgets, etc. The number of CC (credit card) frauds has increased with the increase in number of credit cards. The unethical use of credit cards by hackers or credit cards users unwilling to pay back the amount are known as the major credit card frauds. The credit card frauds can be detected by evaluating the CC purchasing patterns using the historical data in order to detect the frauds. This data evaluation can help the banks or other organizations offering credit cards to minimize their losses due to the credit card frauds. The historical data evaluation with the current purchasing patterns requires the statistical modelling, which can automatically evaluate the fraudulent patterns and alarm the banks about the transactions. This helps the banks for early detection of the frauds, where they can easily eliminate the CC frauds by declining the suspected transactions. Recent Trends in Information Technology and its Application Volume 6 Issue 2 Recent Trends in Information Technology and its Application Volume 6 Issue 2 Dr. Sayed Abdulhayan1, Laiba Firdouse2, Junaina k3, Nishwa Haleema4, Zulaikath Anfeeda5 Department of Computer Science and Engineering, P A College of Engineering, Mangalore, India Corresponding Author E-mail ID:-sabdulhayan.cs@pace.edu.in LITERATURE REVIEW Credit card fraud refers to any type of fraud performed with a payment card, such as a credit card or debit card. The goal might be to get products or services or to transfer funds to another account controlled by a criminal. The Payment Card Industry Data Security Standard (PCI DSS) is a data security standard designed to assist financial institutions in securely processing card payments and reducing card fraud. Credit card fraud can be either authorized, in which the actual customer processes payment to another account controlled by a criminal, or unauthorized, in which the account holder does not grant authorization for the payment to proceed and the transaction is carried out by a third party. In the United Kingdom, unlawful financial fraud losses via payment cards and remote banking were £844.8 million in 2018. In 2018, banks and credit card providers stopped £1.66 billion in illegal fraud. That equates to £2 for every £3 of attempted fraud. Unauthorized individuals may use someone else's credit card information to make purchases, conduct other activities, or create new accounts, which is referred to as credit card fraud. Account takeover fraud, new account fraud, cloned cards, and cards-not-present schemes are a few instances of credit card fraud. Phishing, information skimming, and information sharing by a user-often without their knowledge-lead to this unlawful access. However, this kind of fraud may be stopped by issuers, organizations, and individual cardholders as well as recognized by artificial intelligence and machine learning. More than one in three people who own credit or debit cards have encountered fraud more than once, according to a 2021 annual survey. INTRODUCTION decrease it and defend against such occurrences in the future. Deception in Credit card happens when someone uses someone else's credit card without the owner's knowledge or the authorities who issue the card being used for personal expenses. Fraud detection is monitoring the behaviours of user populations in order to estimate, perceive, or avoid unacceptable behaviour such as fraud, intrusion, and defaulting. This is a pressing issue that requires the attention of groups such as machine learning and data science, where the answer may be automated. This issue is particularly difficult from the standpoint of learning since it is characterised by many aspects such as class imbalance. The number of genuine transactions considerably outnumbers the number of fraudulent transactions. First and foremost, we collected our dataset from Kaggle, a data analysis service that offers datasets. There are 31 columns in this dataset, 28 of which are labelled v1- v28 to safeguard sensitive data. The remaining columns denote Time, Amount, and Class. Time represents the time elapsed between the first and subsequent transactions. Amount refers to the amount of money exchanged. A genuine transaction is represented by class 0, while a fraudulent transaction is represented by class 1. Fraud in credit card transactions is defined as the illegal and undesired use of a credit card account by someone other than the account's owner. To curb this misuse, necessary preventative measures may be implemented, and the behaviour of such fraudulent operations can be analysed to HBRP Publication Page 1-4 2023. All Rights Reserved Page 1 Recent Trends in Information Technology and its Application Volume 6 Issue 2 Recent Trends in Information Technology and its Application Volume 6 Issue 2 Around 50% of all Americans have received a fraudulent charge on their cards. This translates to 127 million Americans who have at least once experienced credit card theft. Credit cards are safer than ever because regulators, card issuers, and banks spend a lot of time and effort working with investigators throughout the world to make sure fraudsters are unsuccessful. Regulations that hold the card supplier and bank accountable typically safeguard cardholders' money against fraudsters. Credit card technology and security procedures are growing more advanced, making it more difficult for thieves to steal money. This translates to 127 million Americans who have at least once experienced credit card theft. Furthermore, transaction patterns frequently modify their statistical features over time. SYSTEM ARCHITECTURE The credit card dataset is initially extracted from the source and cleaned and validated, which involves removing duplicate data, filling in blank columns, and transforming essential variables into factors or classes. The data is then split into two parts: a training dataset and a test dataset. The initial sample is currently randomly divided into the test and train datasets. The categorization of fraudulent and legitimate transactions is then performed using the Random Forest Classifier, and accuracy is also determined. The following are the steps for random forest algorithm: The data is then split into two parts: a training dataset and a test dataset. The initial sample is currently randomly divided into the test and train datasets. The categorization of fraudulent and legitimate transactions is then performed using the Random Forest Classifier, and accuracy is also determined. The following are the steps for random forest algorithm: 1. Choose N records at random from the dataset. 2. Based on these N records, construct a decision tree. 3. Select how many trees you want to include in your algorithm, then repeat steps 1 and 2. CONCLUSION With more training data, the Random Forest algorithm will perform better, but testing and application time will decrease. Applying extra pre-processing methods would also be beneficial. The results given by SVM are wonderful, but they might have been better if additional preprocessing had been done on the data. Other algorithms still struggle with the unbalanced dataset problem and require more preparation to deliver better results. As a consequence, employing a random forest algorithm with new improvements, we were able to obtain the correct value of credit card fraud detection, which is 0.9994802867383512 (99.93 percent). This suggested module offers more accurate findings and is appropriate for a bigger dataset than existing modules. With more data points, the Random Forest algorithm will perform better. EVALUATION subset of all accessible data that you will be working with is the focus of this stage. Ideally, ML challenges begin with a large amount of data (examples or observations) for which you already know the desired solution. Labeled data is information for which you already know the desired outcome. subset of all accessible data that you will be working with is the focus of this stage. Ideally, ML challenges begin with a large amount of data (examples or observations) for which you already know the desired solution. Labeled data is information for which you already know the desired outcome. The process of developing a model includes evaluation. Finding the model that best depicts our data and predicts how well the model will perform in the future is helpful. In data science, it is not acceptable to evaluate model performance using the training data because this can quickly lead to overly optimistic and over fitted models. Both approaches employ a test set (unseen by the model) to assess model performance in order to prevent over fitting. Based on its average, each categorization model's performance is estimated. The percentage of accurate predictions for the test data is what is meant by accuracy. By dividing the number of accurate forecasts by the total number of predictions, it may be simply determined. DATA PRE-PROCESSING MODULE 2 Format, clean, and sample from your chosen data to organise it. There are three typical steps in data pre-processing: The data you have chosen might not be in a format that you can use to work with it. The data may be in a proprietary file format and you would want it in a relational database or text file, or the data may be in a relational database and you would like it in a flat file. Data cleaning is the process of removing or replacing missing data. There can be data instances that are insufficient and lack the information you think you need to solve the issue. These occurrences might need to be eliminated. You could have access to far more carefully chosen data than you need. Algorithms may take significantly longer to perform on bigger amounts of data, and their computational and memory needs may also increase. Before thinking about the entire dataset, you may choose a smaller representative sample of the chosen data that may be much faster for exploring and testing ideas. EXTRACTION OF FEATURES MODULE 3 A process of attribute reduction is feature extraction. Feature extraction actually alters the characteristics as opposed to feature selection, which ranks the current attributes according to their predictive relevance. The original attributes are linearly combined to generate the changed attributes, or features. Finally, the Classifier algorithm is used to train our models. INFORMATION MODULE 1 Product reviews that were obtained from credit card transaction records are the data used in this article. The selection of the HBRP Publication Page 1-4 2023. All Rights Reserved Page 2 Recent Trends in Information Technology and its Application Volume 6 Issue 2 SOFTWARE REQUIREMENTS Operating System : Windows 10 (x64 based processor) Implementation Language : python Engine/framework : Django IDE : Anaconda Tools : Jupyter Notebook SOFTWARE REQUIREMENTS Operating System : Windows 10 (x64 based processor) Implementation Language : python Engine/framework : Django IDE : Anaconda Tools : Jupyter Notebook SOFTWARE REQUIREMENTS Operating System : Windows 10 (x64 based processor) Implementation Language : python Engine/framework : Django IDE : Anaconda Tools : Jupyter Notebook 4. Amlan Kundu, Suvasini Panigrahi, Shamik Sural, Senior Member, IEEE,“BLAST-SSAHA Hybridization for Credit Card Fraud Detection”, vol. 6, no. 4 pp. 309-315, 2009. 5. Y. Sahin and E. Duman, “Detecting Credit Card Fraud by Decision Trees and Support Vector Machines, Proceedings of International Multi Conference of Engineers and Computer Scientists,vol.I,2011 [6]http://www.rbi.org.in/Circular/Cred itCard FUTURE SCOPE We developed a protocol or model for this proposed project to identify fraud activities in credit card transactions. The majority of the key characteristics needed to distinguish HBRP Publication Page 1-4 2023. All Rights Reserved Page 3 BRP Publication Page 1-4 2023. All Rights Rese Recent Trends in Information Technology and its Application Volume 6 Issue 2 Recent Trends in Information Technology and its Application Volume 6 Issue 2 ADVANTAGES OF USING RANDOM FOREST The benefit of employing random forest for regression and classification. 1. Since there are several trees and each tree is educated on a portion of data, the random forest approach is not biased. The random forest algorithm essentially relies on the influence of "the community," which lessens the system's overall bias. HARDWARE REQUIREMENTS HARDWARE REQUIREMENTS Processor : Intel CORE i3 or above. RAM : 4GB or higher. I/O Parts : Monitor (color), Mouse, Keyboard 3. Wei Sun, Chen-Guang Yang, Jian- Xun Qi: Credit Risk Assessment in Commercial Banks Based On Support Vector Machines, vol.6, pp 2430-2433, 2006. Recent Trends in Information Technology and its Application Volume 6 Issue 2 REFERENCE between genuine and fraudulent transactions may be provided by this system. It is harder to follow the behaviour and pattern of fraudulent transactions as technology develops. It is now possible to automate the process and reduce the actual amount of work required to identify credit card fraud thanks to the growth of machine learning, artificial intelligence, and other pertinent information technology sectors. between genuine and fraudulent transactions may be provided by this system. It is harder to follow the behaviour and pattern of fraudulent transactions as technology develops. It is now possible to automate the process and reduce the actual amount of work required to identify credit card fraud thanks to the growth of machine learning, artificial intelligence, and other pertinent information technology sectors. 1. Sudhamathy G: Credit Risk Analysis and Prediction Modelling of Bank Loans Using R, vol. 8, no-5, pp. 1954- 1966. 2. LI Changjian, HU Peng: Credit Risk Assessment for ural Credit Cooperatives based on Improved Neural Network, International Conference on Smart Grid and Electrical Automation vol. 60, no. - 3, pp 227-230, 2017. 2. LI Changjian, HU Peng: Credit Risk Assessment for ural Credit Cooperatives based on Improved Neural Network, International Conference on Smart Grid and Electrical Automation vol. 60, no. - 3, pp 227-230, 2017. 6. https://www.ftc.gov/news- events/press- 6. https://www.ftc.gov/news- events/press- pp The random forest algorithm essentially relies on the influence of "the community," which lessens the system's overall bias. 7. releases/2019/02/imposter-scams-top- complaints-made- ftc-2018 8. https://www.kaggle.com/mlg- ulb/creditcardfraud 2. . This algorithm is incredibly reliable. Since it is exceedingly difficult for fresh data to have an impact on all the trees, even if a new data point is added to the dataset, the overall method is not significantly changed. 9. https://www.kaggle.com/uciml/default- of-credit-card- clients-dataset 10. https://www.kaggle.com/ntnu- testimon/paysim1/home 3. In situations when there are both categorical and numerical features, the random forest approach performs well. 3. In situations when there are both categorical and numerical features, the random forest approach performs well. 4. The random forest technique also performs effectively with incomplete or poorly scaled data. HBRP Publication Page 1-4 2023. All Rights Reserved Page 4 BRP Publication Page 1-4 2023. All Rights Rese
https://openalex.org/W4392835757	http://journal.wima.ac.id/index.php/JAKO/article/download/5099/3551	Indonesian	null	APPLICATION OF ENVIRONMENTAL ACCOUNTING TO HIU PAUS TOURISM IN TOMINI BAY	Jurnal Akuntansi Kontemporer	2,024	cc-by-sa	6,394	A B S T R A C T Article history: Received November 05, 2023 Revised January 14, 2024 Accepted January 16, 2024 g y Research Methods. This research is in the realm of qualitative study using a phenomenological approach. Research Results and Findings. The results showed that Hiu Paus Tourism still needs to implement environmental accounting by PSAK 2015 No. 1 concerning the presentation of financial statements, and there has been no special report related to environmental costs. In environmental management, Hiu Paus Tourism has carried out waste management and maintenance of coral reefs internally. However, the allocation of environmental costs is still carried out and has not been maximized. However, Hiu Paus tours in Tomini Bay have followed general rules of interaction for the safety of visitors in special interest tours. JURNAL AKUNTANSI KONTEMPORER (JAKO) – VOL 16 ISSUE 1 – JANUARY 2024 – Page 45-54 JURNAL AKUNTANSI KONTEMPORER (JAKO) – VOL 16 ISSUE 1 – JANUARY 2024 – Page 45-54 A B S T R A K Tujuan Penelitian. Penelitian ini bertujuan untuk mengkaji penerapan akuntansi lingkungan pada Wisata Hiu Paus di Teluk Tomini. g g p Metode Penelitian. Penelitian ini berada dalam ranah penelitian kualitatif dengan menggunakan pendekatan fenomenologi. gg p f g Hasil Penelitian dan Temuan Penelitian. Hasil penelitian menunjukkan bahwa wisata Hiu Paus belum melakukan penerapan akuntansi lingkungan sesuai PSAK tahun 2015 No. 1 tentang penyajian laporan keuangan dan belum ada laporan khusus terkait biaya lingkungan. Dalam pengelolaan lingkungan Wisata Hiu Paus telah melakukan pengelolaan limbah dan pemeliharaan terumbu karang secara inter- nal, meskipun dalam pengalokasian biaya lingkungannya masih dilakukan secara se- derhana dan belum maksimal. Namun, wisata Hiu Paus di Teluk Tomini telah mengikuti aturan umum berinteraksi untuk keselamatan pengunjung dalam wisata minat khusus. APPLICATION OF ENVIRONMENTAL ACCOUNTING TO HIU PAUS TOURISM IN TOMINI BAY Juwita Kanon Tri Handayani Amaliah* Ronald Soemitro Badu Gorontalo State University, 6 Jenderal Sudirman Street, Gorontalo, Indonesia, 96128 *triamaliah@ung.ac.id practices and sustainable actions. Accounting that is integrated with environmental management plays a vital role in helping organiza- tions achieve business sustainability, especially in terms of attaining ecological efficiency and manag- ing environmental impacts caused (Egan & Schaltegger, 2023). Environmental accounting can help the tourism industry improve corporate social responsibility by identifying costs and benefits (Sa- putra & Siregar, 2019). Based on several descriptions that have been put forward show that the im- portance of environmental accounting as a whole is expected to help organizations plan and make deci- sions in caring for the business environment. Ac- counting allows organizations to achieve sustaina- ble goals and reduce negative impacts on the envi- ronment in achieving organizational goals. In this regard, the Government has issued Undang-Un- dang Republik Indonesia No. 10 (2009) about Envi- ronmental Management in Tourism. It contains pro- visions relating to environmental issues in the con- text of the tourism industry in Indonesia. Several studies have been conducted on apply- ing environmental accounting in the tourism sector. Research from Mahesa & Amna (2022) analyze the application of environmental accounting to wood processing plants. Research on other environmental accounting is conducted by Saputra & Siregar (2019) which examines the application of environmental accounting in the coastal tourism sector in Batam us- ing a quantitative paradigm. Research by Russell et al. (2017) identify interdisciplinary research areas in environmental accounting that contribute to ecolog- ical sustainability and social justice. This research is helpful in developing theories about accounting and conceptualizing humans and nonhumans. Mean- while, Skaf et al. (2019) explore the environmental performance and sustainability capabilities of agri- cultural production. Meanwhile, Campos et al. (2023) conduct a holistic assessment of the environ- mental impact of tourism using the method Life Cy- cle Assessment (LCA) dengan pay attention to all stages of tourism activities. This study provides rec- ommendations for the use of environmentally friendly transportation as the key to reducing over- all environmental impact. On the slightly different side, research Mata et al. (2018) presents a literature review of environmental reports. The results of his research revealed that most environmental account- ing studies offer a longitudinal approach and use content analysis as their methodology. At the same time, the most widely used theories are legitimacy and stakeholder theories. Research on the impact of environmental reporting on financial performance tends to show inconsistent results. practices and sustainable actions. But in reality, based on the observations that re- searchers have made of one of the tourism objects in Gorontalo Province, namely Hiu Paus tourism, it has not made the maximum allocation of costs in activi- ties related to environmental management even though it has received benefits in its activities. This indicates that several types of tourism environmen- tal management costs have yet to be recognized by applicable regulations. This information illustrates that the financial recording of Hiu Paus Tourism in Botubarani Village still needs to be improved be- cause there is still no special treatment of environ- mental costs from the activities carried out. Hiu Paus Tourism is one of the tourism sectors in the Tomini Bay Area, precisely in Botubarani Village. It is nec- essary to mention that this Whale Shark tourist at- traction is the only destination with unique interest characteristics in the Tomini Bay Area, Gorontalo Province. Based on the phenomena previously revealed, this research can contribute to developing theories about environmental accounting and the development of ecological accounting policies for the tourism industry to produce valuable strategies for sustainable tourism development. This study examines the application of environ- mental accounting in tourism Hiu Paus in Tomini Bay. Although there have been many studies exam- ining the phenomenon of ecological accounting, as far as researchers know, there has yet to be research about accounting. The environment in the perspec- tive of tourist destinations is of particular interest character using a phenomenological approach. The phenomenological approach is an approach used in the qualitative research paradigm. Through phe- nomenology, this research will explore environmen- tal awareness and education among managers and visitors of tourist objects. The extent to which this awareness can shape environmental accounting INTRODUCTION of natural damage. Therefore, the role of the govern- ment is needed to assess the company's performance in environmental management. The management of social responsibility equipped with environmental accounting will have a broad and optimal impact and is sustainable, especially in fulfilling economic, ecological, and social aspects (Natalia, 2022; Skaf et al., 2019). Natural damage is one of the global problems that is increasingly concerning in today's modern era. In recent decades, humans have witnessed a rapid increase in various forms of environmental de- struction, including climate change, land degrada- tion, and environmental pollution. Pelu et al., (2022) revealed that environmental damage is caused by industrial development in Indonesia, which is not accompanied by adequate social environmental re- sponsibility. Mahesa & Amna (2022) affirming that humans are the biggest supporters of the destruction of nature. Correspondingly, Rahim & Mus (2020) re- vealed that the lack of awareness of humans and in- dustry towards the environment causes the problem In the current era, environmental accounting has become very important in the tourism industry be- cause of its environmental impact. Tourism exerts pressure on the environment through services of- fered with various components of destination attrac- tion; however, behind this, the tourism sector is very vulnerable to global warming (Miralles et al., 2023). 45 APPLICATION OF ENVIRONMENTAL ACCOUNTING TO HIU PAUS TOURISM IN TOMINI BAY KANON, AMALIAH, & BADU practices and sustainable actions. practices and sustainable actions. Environmental Accounting This environmental issue is essential and com- plex and can affect all aspects, including accounting. Accounting science is often known as economic ac- tivity, namely in the field of business entities that are always related to nominal. In accounting, the nomi- nal is always expressed in the form of money. How- ever, accounting science is not always related to nominal only. Accounting leads to a proactive ap- proach to social and environmental issues, although it is still experimental and in contrast to established financial accounting to consolidate itself by 46 JURNAL AKUNTANSI KONTEMPORER (JAKO) – VOL 16 ISSUE 1 – JANUARY 2024 – Page 45-54 developing international standards. A more proac- tive approach to evolution-conscious culture is therefore needed (Birkin et al., 2005). Environmental and social accounting is an increasingly diverse field of activity and study. The environmental aspect it- self covers a very broad spectrum of concerns (Bebbington et al., 2000). Alassuli (2024) revealed that a serious step in implementing social and envi- ronmental accounting has the potential to improve the company's overall performance. developing international standards. A more proac- tive approach to evolution-conscious culture is therefore needed (Birkin et al., 2005). Environmental and social accounting is an increasingly diverse field of activity and study. The environmental aspect it- self covers a very broad spectrum of concerns (Bebbington et al., 2000). Alassuli (2024) revealed that a serious step in implementing social and envi- ronmental accounting has the potential to improve the company's overall performance. environmental dimensions in which the company operates (Shofia & Anisah, 2020). This is also stipu- lated in the Undang-Undang Republik Indonesia No. 40 (2007) regulate that social and environmental responsibility is the responsibility of the company which is budgeted and calculated as the company's burden. Environmental Costs According to Hansen & Mowen (2009) saying environmental costs are costs incurred for poor en- vironmental quality or poor environmental quality may occur. In most cost accounting systems, envi- ronmental costs are hidden within overhead costs, making them difficult to assess and analyze. There- fore, management accounting information systems are needed for environmental costs. With improved environmental performance in an industry will pro- vide resource saving benefits so that it not only im- proves mood but potentially saves money buying resources and reducing waste or waste of business. According to Mahmud et al. (2023) that disclo- sure of environmental accounting information is an important topic of ecological accounting to report economic benefits and environmental sustainability of companies. The company's income statement should provide information related to ecological ex- penditures and relevant information in addition to financial information. Alassuli (2024) explain the objectives of environ- mental accounting, namely: 1. Environmental accounting aims to produce a sustainable business environment Stages of Environmental Accounting Treatment Due to the lack of uniform 47 APPLICATION OF ENVIRONMENTAL ACCOUNTING TO HIU PAUS TOURISM IN TOMINI BAY KANON, AMALIAH, & BADU regulations on the provision of environmental subsidies, costs may occur annual financial statements of different account names. The presentation of the Financial statements in section 14 states the purpose of the financing report must be met by the needs jointly reported by the user. related to the object of research produced by data from informants. The phenomenological approach aims to obtain a form of informant awareness of the problem under study. p y Basically, phenomenological research aims to analyze and understand data sourced from the ex- perience and awareness of informants to then be translated into forming a meaning from the head of the tourist awareness group, tour guides, members and visitors of the Hiu Paus tour in Tomini Bay. In this study, the stage of data analysis carried out is through identifying informant awareness of envi- ronmental management in Hiu Paus tourism in ac- cordance with Undang-Undang Republik Indonesia No. 32 (2009) on Environmental Management and Undang-Undang Republik Indonesia No. 40 (2007), social and environmental responsibility is an indus- trial responsibility that is budgeted and calculated as an industrial burden. In addition, an analysis of the application of environmental accounting was also carried out in accordance with PSAK No. 1 (Ikatan Akuntan Indonesia, 2015) concerning environmen- tal cost treatment. In conducting an analysis of the data obtained in this study, refer to the keywords contained in the phenomenological approach, namely noema and noesis. Noema and noesis on the application of environmental accounting in the Hiu Paus tourist attraction in Tomini bay. The noema lies in the perceived object as contained in the interview results and documents, while the noesis is a subjec- tive understanding contained in the informant in the activities carried out on the management of Hiu Paus tourism and visitor perceptions of Hiu Paus tourism management. p y 5. Disclosure Environmental accounting requires the allocation of special items in recording accounts on financial statements made by companies so that in financial accounting reporting it will appear that social responsibility carried out by companies is not as simple as rhetoric but is in accordance with the practical management of the remaining operational results of the company. RESEARCH METHODS This research is a qualitative research using a phenomenological approach. The phenomenologi- cal approach in this study refers to Husserl's tran- scendental phenomenology. Husserl's transcenden- tal phenomenology focuses on consciousness. Con- sciousness in question is the consciousness con- tained in the individual and is based on one's expe- rience (Saputro et al., 2020; Tassone, 2017). Kama- yanti (2016); Moleong (2018) revealing that the phe- nomenological approach refers to consciousness from one's perspective. This awareness contained in a person is what makes phenomenological research a unique study because phenomenological research is centered on the individual and how an individual understands a particular context based on the expe- rience they have. The object of this research is Hiu Paus Tourism in Botubarani Village located in Ka- bila Bone District, Bone Bolango Regency. The type of data in this study is in the form of primary data. The informant in this study was the Botubarani Hiu Paus tourism awareness group. Here's a table of re- search informants. Stages of Environmental Accounting Treatment 2. Environmental accounting is responsible for the social environment that aims to provide benefits to the company's organization, society and the environment at large Recording and managing everything related to waste management is preceded by a plan grouped based on certain tasks so that actual needs can be determined each year. In determining the alloca- tion of environmental costs on Pernyataan Standar Akuntansi Keuangan (PSAK) No. 1 (Ikatan Akuntan Indonesia, 2015) about Presentation of Fi- nancial Statements, the steps in allocating costs are as follows: According to Ikhsan (2009) suggests that the roles and functions of environmental accounting in a company or organization are divided into 2, namely as follows: y 1. Internal factors. The main factor affecting internal operations is the management of the enterprise. 1. Identification Based on the type of environmental costs according to Hansen & Mowen (2009) environmental costs are divided into four types, namely: (1) Environmental prevention costs, (2) Environmental detection costs, (3) Internal failure costs, (4) External failure costs. 2. External factors. The external function is a function that is closely related to stakeholders who use financial statements, the main focus of this function is the environmental cost report in financial reporting. 2. 2. Confession. After identification of the negative impact of an activity on finances, the component is recognized as profit and a certain amount in the account or expense account. The form of implementation of industrial social responsibility is called industrial social responsibil- ity, which is carried out by the company with the aim of restoring and balancing what the industry has done so that it has a negative impact on the environ- ment and society. By paying more attention to the surrounding environment, companies can partici- pate in environmental protection to achieve a main- tained quality of life in the future. The concept of corporate responsibility is closely related to the sus- tainable development of the company. Corporate sustainability is realized when the company is not only responsible to shareholders, but the company must also pay attention to the social and 3. Measurement Measurement (measuring) is the giving of numbers or units of measure on an object to indicate a certain meaning dari benda tersebut. 4. Presentation Expenses incurred during the year are generally environmental management and management and other similar creature expenses. Botubarani Whale Shark Tourism Operational Activities Regarding the operational activities of Botuba- rani Hiu Paus Tourism that is currently taking place, information has been obtained through interviews. Researchers searched for information about Botuba- rani Hiu Paus Tourism activities. Informant Wahab Matoka stated: The results of the interview showed that Botu- barani Hiu Paus Tourism is the general rules regard- ing activities on Botubarani Hiu Paus Tourism. This is characterized by aspects of security, safety, service and environmental protection have been carried out. “Berinteraksi dengan hiu paus melibatkan be- berapa regulasi tertentu untuk memastikan keselamatan. Regulasi-regulasi yang kami terapkan dalam berinteraksi dengan hiu paus selalu diumumkan melalui pengeras suara. Kami menyampaikan aturan-aturan tersebut kepada pengunjung, baik yang berpartisipasi dalam snorkeling maupun diving. Hingga ta- hun 2023, kami terus memantau keamanan para pengunjung dengan cermat. Sebelum terlibat dalam kegiatan tersebut, setiap pengunjung diberikan edukasi. Hal serupa juga dilakukan bagi mereka yang berinteraksi dengan hiu paus melalui perahu-perahu di sini. Kami tidak hanya memberikan edukasi secara langsung, tetapi juga melalui pengeras suara, guna memastikan keamanan pengunjung dengan cara yang konsisten.” (Wahab) RESULTS AND DISCUSSION Increasing public awareness of environmental problems has led accounting to experience a para- digm shift from conventional accounting to environ- mental accounting. Natalia (2022) revealing that the paradigm shift in environmental accounting is in- tended to translate organizational actions in safe- guarding the environment in various aspects of business organizations. Similarly, Hiu Paus Tourism in Tomini Bay Area, Botubarani Village, Kabila Bone District, Gorontalo Province, in the process of its ac- tivities in addition to generating profits, also leaves an impact on the environment from tourism activi- ties carried out. Therefore, in this section will be de- scribed the analysis of environmental management on Hiu Paus tourism in Tomini Bay in phenomeno- logical rules. Table 1. Research Informants No Name Position 1 Wahab Matoka Group Leader Sadar Wisata 2 Mimin Badu Tour Guide 3 Teti Ismail Member 4 Lina Visitors Data collection techniques in this study through observation, interviews and documentation. Quali- tative research makes researchers play a key instru- ment, because only researchers have knowledge 48 JURNAL AKUNTANSI KONTEMPORER (JAKO) – VOL 16 ISSUE 1 – JANUARY 2024 – Page 45-54 Waste Analysis of Botubarani Hiu Paus Tourism Activities In addition to operational activities on Botuba- rani Hiu Paus Tourism, researchers also obtained in- formation about the waste of Botubarani Hiu Paus Tourism activities. Researchers searched for infor- mation about waste Hiu Paus Tourism activities. In- formant Wahab Matoka stated: “Disini, kami banyak menerima sampah teru- tama dari Kota. Saat arus membawa sampah dari Kota, baik yang berada di atas air maupun di bawah air, sampah tersebut akhirnya men- capai daratan di lokasi kami. Ini menjadi ken- dala utama karena kelompok kami kesulitan mengatasi sampah. Kami menyampaikan bahwa sampah yang kami tangani ada juga yang berasal dari pengunjung” (Wahab) Wahab Matoka's statement shows a noema that regulation and education are a way to effectively serve and keep visitors safe. This will make tourism activities run effectively. The statement also shows noesis that safety, comfort, preservation of natural re- sources and the safety of visitors are the main things that must be considered by the management of Botu- barani Hiu Paus Tourism. Teti Related to tourist waste explained by Mrs. Teti Ismail: and fauna in the region. Botubarani Hiu Paus Tourism Waste Manage- ment Regarding waste management in the Hiu Paus Tourism environment, Wahab Matoka stated: “Limbah di air juga sangat banyak, terutama kantong plastik yang terapung di atas per- mukaan air. Meskipun demikian, saya tetap memberikan edukasi kepada pemilik perahu yang melayani pengunjung, untuk tolong memungut sampah yang ada di atas air. Kami berharap agar mereka dapat mengambil ini- siatif untuk mengumpulkan sampah tersebut, menaruhnya di perahu masing-masing, dan di sini sudah disediakan tempat sampah khusus. Jadi, kami mengajak untuk membuang sam- pah di tempat sampah yang telah disediakan.” (Wahab) Related to environmental conservation efforts, the following are presented the results of an inter- view with the Chairman of the pokdarwis. ”Sudah ada spot-spot yang bisa dinikmati oleh pengunjung yang akan melakukan diving. usaha dari BPSPL Makassar Pada Tahun 2017 Itu mereka membuat 1 eee bagaimana caranya supaya karang-karang ini ia bisa tumbuh dan berkembang dia. Karena menurut penelitian mereka karang-karang yang ada di sini ini sudah mulai berkurang. nah mereka membuat 1 pola, bagaimana caranya untuk menghidupkan ee karang-karang ini. Nah sekarang di tempat sana dibawah itu , dibawah dari perahu yang merah ini, itu ada disitu ke- bun dari ee apa ee karang-karang yang mereka pasang. Sekarang sudah mulai, karang-ka- rangnya sudah mulai ada. Itu, itu usaha dari mereka, dan kemudian juga ee banyak bantuan loh dari BPSPL Makassar.”(Wahab) Waste management on Botubarani Hiu Paus Tourism was also explained by Mrs. Mimin Badu, namely" “Pengelolaan limbah di sini melibatkan be- berapa upaya yang dilakukan untuk menjaga citra wisata ini. Anggota kelompok sadar wisata di sini memanfaatkan limbah untuk membuat souvenir yang kemudian dijual kepada wisatawan. Souvenir-souvenir ini dipajang di dekat pintu masuk sebagai salah satu upaya yang kami lakukan dan juga menambah pemasukan.” (Mimin) “Pengelolaan limbah di sini melibatkan be- berapa upaya yang dilakukan untuk menjaga citra wisata ini. Anggota kelompok sadar wisata di sini memanfaatkan limbah untuk membuat souvenir yang kemudian dijual kepada wisatawan. Souvenir-souvenir ini dipajang di dekat pintu masuk sebagai salah satu upaya yang kami lakukan dan juga menambah pemasukan.” (Mimin) The results of an interview with Mimin, a tour guide, revealed about the waste problem which is basically no longer a problem. The problem of waste, both shipment waste and tourist waste can be over- come because of the form of mutual assistance from tourism managers and local communities. Related to tourist waste explained by Mrs. Teti Ismail: “Kalau limbah di sini kebanyakan sampah plastik dan sisa kayu di tengah laut, tentu ini jadi masalah bagi kami. Apalagi limbah terse- but bukan berasal dari masyarakat sini, melainkan limbah yang terbawa arus dari Ta- lumolo. Meskipun demikian kami berusaha untuk menanggulanginya kami mem- bersihkan setiap hari, ini merupakan hal yang harus kami perhatikan demi kebaikan wisata ini dan bagaimana kedepannya agar wisata ini lebih baik lagi.” (Teti) This statement is also supported by the inter- view of tour guide mimin badu as follows. “Operasional Wisata Hiu Paus melibatkan partisipasi pengunjung dalam beberapa aspek. Persiapannya mencakup kegiatan seperti per- siapan sebelum turun ke air, dan keamanan pengunjung menjadi prioritas utama. Ke- lompok sadar wisata, termasuk pemandu wisata, memberikan pelayanan kepada pengunjung dengan mengikuti aturan dan prosedur keselamatan. Dalam kegiatan ber- interaksi dengan hiu, terdapat aturan umum yang harus diperhatikan, dan pemandu wisata memiliki peran penting dalam menjelaskan aturan tersebut kepada pengunjung, terutama mereka yang berasal dari luar negeri. Selain itu, upaya pelestarian juga dilakukan oleh ke- lompok ini, dengan berbagai inisiatif untuk Based on data obtained in interviews show that Botubarani Hiu Paus Tourism has problems related to the waste produced, which can have a negative impact on the sustainability of the tourism environ- ment. During the process of tourism activities, waste comes from various types of waste, both from tour- ists and local communities in Gorontalo City which are often carried away by sea currents and eventu- ally dragged on Botubarani beach. The impact of the presence of this waste is not only felt on land, but also affects the underwater ecosystem and the flora 49 APPLICATION OF ENVIRONMENTAL ACCOUNTING TO HIU PAUS TOURISM IN TOMINI BAY KANON, AMALIAH, & BADU APPLICATION OF ENVIRONMENTAL ACCOUNTING TO HIU PAUS TOURISM IN TOMINI BAY KANON, AMALIAH, & BADU Based on the results of observations that re- searchers made as far as the eye can see the tourist environment of Hiu Paus in Tomini Bay, it gives the impression that tour managers are very concerned in maintaining the cleanliness of the tourist environ- ment. What was revealed by the informant also ex- plained that the beauty of Hiu Paus tourism is a spe- cial attraction so that it is not only once but can be visited many times because of the beauty of the tour- ist destinations offered. Environmental cleanliness is one of the uniqueness of this Hiu Paus tour. The maintained natural environment makes the distinc- tive value possessed by Hiu Paus tourism in Tomini Bay. and fauna in the region. and fauna in the region. and fauna in the region. In envi- ronmental management, the value of local wisdom, mutual assistance, is an important point in realizing a clean and comfortable environment. This is also re- inforced by the statement of one of the visitors to the Hiu Paus tour, the following: Based on interviews from the Hiu Paus tourism awareness group in Tomini Bay, explained that in the management of the tourist environment, espe- cially in the management of waste generated by Hiu Paus tourism activities, pokdarwis strive to make environmental conservation efforts so that environ- mental cleanliness and sustainability are main- tained. In this case, the activities carried out by the tourism awareness group are cleaning around the beach which is carried out in a mutual cooperation, preservation care of underwater areas, underwater environmental care of Hiu Paus tourism. In this case, the tourism awareness group collaborates with BPSPL Makassar to maintain and make an effort so that the preservation of the underwater environ- ment is also maintained. This is done by making pat- terns so that coral reefs do not die, so that they can be enjoyed by tourist visitors who do diving. “Selama ini saya sering ke sini dengan te- man-teman. Kami sering diving menikmati keindahan panorama alam yang ada di bawah laut tidak hanya bertemu dengan ikan-ikan yang warna warni juga terumbu karang dan tanaman laut yang bikin tambah semangat untuk menjelajahi panorama indahnya cip- taan Tuhan.” (Lina) 50 50 JURNAL AKUNTANSI KONTEMPORER (JAKO) – VOL 16 ISSUE 1 – JANUARY 2024 – Page 45-54 tourist site and the preservation of the underwater environment. According to the analysis of environ- mental costs on Hiu Paus tourism on waste gener- ated from tourist activities. This waste is organic and non-organic waste. To maintain the sustainability of tourist attractions in the underwater environment, tourism awareness groups collaborate with BPSPL Makassar to make patterns so that coral reefs in tour- ist objects continue to grow and maintenance of Hiu Paus, tourism awareness groups provide feed to Hiu Paus. Hiu Paus environmental cost identification is divided into several parts. Furthermore, in maintaining the cleanliness of the Hiu Paus tourist attraction, garbage carts are pro- vided as containers to clean tourist waste before be- ing thrown into the final landfill. Furthermore, in the management of community waste carried by water currents to the Whale Shark tourist attraction, it is used by pokdarwis to be used as a soevenir. Environmental Cost Recognition of Botubarani Hiu Paus Tourism The recognition of environmental costs on Hiu Paus tourism has not been recognized in its entirety in the Financial statements, this is evidenced by the financial report of Hiu Paus tourism in the last 2 years. Based on data in the Hiu Paus tourism finan- cial report, it is explained that environmental costs have not been covered in their entirety in the tour- ism financial statements. And there has been no spe- cific reporting on environmental costs on Hiu Paus Tourism in Tomini Bay. and fauna in the region. Based on an interview with the treas- urer of the tourism awareness group dated July 22, 2023: “Untuk limbah disinikan hanya sampah-sam- pah ada kami manfaatkan untuk dibuat souve- nir, jadi ini sampah di daur ulang menjadi ke- rajinan, seperti souvenir. Eee bahannya itu hanya dari sampah-sampah kemudian kami daur ulang menjadi souvenir yang hiu paus ini terbuat dari sampah kayu yang anyor dari sungai, baru dpe sirip itu dari sampah-sampah plastik biasanya, bukan hanya itu saja ada juga beberapa souvenir laun seperti gelang dan kalung. Untuk gelang dan kalung ini juga eee kami pakai bahan-bahannya itu dari sam- pah-sampah plastik. Dan yang juga torang pa- kai ee karang macam untuk depe mata kalung. Biasa yang buat itu orang-orang di sini.” (Dharma) Environmental Cost Measurement of Botubarani Hiu Paus Tourism Furthermore, as previously revealed that Hiu Paus tourism is the only tourist attraction with Spe- cial Interest character owned by Gorontalo Province located in the Tomini Bay Area. The character of spe- cial interest tourism makes the management of the natural environment inseparable from the corridor of safety signs that must be considered and facili- tated by tour managers. Safety is not only the safety of visitors who will interact with Hiu Paus but also the safety of the Hiu Paus itself. Related to the safety of Hiu Paus and tour party visitors Tour managers in this case the Tourism Aware Group have imple- mented the rules. Not only that, Hiu Paus tours have also prepared tour guides for visitors so that safety is maintained and also provides snorkeling and div- ing equipment for tourist visitors. Hiu Paus Tourism in Tomini Bay in measuring the amount and value used for environmental costs in environmental management uses a monetary unit of costs incurred based on the sum of the amount of environmental management of Hiu Paus tourism. Hiu Paus tourist attraction also does not have its own budget in waste management from the results of tourist attraction activities. Presentation of Environmental Costs of Botubarani Hiu Paus Tourism Presentation related to a financial information will be presented in the financial statements. The costs incurred in terms of environmental cost man- agement for the management of environmental costs on Hiu Paus tourism are presented on simple finan- cial statements that are presented monthly and re- ported quarterly. Application of Environmental Accounting to Hiu Paus Attractions Based on data obtained by researchers in the field, it shows that environmental costs incurred di- rectly by Botubarani Hiu Paus tourism are presented in the general financial statements of tourism, and included in credits. In the tourism financial state- ments there are several environmental costs that are internalized and budgeted as a whole in the finan- cial statements. Disclosure of Botubarani Hiu Paus Tourism Environmental Costs Paus attraction's financial statements, but environ- mental exaggerations have not been presented in their entirety. In Hiu Paus tourism there is also no report explaining information about the responsibil- ity of tourist attractions to the environment, this is evidenced by the absence of specific reports on envi- ronmental costs. In environmental management, Hiu Paus Tourism has carried out responsibility for the environment in accordance with applicable reg- ulations, but in the presentation of financial state- ments of tourist attractions have not been presented in accordance with PSAK No. 1 (Ikatan Akuntan In- donesia, 2015) concerning the presentation of finan- cial statements. Disclosures related to financial information or accounting policies on Botubarani Hiu Paus Tour- ism have not been disclosed. According to the re- sults of research conducted by researchers at Botu- barani Hiu Paus Tourism. Environmental costs in- curred related to environmental management are treated as environmental costs. Based on the tourism financial statements, the financial statements only include the cost of cleaning wages. Based on the re- sults of the study, it is explained that there are sev- eral environmental costs that should be incurred and budgeted have not been covered as a whole. Based on research Sukirman & Suciati, (2019) explained that the application of environmental accounting is explained that in the application of good environ- mental accounting evidenced by the results of iden- tification, recognition, measurement, presentation and disclosure that have been made. In the identifi- cation process there are environmental and social costs. Recognition in environmental costs is car- ried out by the method of accrual recognition basis. Cost measurement uses monetary units based on costs that have been incurred. The presentation of costs regarding environmental management has been presented with similar units. Disclosure of in- formation on environmental management costs has been reported. Based on existing data in the field, it is explained that the Botubarani whale shark tourist attraction has not applied environmental accounting in terms of reporting This research provides important implications for tourism objects with special interest characteristics to prepare financial statements in accordance with PSAK No. 1 (Ikatan Akuntan Indonesia, 2015) concerning the presentation of financial statements. This is because so far there have been no accounting standards that regulate the reporting of environmental costs for tourism objects of special interest character. Disclosure of Botubarani Hiu Paus Tourism Environmental Costs However, this study has limitations, namely that the design of a more structured and comprehensive environmental accounting system has not been presented. Future research is expected to design and implement a more structured and comprehensive environmental accounting system in Botubarani Whale Shark Tourism. 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https://openalex.org/W4306874534	https://chemrxiv.org/engage/api-gateway/chemrxiv/assets/orp/resource/item/63511a8adfbd2bb7d45e41be/original/dual-nhc-photoredox-catalytic-synthesis-of-1-4-diketones-using-an-mr-tadf-photocatalyst-di-k-ta.pdf	English	null	Dual NHC/Photoredox Catalytic Synthesis of 1,4-Diketones Using an MR-TADF Photocatalyst (DiKTa)	null	2,022	cc-by	4,369	Abstract The use of the recently reported organic multi-resonant thermally activated delayed fluorescence (MR-TADF) photocatalyst DiKTa allows for the modular synthesis of 1,4- diketones under mild and metal-free conditions. The reaction proceeds via a three- component relay process in the presence of an N-heterocyclic carbene (NHC) organocatalyst. Callum Prentice,[a],[b] James Morrison,[c] Andrew D. Smith,[b]* and Eli Zysman-Colman[a]* Callum Prentice,[a],[b] James Morrison,[c] Andrew D. Smith,[b]* and Eli Zysman-Colman[a]* aOrganic Semiconductor Centre, EaStCHEM School of Chemistry, University of St Andrews, St Andrews, Fife, U.K., KY16 9ST. E-mail: eli.zysman-colman@st-andrews.ac.uk; URL: http://www.zysman-colman.com bEaStCHEM, School of Chemistry, University of St Andrews, St Andrews, Fife, U.K., KY16 9ST E-mail: http://www.ads-group.squarespace.com URL htt // d Zysman Colman aOrganic Semiconductor Centre, EaStCHEM School of Chemistry, University of St Andrews, St Andrews, Fife, U.K., KY16 9ST. E-mail: eli.zysman-colman@st-andrews.ac.uk; URL: http://www.zysman-colman.com bEaStCHEM, School of Chemistry, University of St Andrews, St Andrews, Fife, U.K., KY16 9ST E-mail: http://www.ads-group.squarespace.com URL: http://www.ads-group.squarespace.com cPharmaceutical Sciences, IMED, Biotech Unit, AstraZeneca, Macclesfield SK102NA (U.K.) aOrganic Semiconductor Centre, EaStCHEM School of Chemistry, University of St Andrews, St Andrews, Fife, U.K., KY16 9ST. E-mail: eli.zysman-colman@st-andrews.ac.uk; URL: http://www.zysman-colman.com bEaStCHEM, School of Chemistry, University of St Andrews, St Andrews, Fife, U.K., KY16 9ST E-mail: http://www.ads-group.squarespace.com aOrganic Semiconductor Centre, EaStCHEM School of Chemistry, University of St Andrews, St Andrews, Fife, U.K., KY16 9ST. E-mail: eli.zysman-colman@st-andrews.ac.uk; URL: http://www.zysman-colman.com bEaStCHEM, School of Chemistry, University of St Andrews, St Andrews, Fife, U.K., KY16 9ST E il h // d E-mail: http://www.ads-group.squarespace.com URL: http://www.ads-group.squarespace.com cPharmaceutical Sciences, IMED, Biotech Unit, AstraZeneca, Macclesfield SK102NA (U.K.) Dual NHC/Photoredox Catalytic Synthesis of 1,4-Diketones Using an MR-TADF Photocatalyst (DiKTa) Callum Prentice,[a],[b] James Morrison,[c] Andrew D. Smith,[b]* and Eli Zysman-Colman[a]* Introduction The development of synthetic methodologies to produce 1,4-diketones is a well-studied area of research,1–3 in large part due to their utility as precursors of various heterocycles such as furans,4 thiophenes,5 and pyrroles.6 While a number of disconnections are possible, perhaps the most commonly used synthetic strategy follows the umpolung approach of disconnecting the 1,4-diketone into a nucleophilic acyl group equivalent and an α,ß-unsaturated ketone, such as employed in the Stetter reaction.1 The Stetter reaction uses substoichiometric amounts of an N-heterocyclic carbene (NHC) in the presence of an aldehyde and an α,ß-unsaturated ketone to furnish the desired 1,4- diketones via a nucleophilic Breslow intermediate. Another possible disconnection would involve generating the 1,4-diketone from three distinct fragments. While such processes have been developed employing conventional two electron mechanisms,7,8 they are currently limited to the use of 1,3-diketones in combination with an appropriate α-ketoaldehyde. As a more general approach, the 1,4-diketone may be 1 1 disconnected into two acyl radical components and an alkene, which in the forward direction would involve a radical addition of one acyl group to the alkene, followed by a subsequent radical-radical coupling. This strategy has been executed successfully by the groups of Ackermann,9 Gilmour,10 Larionov,11 Li,12 and Wu.11 However, these examples are either limited to only symmetric 1,4-diketones or are restricted in terms of the scope of the acyl radical or alkenes available. The challenge of making unsymmetric 1,4-diketones using this strategy is significant, as the two acyl radicals must offer distinct reactivity to avoid forming a mixture of symmetric and unsymmetric products. Acyl radical generation and subsequent addition to alkenes using photoredox catalysis from commercially available, or simple to prepare, α-ketoacids, is well- known.10–12 Application to achieve selective formation of the desired unsymmetric 1,4- diketones requires another acyl radical, or equivalent, that must be generated simultaneously but itself will not react with the alkene. In a recent report Wang and Ackermann accomplished this transformation selectively through the use of metallaphotoredox catalysis using a tungsten polyoxometalate photocatalyst and a nickel co-catalyst.9 As an alternative, we envisaged that an acyl azolium intermediate, generated from an NHC catalyst and an appropriate electrophile, could undergo reduction through single electron transfer (SET) to the corresponding NHC-stabilised ketyl radical,13–15 which could act as the second acyl radical required for this transformation. Introduction During the course of our investigations Feng et al.16 published the use of triphenylphosphine in combination with NHC/photoredox dual catalysis using an iridium(III) photocatalyst for the generation of acyl radicals from benzoic acids generated in situ. However, to achieve the challenging synthesis of unsymmetric Scheme 1. (A) Previous work using stoichiometric imidazolium species.18 (B) Present work using catalytic NHCs and DiKTa as the photocatalyst. A. Photocatalytic 1,4-Dicarbonyl Synthesis - Stoichiometric Imidazoliums (Feng) B. This work: Dual NHC/Photoredox Catalytic Synthesis of 1,4-Diones ● Modular 3-component 1,4-dione synthesis ● Catalytic in NHC ● Metal-free MR-TADF Ar2 F O R CO2H O Ar1 Ar2 Ar1 O Ar2 O Ar1 R O O R Ar1 OH O Ar3 O Ar2 [Ir], PPh3 Ar3 O O Ar1 N N Me Me OTf Ar2 R O N N Me Me Ar2 R O (cat) ! ! N N N Ar MR-TADF DiKTa N N N Ar Scheme 1. (A) Previous work using stoichiometric imidazolium species.18 (B) Present work using catalytic NHCs and DiKTa as the photocatalyst. 2 2 1,4-diketones, the use of stoichiometric acyl azoliums was required (Scheme 1A). Building upon this work, we considered that using an α-keto acid in combination with the organic multi-resonant thermally activated delayed fluorescent (MR-TADF) photocatalyst DiKTa, recently reported by us,19 would allow for the preparation of unsymmetric 1,4-diketones in a modular, metal-free, three-component relay reaction using NHC/photoredox dual catalysis (Scheme 1B). During the preparation of this manuscript Zhang et al.20 published a similar study that relied upon the use of an Ir-based photocatalyst. Results and Discussion Initial exploration used the reaction of commercially available benzoyl fluoride 1, styrene 2 and phenylglyoxylic acid 3 (Table 1 and ESI). Using azolium salt precatalyst 4 in the presence of Cs2CO3 as a base and DiKTa, under photoexcitation using a 427 nm LED, the corresponding 1,4-dione 5 was isolated in good yield (entry 1). A range of alternative NHC catalysts such as 6 were tested but resulted in decreased product yield (entry 2 and ESI). The use of Cs2CO3 as the base was found to be decisive, with substitution by alternative inorganic bases leading to diminished product yield (see ESI). Various photocatalysts were also evaluated (see ESI), with for example [Ir(ppy)2(dtbbpy)](PF6) giving 5 in a slightly reduced yield (entry 3). The use of alternative solvents such as toluene and acetonitrile were found to be mildly detrimental to product yield (entries 4-5 and ESI). Control experiments verified the co-requirement of photocatalyst, NHC and light (entries 6-8). Observed side-products included a dimerization product of the initial radical addition intermediate, and an esterification product of benzoin (see ESI). 3 3 Table 1. Optimization of the NHC/photoredox catalysed 1,4-dione synthesis.a a. Conditions: 1 (0.40 mmol), 2 (0.10 mmol), 3 (0.15 mmol), DiKTa (2 mol%), azolium salt 4 (20 mol%), CH2Cl2 (0.10 M), rt, N2, LED (λexc = 427 nm), rt, 16 h. b 1H-NMR yield with 1,3,5-trimethoxybenzene as the internal standard. Isolated yield in parentheses. Cs2CO3 (2 equiv.) CH2Cl2 (0.1 M) LED (λexc = 427 nm) rt, 16 ha Ph F O Ph Ph Ph O Ph O OH O Ph O + 1 2 3 5 N O O N N N O Mes N N N Mes BF4 Cl azolium salt 4 (20 mol%) DiKTa (2 mol%) DiKTa 4 6 Entry Deviation from optimized conditions Yield (%)b 1 5 no deviation 61 (58) 3 5 6 7 0 8 azolium salt 6 instead of 4 42 2 No DiKTa 0 CH3CN instead of CH2Cl2 48 No light 4 Toluene instead of CH2Cl2 54 [Ir(ppy)2(dtbbpy)](PF6) instead of DiKTa No azolium salt 4 54 Table 1. Optimization of the NHC/photoredox catalysed 1,4-dione synthesis.a Table 1. Optimization of the NHC/photoredox catalysed 1,4-dione synthesis.a a. Conditions: 1 (0.40 mmol), 2 (0.10 mmol), 3 (0.15 mmol), DiKTa (2 mol%), azolium salt 4 (20 mol%), CH2Cl2 (0.10 M), rt, N2, LED (λexc = 427 nm), rt, 16 h. b 1H-NMR yield with 1,3,5-trimethoxybenzene as the internal standard. Isolated yield in parentheses. Cs2CO3 (2 equiv.) CH2Cl2 (0.1 M) LED (λexc = 427 nm) rt, 16 ha Ph F O Ph Ph Ph O Ph O OH O Ph O + 1 2 3 5 N O O N N N O Mes N N N Mes BF4 Cl azolium salt 4 (20 mol%) DiKTa (2 mol%) DiKTa 4 6 Entry Deviation from optimized conditions Yield (%)b 1 5 no deviation 61 (58) 3 5 6 7 0 8 azolium salt 6 instead of 4 42 2 No DiKTa 0 CH3CN instead of CH2Cl2 48 No light 4 Toluene instead of CH2Cl2 54 [Ir(ppy)2(dtbbpy)](PF6) instead of DiKTa No azolium salt 4 54 Ph F O Ph Ph O OH O + 1 2 3 N O O DiKTa N N N O Mes Cl 4 N Mes a. Conditions: 1 (0.40 mmol), 2 (0.10 mmol), 3 (0.15 mmol), DiKTa (2 mol%), azolium salt 4 (20 mol%), CH2Cl2 (0.10 M), rt, N2, LED (λexc = 427 nm), rt, 16 h. 3 b 1H-NMR yield with 1,3,5-trimethoxybenzene as the internal standard. Isolated yield in parentheses. With the optimized conditions in hand (Table 1, entry 1), the scope and limitations of this NHC/photoredox dual catalysed synthesis of 1,4-diketones was developed. Using benzoyl fluoride 1 and phenylglyoxylic acid 3 a variety of terminal alkene derivatives was explored in this process (Table 2). Incorporation of halogenated (para-Br, para-F) as well as electron-withdrawing (para-CF3) substituents within the styrene component were tolerated, giving the desired 1,4- diketones in 42-46% yield (7-9). Incorporation of the heteroaromatic 2-vinyl pyridine variant produced comparable yield of product (10). The incorporation of alkyl substituents at the para position were well tolerated (11-13), while ortho substitution (to give 14) or electron-donating (para-MeO) substituents delivered the 1,4-diketone (15) in lower yields. However, 15 could be obtained in an improved yield when using an Ir-based photocatalyst and changing reaction stoichiometries.20 Substrates that proved unsuccessful under the developed conditions included α- and ß-methyl styrene, 2-vinylthiophene, oct-1-ene and 4- vinylpyridine. 4 4 Table 2. Scope and limitations of the styrenes used in the synthesis of 1,4- diones.a Table 2. Scope and limitations of the styrenes used in the synthesis of 1,4 diones.a diones. a All reactions performed using 1 (0.40 mmol), 3 (0.15 mmol) and an alkene (0.10 mmol). b Conditions: 1 (0.40 mmol), alkene (0.20 mmol), 3 (0.40 mmol), [Ir(ppy)2(dtbbpy)](PF6) (1.5 mol%), azolium salt 4 (15 mol%), toluene (0.05 M), rt, N2, LED (λexc = 456 nm), rt, 16 h. Cs2CO3 (2 equiv.) CH2Cl2 (0.1 M) LED (λexc = 427 nm) rt, 16 ha Ph F O R Ph R O Ph O OH O Ph O + 1 3 azolium salt 4 (20 mol%) DiKTa (2 mol%) Ph O Ph O 5 58% Ph O Ph O Ph O Ph O Ph O Ph O Ph O Ph O Ph O Ph O Ph O Ph O Ph O Ph O Ph O Ph O Ph O Ph O 8 46% 15 26%, 58%b 9 42% 12 43% 7 45% 11 57% 13 41% 14 39% 10 55% N S Unsuccessful alkenes Br F CF3 N Me Me t-Bu Cl OMe Variation of styrenyl component 5, 7-15 a All reactions performed using 1 (0.40 mmol), 3 (0.15 mmol) and an alkene (0.10 mmol). 3 b Conditions: 1 (0.40 mmol), alkene (0.20 mmol), 3 (0.40 mmol), [Ir(ppy)2(dtbbpy)](PF6) (1.5 mol%), azolium salt 4 (15 mol%), toluene (0.05 M), rt, N2, LED (λexc = 456 nm), rt, 16 h. With the alkene scope established, attention turned to variation of the benzoyl fluoride component and the synthesis of unsymmetric 1,4-diketones (Table 3). In each case ~5 % of the symmetrical 1,4-diketone product was also observed, likely formed through the same process as reported by Wu and co-workers.11 Trace amounts of the corresponding 1,2-dione, where effectively the styrene component has been excluded, were also detected. Larger π-systems such as biphenyl (16) and 2-naphthyl (17) were well tolerated, although lower yields resulted from the use of 1-naphthyl (18), perhaps due to increased steric congestion. para-Fluoro- (19) and para-chloro- (20) substituted benzoyl fluorides worked well; however, para-bromo (21) and para-iodo (22) derivatives gave reduced product yield. However, 21 and 22 could be obtained in improved yields using alternative conditions with the Ir-based photocatalyst.20 Pleasingly, 5 electron-donating groups such as para-methoxy (23) and para-t-Bu (24) gave good yields of the corresponding unsymmetric 1,4-diketones. Unsuccessful substrates for the benzoyl fluoride component included para-cyanobenzoyl fluoride, cyclohexane carbonyl fluoride and 2-furanoyl fluoride. Table 3. Scope of the Benzoyl fluorides used in the synthesis of 1,4-diones.a a All reactions performed using 2 (0.1 mmol), 3 (0.15 mmol) and a benzoyl fluoride (0.40 mmol). b Conditions: 1 (0.60 mmol), alkene (0.20 mmol), 3 (0.60 mmol), [Ir(ppy)2(dtbbpy)](PF6) (1.5 mol%), azolium salt 4 (15 mol%), toluene (0.05 M), rt, N2, LED (λexc = 456 nm), rt, 16 h. O Ph O 5 58% O Ph O O Ph O O Ph O O Ph O O Ph O O Ph O O Ph O O Ph O O Ph O 17 41% 22 20%, 50%b 18 27% 19 51% 16 53% 23 57% 20 49% 21 25%, 39%b 24 36% O F CN O F O O F Ph F Cl Br I MeO t-Bu Ph Ph Ph Ph Ph Ph Ph Ph Ph Ph Cs2CO3 (2 equiv.) CH2Cl2 (0.1 M) LED (λexc = 427 nm) rt, 16 ha Ar F O Ph Ar Ph O Ph O OH O Ph O + 2 3 5, 16-24 azolium salt 4 (20 mol%) DiKTa (2 mol%) Unsuccessful fluorides Variation of aroyl fluoride component Table 3. 3 Scope of the Benzoyl fluorides used in the synthesis of 1,4-diones.a Ar Ph O Ph O 5, 16-24 2 3 17 41% O F CN O F O O F Unsuccessful fluorides a All reactions performed using 2 (0.1 mmol), 3 (0.15 mmol) and a benzoyl fluoride (0.40 mmol). b Conditions: 1 (0.60 mmol), alkene (0.20 mmol), 3 (0.60 mmol), [Ir(ppy)2(dtbbpy)](PF6) (1.5 mol%), azolium salt 4 (15 mol%), toluene (0.05 M), rt, N2, LED (λexc = 456 nm), rt, 16 h. Given these promising results in terms of the breadth of both the alkene and benzoyl fluoride components, the scope of possible α-ketoacids was evaluated next. Surprisingly, even seemingly small changes to the structure of the α-ketoacid such as the addition of a para-methyl substituent gave significantly lower yields of the corresponding 1,4-dione (25). This also proved to be the case for other substituents such as para-bromo, para-methoxy, para-fluoro and para-trifluoromethyl (26-29). However, using the alternative Ir photocatalyst-based catalytic conditions the yields for each of these substrates could be improved. Pleasingly, the use of pyruvic acid under Given these promising results in terms of the breadth of both the alkene and benzoyl fluoride components, the scope of possible α-ketoacids was evaluated next. Surprisingly, even seemingly small changes to the structure of the α-ketoacid such as the addition of a para-methyl substituent gave significantly lower yields of the corresponding 1,4-dione (25). This also proved to be the case for other substituents such as para-bromo, para-methoxy, para-fluoro and para-trifluoromethyl (26-29). However, using the alternative Ir photocatalyst-based catalytic conditions the yields for each of these substrates could be improved. Pleasingly, the use of pyruvic acid under 6 6 standard conditions gave the corresponding 1,4-diketone (30) while the use of alkyl α- keto acids could be extended to the cyclopentyl variant with the use of 2-vinyl pyridine to give the corresponding 1,4-diketone (31). Table 4. Scope of the α-keto acids used in the synthesis of 1,4-diones.a a All reactions performed using 1 (0.4 mmol), an α-keto acid (0.15 mmol) and an alkene (0.1 mmol). b Conditions: 1 (0.60 mmol), alkene (0.20 mmol), 3 (0.60 mmol), [Ir(ppy)2(dtbbpy)](PF6) (1.5 mol%), azolium salt 4 (15 mol%), toluene (0.05 M), rt, N2, LED (λexc = 456 nm), rt, 16 h. 3 Ph Ph O O Ph Ph O O Me Ph Ph O O Br Ph Ph O O OMe Ph Ph O O CF3 Ph Ph O Me O 5 58% 25 30%, 50%b 26 27%, 54%b 27 22%, 44%b 29 17%, 29%b 30 45% Ph O O N 31 40% Ph Ph O O F 28 22%, 71%b Cs2CO3 (2 equiv.) CH2Cl2 (0.1 M) LED (λexc = 427 nm) rt, 16 ha Ph F O Ar Ph Ar O R O OH O R O + 1 5, 25-31 azolium salt 4 (20 mol%) DiKTa (2 mol%) Variation of α-keto acid component Table 4. Scope of the α-keto acids used in the synthesis of 1,4-diones.a Cs2CO3 (2 equiv.) CH2Cl2 (0.1 M) LED (λexc = 427 nm) rt, 16 ha Ph F O Ar Ph Ar O R O OH O R O + 1 5, 25-31 azolium salt 4 (20 mol%) DiKTa (2 mol%) Variation of α-keto acid component Ph F O Ar R O OH O + 1 Variation of α-keto acid c Ph Ph O O Ph Ph O O Me Ph Ph O O Br 5 58% 25 30%, 50%b 26 27%, 54%b Variation of α keto acid component Ph Ph Ph O O OMe Ph Ph O O CF3 Ph Ph O Me O 27 22%, 44%b 29 17%, 29%b 30 45% Ph O O N 31 40% Ph Ph O O F 28 22%, 71%b a All reactions performed using 1 (0.4 mmol), an α-keto acid (0.15 mmol) and an alkene (0.1 mmol). b Conditions: 1 (0.60 mmol), alkene (0.20 mmol), 3 (0.60 mmol), [Ir(ppy)2(dtbbpy)](PF6) (1.5 mol%), azolium salt 4 (15 mol%), toluene (0.05 M), rt, N2, LED (λexc = 456 nm), rt, 16 h. A mechanism is proposed for the three-component transformation (Scheme 2A). Acylation of the in situ generated NHC by benzoyl fluoride [or by a bis(acyl) carbonate intermediate as proposed by Feng and co-workers16] leads to the acyl azolium ion pair intermediate I. Simultaneously the MR-TADF photocatalyst DiKTa (Ered(PC/PC•–) = 1.49 V vs SCE) is generated in its excited state by absorption. This oxidizes the α-keto acid via SET (Eox([PhCOCO2][/K]) = 1.03 vs SCE in DMSO),21 which undergoes decarboxylation to generate the key acyl radical intermediate. Addition of this acyl species to the least substituted terminus of the styrene generates the corresponding stabilised radical II. 3 The reduced photocatalyst is oxidized via single electron reduction of I to generate NHC-stabilised ketyl intermediate III, closing the photocatalytic cycle. Subsequent radical-radical coupling of II and III give intermediate IV. Release of the A mechanism is proposed for the three-component transformation (Scheme 2A). Acylation of the in situ generated NHC by benzoyl fluoride [or by a bis(acyl) carbonate intermediate as proposed by Feng and co-workers16] leads to the acyl azolium ion pair intermediate I. Simultaneously the MR-TADF photocatalyst DiKTa (Ered(PC/PC•–) = 1.49 V vs SCE) is generated in its excited state by absorption. This oxidizes the α-keto acid via SET (Eox([PhCOCO2][/K]) = 1.03 vs SCE in DMSO),21 which undergoes decarboxylation to generate the key acyl radical intermediate. Addition of this acyl species to the least substituted terminus of the styrene generates the corresponding stabilised radical II. The reduced photocatalyst is oxidized via single electron reduction of I to generate NHC-stabilised ketyl intermediate III, closing the photocatalytic cycle. Subsequent radical-radical coupling of II and III give intermediate IV. Release of the 7 7 NHC catalyst furnishes the desired 1,4-diketone product and completes the catalytic cycle. To support this mechanistic proposal, Stern-Volmer quenching studies of benzoyl fluoride 1, styrene 2 and phenylglyoxylic acid 3 were undertaken. These studies revealed that quenching occurs in the presence of 3 with a quenching rate constant, kq, of 3.8x109. However, fluorescence quenching was not observed in the presence of 1 or 2. While these investigations support the plausibility of the proposed mechanism, they do not rule out the possibility of an alternative oxidative quenching mechanism. Scheme 2. (A) Proposed mechanism of NHC/photoredox dual catalysed synthesis of 1,4- diones. (B) Stern-Volmer quenching studies. A. Proposed catalytic cycle R2 PC PC* PH2CH3 R1 F O R1 O R1 O O N N N Mes R N N N R Mes F N N N R Mes N N N R Mes R2 R1 O R2 R1 R2 R3 O SET R3 O – CO2, H+ R3 O R3 O Acylation SET Reduction NHC Turnover Radical Coupling R3 O OH O F O Ph O OH O 3 kq = 3.8x109 M-1s-1 2 1 I II III + ! B. Stern-Volmer quenching plots PC = DiKTa Scheme 2. (A) Proposed mechanism of NHC/photoredox dual catalysed synthesis of 1,4 diones. (B) Stern-Volmer quenching studies. Conclusions In summary, we have developed a modular synthetic route to unsymmetric 1,4-diketones through the combination of benzoyl fluorides, styrenes and α-keto 8 acids catalysed by a dual catalytic NHC/photoredox system using the recently developed MR-TADF organophotocatalyst DiKTa.22 During the development of this system Zhang and co-workers published a similar methodology requiring an iridium-containing photocatalyst.20 Certain substrates performed better under their conditions, although most 1,4-diones were obtained in comparable yields using the metal-free conditions described herein. Further work will probe alternative strategies to utilise the reactivity of DiKTa in other photocatalytic transformations. Acknowledgements The authors thank AstraZeneca and the University of St Andrews for funding (Case studentship to C.P.). References 1 M. M. Heravi, V. 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Smith, 2022, data underpinning: " Dual NHC/Photoredox Catalytic Synthesis of 1,4-Diketones Using an MR-TADF Photocatalyst (DiKTa)". 22 The research data supporting this publication can be accessed at: C. Prentice, J. Morrison, E. Zysman-Colman, A. D. Smith, 2022, data underpinning: " Dual NHC/Photoredox Catalytic Synthesis of 1,4-Diketones Using an MR-TADF Photocatalyst (DiKTa)". University of St Andrews Research Portal. https://doi.org/10.17630/88844362-7585-4c97-905e-2ad1f580b68f References University of St Andrews Research Portal. https://doi.org/10.17630/88844362-7585-4c97-905e-2ad1f580b68f 10
W4391056583.txt	https://www.medrxiv.org/content/medrxiv/early/2024/01/17/2024.01.17.24301405.full.pdf	en		Titin-Truncating variants Predispose to Dilated Cardiomyopathy in Diverse Populations	medRxiv (Cold Spring Harbor Laboratory)	2,024	cc-by	6,755	medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 TITLE: Titin-Truncating variants Predispose to Dilated Cardiomyopathy in Diverse Populations Authors: John DePaolo, MD, PhD,1 Marc Bornstein, AB,2 Renae Judy, MS,1 Sarah Abramowitz, BA,1 Shefali S. Verma, PhD,3 Michael G. Levin, MD,2,4 Zoltan Arany, MD, PhD,2* Scott M. Damrauer, MD,1,2,4,5* Affiliations: 1 Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 2 Cardiovascular Institute, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, PA 19104, USA. 3 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, PA 19104, USA. 4 Corporal Michael J. Crescenz VA Medical Center, Philadelphia, PA 19104, USA. 5 Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. * Jointly supervised this work Text Word Count: References: Tables: Figures: 2704 25 1 3 Address for correspondence: Scott Damrauer, MD Perelman School of Medicine Division of Vascular Surgery Hospital of the University of Pennsylvania 3400 Spruce Street, 14th Floor South Perelman Center Philadelphia, PA 19104 Office: 215-615-1698 scott.damrauer@pennmedicine.upenn.edu 36 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 2 37 Key Points 38 Question: Do high percentage spliced in (hiPSI) titin truncating variants (TTNtvs) confer 39 similar levels of risk for dilated cardiomyopathy (DCM) across diverse populations? 40 Findings: In a cohort study that comprised 43,731 individuals of diverse genetic 41 background with electronic health records linked to whole exome sequencing data, 42 hiPSI TTNtvs conferred increased risk of DCM across all individuals irrespective of 43 genetic background as measured by genetic distance from the 1000 Genomes Project 44 European centroid. 45 Meaning: The findings of this study suggest that TTNtvs increase risk of DCM among 46 individuals independent of genetic background and that genetic similarity to a reference 47 population should not play a role in screening for genetic causes of dilated 48 cardiomyopathy. 49 medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 3 50 Abstract 51 Importance: The effect of high percentage spliced in (hiPSI) TTN truncating variants 52 (TTNtvs) on risk of dilated cardiomyopathy (DCM) has historically been studied among 53 population subgroups defined by genetic similarity to European reference populations. 54 This has raised questions about the effect of TTNtvs in diverse populations, especially 55 among individuals genetically similar to African reference populations. 56 Objective: To determine the effect of TTNtvs on risk of DCM in diverse population as 57 measured by genetic distance (GD) in principal component (PC) space. 58 Design: Cohort study 59 Setting: Penn Medicine Biobank (PMBB) is a large, diverse biobank. 60 Participants: Participants were recruited from across the Penn Medicine healthcare 61 system and volunteered to have their electronic health records linked to biospecimen 62 data including DNA which has undergone whole exome sequencing. 63 Main Outcomes and Measures: Risk of DCM among individuals carrying a hiPSI 64 TTNtv. 65 Results: Carrying a hiPSI TTNtv was associated with DCM among PMBB participants 66 across a range of GD deciles from the 1000G European centroid; the effect estimates 67 ranged from odds ratio (OR) = 3.29 (95% confidence interval [CI] 1.26 to 8.56) to OR = 68 9.39 (95% CI 3.82 to 23.13). When individuals were assigned to population subgroups 69 based on genetic similarity to the 1000G reference populations, hiPSI TTNtvs conferred medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 4 70 significant risk of DCM among those genetically similar to the 1000G European 71 reference population (OR = 7.55, 95% CI 4.99 to 11.42, P<0.001) and individuals 72 genetically similar to the 1000G African reference population (OR 3.50, 95% CI 1.48 to 73 8.24, P=0.004). 74 Conclusions and Relevance: TTNtvs are associated with increased risk of DCM 75 among a diverse cohort. There is no significant difference in effect of TTNtvs on DCM 76 risk across deciles of GD from the 1000G European centroid, suggesting genetic 77 background should not be considered when screening individuals for titin-related DCM. 78 medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 5 79 80 Introduction Titin, the protein encoded by the TTN gene, is the largest protein in the human 81 body. It is found in the sarcomere where it spans from the Z-disk to the M-band, and is 82 critical for sarcomere assembly, contraction and relaxation in striated cardiac muscle.1 83 In populations largely composed of individuals genetically similar to the 1000 Genomes 84 Project (1000G) European reference population (EUR), heterozygous TTN truncating 85 variants (TTNtvs) that encode for shortened forms of the titin protein have been 86 identified as a common genetic cause of dilated cardiomyopathy (DCM). These TTNtv 87 have been associated with 25% of familial cases of DCM, and 10-20% of sporadic 88 cases.2-7 Only variants located in exons that are highly likely to be spliced into adult 89 cardiac TTN transcripts, known as high percentage spliced in (hiPSI) variants, are 90 pathogenic.3 hiPSI TTNtvs may cause DCM by reducing abundance of full-length TTN 91 protein (haploinsufficiency) and/or through dominant negative effects.8,9 92 We previously demonstrated the association of hiPSI TTNtvs with DCM 93 exclusively in EUR individuals, but were unable to detect an association between hiPSI 94 TTNtvs and DCM among Penn Medicine Biobank (PMBB) participants genetically 95 similar to the 1000G African reference population (AFR; odds ratio [OR] 1.8, 95% CI 0.2 96 to 13.7, P=0.57), or among AFR participants of the Jackson Heart Study.7 Recently, a 97 cross-sectional analysis of individuals with DCM demonstrated a statistically significant 98 but attenuated effect of predicted loss-of-function (pLOF) variants in TTN among AFR 99 individuals compared to EUR individuals, potentially underscoring the limitations of 100 applying genetic understanding of disease derived from a single ancestry group to 101 diverse populations.10 medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 6 102 Common and rare genetic risk factors for disease have historically been 103 analyzed among different populations separated by ancestry.11-14 This reflects the belief 104 that the artificial grouping of individuals into genetically common cohorts allows 105 improved assignment of genetic risk. However, greater understanding of genetic 106 similarities between groups of individuals combined with the results of genetic admixture 107 within different populations suggests that instead of strict dichotomization into groups, 108 genetic ancestry is better thought of as a continuum of relative similarity.12 109 Here we describe the assessment of DCM risk conferred by hiPSI TTNtvs in a 110 large, diverse biobank. Instead of using genetic similarity to group individuals together, 111 each participant’s genetic distance (GD) from the 1000G European centroid was 112 calculated and risk of DCM was assessed across a range of genetic distances. As a 113 sensitivity analysis, participants were assigned an “population group” according to their 114 genetic similarity to one of the 1000G continental-level reference populations and risk of 115 DCM was stratified by “genetic similar” group. Due to previous research identifying 116 TTNtvs risk factors for atrial fibrillation (Afib),15-17 we performed a similar investigation 117 into the link between TTNtvs and Afib. 118 119 Methods 120 Study Population 121 The Penn Medicine BioBank is a genomic and precision medicine cohort 122 comprising participants who receive care in the Penn Medicine health system and who 123 consent to linkage of electronic health records with biospecimens, including 43,731 with 124 DNA which has undergone whole exome sequencing (pmbb.med.upenn.edu). As medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 7 125 previously described,7 DCM was defined either as ≥2 outpatient or ≥1 inpatient 126 encounters with: 1) the International Classification of Diseases, 10th Revision (ICD10) 127 diagnosis code of I42.0; or 2) ICD10 codes I42.8 or I42.9 or the International 128 Classification of Diseases, Ninth Revision (ICD9) codes 425.4, 425.8, or 425.9, and 129 mention of “dilated cardiomyopathy” or “DCM” in free text encounter notes. Ischemic 130 cardiomyopathy (ICM) was defined as ≥2 outpatient or ≥1 inpatient encounters with 131 ICD10 codes I24 or I25, or ICD9 codes 411 or 414. Afib was defined as ≥2 outpatient or 132 ≥1 inpatient encounters with ICD10 codes I48, I48.1, I48.2, or I48.9, or ICD9 codes 133 427.2 or 427.21. A total of 9,020 individuals in PMBB had transthoracic 134 echocardiography (TTE) data available that included left ventricular ejection fraction 135 (LVEF). 136 Genetic Data 137 Whole exome sequencing was performed as previously described by Regeneron 138 Genomics Center.18 Individual patient DNA samples were processed and sequenced on 139 the Illumina NovaSeq 6000 (Albany, NY, USA). WeCall variant caller (v2.0.0) was 140 employed for sequence alignment (GRCh38), variant identification, and genotype 141 assignment. Quality control exclusions included sex errors, high rates of heterozygosity 142 (D-statistic > 0.4), low sequence coverage, and genetically identified sample duplicates. 143 Single nucleotide variants (SNVs) were filtered for a read depth 144 they either had at least one heterozygous variant genotype with an allele balance ratio 145 7 and were retained if 0.15, or a homozygous variant genotype.19 Insertion-deletion variants (INDELs) were 146 filtered for a read depth 147 balance 10 and either a heterozygous variant genotype with an allele 0.20, or a homozygous variant genotype. medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 8 148 TTN Truncating Variant Calls 149 TTNtvs were filtered for minor allele frequency < 0.001 and selected based on 150 predicted loss of function, truncating variants using ANNOVAR.20 Splice site variants 151 were screened for those affecting canonical donor or acceptor splice sites (two bases 152 flanking either exon). Variants were considered hiPSI if percent spliced in was greater 153 than 90% (PSI>0.9).3 154 Genetic Distance To consider differences in genetic ancestry along a continuous scale, genetic 155 156 distance (GD) from the 1000G European centroid was calculated as previously 157 decribed.14 Briefly, the 43,731 individuals in the PMBB were projected onto the 1000G21 158 principal component (PC) space. The GD was calculated by taking the Euclidean 159 distance for each individual from the 1000G European centroid using the equation 160 ∑ 161 European mean of the jth PC, and J is set to 10. GD was used to bin each individual in 162 PMBB into deciles from the closest 10% to the European centroid to the furthest 10% 163 from the European centroid. 164 Statistical Analysis ௃ୀଵ଴ ௜ ௝ୀଵ ௜௝ ா௝ ଶ where pcij is the jth PC of individual I, pcEj is the 1000G 165 Logistic regression was employed to evaluate the association of hiPSI TTNtvs 166 with prevalent DCM adjusting for age and sex. Linear regression was used to assess 167 the effect of hiPSI TTNtvs on minimum left ventricular ejection fraction (LVEF) adjusting 168 for age and sex. Statistical analyses were performed using R (version 4.2.0). 169 170 medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 9 171 Results 172 Study Population 173 There were 43,371 participants in the analytic cohort (Table 1). The median age 174 at analysis was 57 years (IQR: 45-69 years) and 21,907 (50%) were female. One 175 percent (436 individuals) carried a hiPSI TTNtv, 1,112 individuals (2.5%) had a DCM 176 diagnosis, and 4,920 individuals (11%) had an Afib diagnosis. There were 365 unique 177 TTNtvs identified including 174 stop-gains, 44 frameshifting insertion-deletions, and 147 178 essential splice site variants. 179 Calculating genetic distance 180 GD as the calculated Euclidean distance provides a method to assess the risk of 181 DCM conferred by TTNtvs across a genetically diverse population without relying on 182 artificial grouping based on genetic similarity to a reference panel. Euclidean distance is 183 the distance between two points in nth-dimensional space. The vast majority of 184 understanding of the genetic risk of DCM, including that conferred by TTNtvs, was 185 derived from investigation of cohorts primarily comprised of individuals genetically 186 similar to the 1000G European reference population. Integrating the genetic distance 187 from the 1000G European centroid into the analysis of DCM risk among TTNtv carriers 188 accounts for population differences between individuals and the cohort from which 189 genetic understanding of disease has been derived. Therefore, GD provides two useful 190 tools in the current analysis: 1) a method to treat genetic similarity as a continuum 191 rather than an artificially dichotomized value; and 2) a way to test if decreasing genetic 192 similarity indicates a change in TTNtv-derived DCM risk that might support previous 193 findings of differing effects among genetically dissimilar groups. medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 10 194 GD was calculated for each individual in PMBB and genetic diversity by GD was 195 compared to categorical assignment to population subgroups similar to the 1000G 196 reference populations based on PC analysis (Figure 1A-D). To demonstrate the extent 197 of genetic admixture and the arbitrary nature of grouping individuals genetically similar 198 to reference populations, PMBB individuals were compared to 1000G reference panel 199 individuals based on PC analysis (Figure 1E-G). Individuals were then binned by GD 200 decile; each decile included a range of individuals genetically similar to a 1000G 201 reference population (Supplemental Table 1) and PC analysis by GD decile 202 redemonstrated the extent of genetic admixture (Supplemental Figure 1). Together 203 these analyses characterize the genetic heterogeneity in the PMBB population. 204 205 206 Effect of TTNtvs on risk of DCM across genetic distances In the population overall, there is a 5.6-fold increased risk of DCM conferred by 207 carrying a TTNtv when adjusting for age, sex, and the first five genetic PCs 208 [Supplemental Table 2]. To determine the effect of TTNtvs on DCM risk at different 209 GDs from the European centroid, logistic regression analysis was performed among 210 PMBB individuals binned by GD decile adjusting for age and sex within each bin. 211 Among those individuals closest to the 1000G European centroid by GD, hiPSI TTNtvs 212 were associated with DCM (Decile 1 OR = 5.82, 95% CI 2.42 to 14.01, P<0.001) 213 [Figure 2A]; there was no significant effect decomposition as GD decile increased 214 (Decile 10 OR = 9.39, 95% CI 3.82 to 23.13, P<0.001). Meta-analysis of binned 215 results demonstrated an overall OR = 5.42 (95% CI 4.01 to 7.32, P<0.001); there 216 was no evidence of significant heterogeneity (I2=0%, χ2=4.59, P=0.87). This was medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 11 217 supported by a second analysis among all participants in which either GD from the 218 1000G European centroid alone nor the interaction between GD and hiPSI TTNtvs had 219 a statistically significant effect on the risk of DCM (Supplemental Table 2). Taken 220 together these results suggest that GD does not alter DCM risk. Similarly, there was no 221 evident trend of greater or lesser effect on minimum LVEF as GD increased (Figure 222 2B), though there was a trend towards increased heterogeneity (I2=42%, χ2=15.48, 223 P=0.08). We conclude that hiPSI TTNtvs associate with increased DCM risk and 224 reduced minimum LVEF across diverse populations. 225 226 227 Comparison to categorical assignment of genetic diversity We previously demonstrated a significant effect on DCM risk conferred by 228 carrying TTNtvs among individuals genetically similar to the 1000G EUR reference 229 population, however could not detect an effect among individuals genetically similar to 230 the 1000G AFR reference population.7 In this updated study, encompassing a 231 substantially larger number of participants, we evaluated the effect of TTNtvs among 232 individuals artificially separated into groups to compare both to our previous published 233 results and to the results above. Carrying a hiPSI TTNtv was associated with DCM 234 among individuals genetically similar to the 1000G EUR reference population, whether 235 including (Supplemental Figure 2) or excluding individuals with ischemic 236 cardiomyopathy [ICM] (odds ratio [OR] 7.55, 95% CI 4.99 to 11.42, P<0.001) [Figure 237 3A]. Similar, but attenuated, results were observed among individuals genetically similar 238 to the 1000G AFR reference population (OR 3.50, 95% CI 1.48 to 8.24, P=0.004). 239 Linear regression analysis demonstrated a consistent effect on minimum LVEF medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 12 240 conferred by carrying a TTNtv without attenuation in the AFR population group [Figure 241 3B]. We conclude that when individuals are artificially grouped by genetic similarity to a 242 reference population, hiPSI TTNtvs continue to associate with a diagnosis of DCM and 243 negatively associate with decreased minimum LVEF across diverse groups. 244 245 246 Effect of titin truncating variants on risk of atrial fibrillation To test the effect of TTNtvs on risk of Afib, another cardiovascular diagnosis 247 previously shown to associate with TTNtvs, we used logistic regression analysis by GD 248 and by genetically similar group. Among those individuals closest to the 1000G 249 European centroid by GD, hiPSI TTNtvs were associated with Afib (Decile 1 OR = 2.68, 250 95% CI 1.40 to 5.16, P=0.003) [Supplemental Figure 3A]. While there was increased 251 variability across deciles (deciles 2,4, and 5 had statistically insignificant associations 252 between hiPSI TTNtvs and Afib, I2=40%, χ2=14.96, P=0.09), there was no significant 253 trend in effect as GD from the 1000G European centroid increased (Decile 10 OR = 254 4.36, 95% CI 2.21 to 8.61, P<0.001). When PMBB individuals were grouped by genetic 255 similarity to a reference population, the effect estimate among individuals genetically 256 similar to the 1000G EUR reference population (OR = 2.11, 95% CI 1.62 to 2.77, 257 P<0.001) was similar to the effect estimate among individuals genetically similar to the 258 1000G AFR reference population (OR = 1.98, 95% CI 1.02 to 3.85, P=0.04) 259 [Supplemental Figure 3B]. We conclude that TTNtvs associate with increased risk of 260 Afib across diverse populations. 261 262 medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 13 263 264 Discussion Using continuous genetic distance as an alternative method to investigate the 265 effect of genetic similarity on rare variant cardiovascular disease risk, we demonstrated 266 that hiPSI TTNtvs associated with increased risk of DCM and reduced minimum LVEF 267 across diverse populations. These results were supported when individuals were 268 artificially dichotomized by genetic similarity to reference populations, although the 269 effect estimate among individuals genetically similar to the AFR reference population 270 may be attenuated. We also demonstrated a consistent though less robust effect of 271 hiPSI TTNtvs on risk of Afib across diverse populations. 272 Although genetic epidemiology studies have historically relied on arbitrary cutoffs 273 to differentiate and label populations of similar ancestry, genetic ancestry is more 274 appropriately considered as a continuum due to the degree of admixture even among 275 similar populations.22-24 The question of TTNtv effect among individuals genetically 276 similar to the 1000G AFR reference population provided an opportunity to reassess the 277 role ancestry plays in how we approach individual risk in cardiovascular disease. The 278 homogeneity of effect of TTNtvs across deciles of GD from the 1000G European 279 centroid removes barriers constructed by presenting results separated by artificially 280 constructed population subgroups, such as within group variation and small sample 281 size. Future evaluation of monogenic risk of cardiovascular disease may benefit from 282 utilizing a similar approach, which may be more broadly applicable than analyses using 283 genetically similar group dichotomization alone. 284 285 These results refine our previous findings that hiPSI TTNtvs are associated with DCM among individuals genetically similar to EUR reference populations in PMBB. medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 14 286 Previously, we reported an OR of 18.7 (95% CI 9.1 to 39.4) for DCM among EUR 287 individuals with hiPSI TTNtvs.7 The range of effect estimates in our analysis of risk by 288 GD from the 1000G European centroid was OR 3.3 (95% CI 1.3 to 8.6) to OR 9.4 (95% 289 CI 3.8 to 23.1), and the effect estimate for individuals genetically similar to EUR 290 reference panels was contained within that range (OR 7.6, 95% CI 5.0 to 11.4, 291 P<0.001). The attenuation of effect we observed between our initial publication and the 292 present study may reflect a winner’s curse in our initial study, where the initially-reported 293 effect size tends to be over-estimated in genetic association studies.25 However, the 294 makeup of PMBB has also changed substantially in the past five years such that it is 295 less enriched for cardiovascular disease (6.2% prevalence of DCM previously 296 compared to 2.5% now) due to increasingly broad regional enrollment from centers of 297 primary care which may have had a more meaningful impact on our current results. 298 Our findings also clarify the effect of hiPSI TTNtvs in individuals genetically 299 similar to the AFR reference population. We previously identified no statistically 300 significant effect among PMBB participants genetically similar to the AFR reference 301 population.7 This corroborated other evidence suggesting the possibility of differing 302 effects across diverse populations.3 Notably, our previous PMBB cohort included only 303 2,123 participants genetically similar to the AFR reference population, 20 of whom 304 carried a hiPSI TTNtv, representing a power of 55% to identify an OR of 3.3, suggesting 305 we were previously underpowered to identify an effect similar to what we presently 306 report. We now identify a significant effect of TTNtvs (OR = 3.5, 95% CI 1.5 to 8.2, 307 P=0.004) in this population. Moreover, when we consider the extent of admixture that 308 exists in this population, the dispersion of PMBB participants genetically similar to the medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 15 309 1000G AFR reference population across a range of GD from the 1000G European 310 centroid, and the similar effect estimates across those distances, these findings are 311 convincing that hiPSI TTNtvs confer increased DCM risk among individuals within these 312 GD deciles corresponding to those previously labeled genetically similar to the 1000G 313 AFR reference population. 314 315 316 Limitations This work has some limitations. Reliance on electronic health records to establish 317 a diagnosis of DCM may have misclassified a subset of individuals. Similarly, TTEs 318 were not interpreted by a uniform reader. Our cohort is also largely composed of EUR 319 and AFR individuals, limiting generalizability of the GD continuum concept to other 320 genetically similar groups. 321 322 323 Conclusion In summary, our updated analysis demonstrates a strong association between 324 hiPSI TTNtvs and DCM across a range of genetic backgrounds, including subjects 325 genetically similar to African reference population. These data suggest that 326 recommendations for genetic testing and counseling should not differ between diverse 327 populations. 328 329 Acknowledgements, Conflicts of Interest and Disclosures 330 We acknowledge the Penn Medicine BioBank (PMBB) for providing data and 331 thank the patient-participants of Penn Medicine who consented to participate in this medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 16 332 research program. We would also like to thank the Penn Medicine BioBank team and 333 Regeneron Genetics Center for providing genetic variant data for analysis. The PMBB is 334 approved under IRB protocol# 813913 and supported by Perelman School of Medicine 335 at University of Pennsylvania, a gift from the Smilow family, and the National Center for 336 Advancing Translational Sciences of the National Institutes of Health under CTSA 337 award number UL1TR001878. 338 M.G.L. receives research support to his institution from MyOme outside of this 339 work. S.M.D receives research support to his institution from RenalytixAI and in kind 340 support from Novo Nordisk, both outside of this work. 341 342 343 Sources of Funding J.D. is supported by the American Heart Association (23POST1011251). M.G.L. 344 received support from the Institute for Translational Medicine and Therapeutics of the 345 Perelman School of Medicine at the University of Pennsylvania, the NIH/NHLBI National 346 Research Service Award postdoctoral fellowship (T32HL007843), the Measey 347 Foundation, and the Doris Duke Foundation (2023-0224). Z.A. is supported by the 348 NIH/NHLBI (R01-HL152446) and the Department of Defense (W81XWH18-1-0503). 349 350 medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 17 351 References 352 353 1. Freiburg A, Trombitas K, Hell W, Cazorla O, Fougerousse F, Centner T, 354 Kolmerer B, Witt C, Beckmann JS, Gregorio CC, et al. Series of exon-skipping 355 events in the elastic spring region of titin as the structural basis for myofibrillar 356 elastic diversity. Circ Res. 2000;86:1114-1121. doi: 10.1161/01.res.86.11.1114 357 2. Herman DS, Lam L, Taylor MR, Wang L, Teekakirikul P, Christodoulou D, 358 Conner L, DePalma SR, McDonough B, Sparks E, et al. Truncations of titin 359 causing dilated cardiomyopathy. N Engl J Med. 2012;366:619-628. doi: 360 10.1056/NEJMoa1110186 361 3. Roberts AM, Ware JS, Herman DS, Schafer S, Baksi J, Bick AG, Buchan RJ, 362 Walsh R, John S, Wilkinson S, et al. Integrated allelic, transcriptional, and 363 phenomic dissection of the cardiac effects of titin truncations in health and 364 disease. Sci Transl Med. 2015;7:270ra276. doi: 10.1126/scitranslmed.3010134 365 4. 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The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 22 453 Tables and Figure Legends 454 Table 1: Clinical characteristics of individuals in the Penn Medicine Biobank with 455 and without high percentage spliced-in TTN truncating variants. Demographic hiPSI TTNtv (436) non-hiPSI TTNtv (43,295) P-value Difference Age, median (IQR) 56 (45-67) 57 (45-69) 0.65 Female Sex, N (%) 184 (42%) 21,723 (50%) <0.001 EUR 330 (75%) 29,626 (68%) 0.002 AFR 88 (20%) 11,048 (26%) 0.008 AMR 2 (0.4%) 564 (1.3%) 0.12 EAS 4 (0.9%) 466 (1.1%) 0.74 SAS 5 (1.1%) 554 (1.3%) 0.79 Unknown 7 (1.6%) 1,037 (2.4%) 0.28 Dilated Cardiomyopathy, N (%) 52 (12%) 1,060 (2.4%) <0.001 Ischemic Cardiomyopathy, N (%) 64 (15%) 5,305 (12%) 0.13 Atrial Fibrillation, N (%) 89 (20%) 4,831 (11%) <0.001 "Genetically Similar" Group, N (%) 456 457 medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 23 458 459 Figure 1: Principal component based clusters of individuals in the Penn Medicine 460 Biobank compared to genetic distance from the 1000 Genomes Project European 461 centroid and compared to continental-level clusters of 1000 Genomes Project 462 individuals demonstrating the degree of overlap of different genetically similar 463 groups. A-C: Discrete labelling of the position of each individual within the Penn 464 Medicine Biobank (PMBB) colored by genetically similar group based on 1000 465 Genomes Project (1000G) continental-level reference populations in a plot of (A) 466 principal component (PC) 1 versus PC2, (B) PC2 versus PC3, and (C) PC1 versus PC3 467 with 1000G European centroid (black dot) identified in each plot. (D) the genetic 468 distance from the 1000G European centroid of each individual within PMBB again 469 colored by genetically similar group based on 1000 Genomes Project (1000G) 470 continental-level reference populations. E-G: Discrete labelling of the position of each 471 1000G reference panel individuals colored by continental region with PMBB individuals medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 24 472 represented by grey dots in a plot of (E) PC1 versus PC2, (F) PC2 versus PC3, and (G) 473 PC1 versus PC3 with 1000G European centroid (red dot) identified in each plot. 474 medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 25 475 476 Figure 2: Effect of TTN truncating variants on risk of dilated cardiomyopathy and 477 on minimum left ventricular ejection fraction, stratified by genetic distance from 478 e the 1000 Genomes Project European centroid. (A) Logistic regression analysis of the 479 association between hiPSI TTNtvs on risk of DCM stratified by genetic distance (GD) 480 from the 1000 Genomes Project (1000G) European centroid. (B) Linear regression 481 analysis of the association between hiPSI TTNtvs and minimum left ventricular ejection 482 fraction stratified by GD from the 1000G European centroid. OR = odds ratio; CI = 483 confidence interval; EUR = European; LVEF = left ventricular ejection fraction. medRxiv preprint doi: https://doi.org/10.1101/2024.01.17.24301405; this version posted January 17, 2024. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license . 26 484 485 486 Figure 3: Effect of high percentage spliced in TTN truncating variant on risk of 487 dilated cardiomyopathy diagnosis and minimum left ventricular ejection fraction 488 reduction stratified by genetically similar group excluding those with ischemic 489 cardiomyopathy in the Penn Medicine Biobank. (A) Logistic regression analysis of 490 the association between hiPSI TTNtvs and DCM diagnosis, and (B) linear regression 491 analysis of the association between hiPSI TTNtvs and minimum left ventricular ejection 492 fraction among individuals genetically similar to the 1000 Genomes Project European 493 e and African reference population, and meta-analyzed. OR = odds ratio; CI = confidence 494 interval; EUR = individuals genetically similar to the European reference population; 495 AFR = individuals genetically similar to the African reference population.
https://openalex.org/W4386199384	https://zenodo.org/records/8286941/files/Research%20and%20Publication%20Output%20of%20City%20University.pdf	English	null	Research and publication output of Md. Anowar Hossain on Textile Engineering under affiliation of City University, Dhaka, Bangladesh, Year 2006-2010 (academic), 2017-2019 (professional)	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	3,071	Research and publication output of Md. Anowar Hossain on Textile Engineering under affiliation of City University, Dhaka, Bangladesh, Year 2006-2010 (academic), 2017-2019 (professional) Md. Anowar Hossain Department of Textile Engineering, City University, Savar, Dhaka, Bangladesh (engr. anowar@yahoo.com) Research and publication output of Md. Anowar Hossain on Textile Engineering under affiliation of City University, Dhaka, Bangladesh, Year 2006-2010 (academic), 2017-2019 ( f l) (professional) Md. Anowar Hossain Department of Textile Engineering, City University, Savar, Dhaka, Bangladesh (engr. anowar@yahoo.com) BSc Thesis and Internship report on Textile Engineering (Hossain, 2010b, 2010e), Publication on Textile Engineering (A. Hossain, A. S. Islam, et al., 2018; A. Hossain & A. K. Samanta, 2018; A. Hossain, A. K. Samanta, et al., 2018; A. Hossain et al., 2019; Hossain, 2009, 2010c, 2010d, 2019b, 2019c; M. A. Hossain & A. Samanta, 2018; M. A. Hossain et al., 2019; M. A. Hossain et al., 2018; Md. Anowar Hossain, 2019), Preprint(Hossain, 2019a, 2023a) (Hossain, 17 May 2023, 2010a, 2023a, 2023b, 2023c, 2023d, 2023e, 2023f, 2023g, 2023h, 2023i, 2023j, 2023k, 2023l, 2023m, 2023n, 2023o, 2023p, 2023q, 2023r, 2023s, 2023t, 2023u, 2023v, 2023w, 2023x, 2023y, 2023z, 2023aa) have been attached for contribution on Textile Engineering under affiliation of City University, Dhaka, Bangladesh , Year 2006-2010 (academic), 2017-2019 (professional). BSc Thesis and Internship report on Textile Engineering (Hossain, 2010b, 2010e), Publication on Textile Engineering (A. Hossain, A. S. Islam, et al., 2018; A. Hossain & A. K. Samanta, 2018; A. Hossain, A. K. Samanta, et al., 2018; A. Hossain et al., 2019; Hossain, 2009, 2010c, 2010d, 2019b, 2019c; M. A. Hossain & A. Samanta, 2018; M. A. Hossain et al., 2019; M. A. Hossain et al., 2018; Md. Anowar Hossain, 2019), Preprint(Hossain, 2019a, 2023a) (Hossain, 17 May 2023, 2010a, 2023a, 2023b, 2023c, 2023d, 2023e, 2023f, 2023g, 2023h, 2023i, 2023j, 2023k, 2023l, 2023m, 2023n, 2023o, 2023p, 2023q, 2023r, 2023s, 2023t, 2023u, 2023v, 2023w, 2023x, 2023y, 2023z, 2023aa) have been attached for contribution on Textile Engineering under affiliation of City University, Dhaka, Bangladesh , Year 2006-2010 (academic), 2017-2019 (professional). Hossain, A., Islam, A. S., & Samanta, A. K. (2018). Pollution Free Dyeing on Cotton Fabric Extracted from Swietenia macrophylla and Musa Acuminata as Unpolluted Dyes and Citrus. Limon (L.) as Unpolluted Mordanting Agent. Trends in Textile Engineering & Fashion Technology, 3(2), 1-8. https://crimsonpublishers.com/tteft/fulltext/TTEFT.000558.php Hossain, A., & Samanta, A. K. (2018). Cost Minimization in Sample Development and Approval Process by Proper Merchandising Action for kids and Ladies Garments. Trends in Textile Engineering and Fashion Technology (Online), USA. https://www.semanticscholar.org/paper/Cost-Minimisation- in-Sample-Development-and-Process-Hossain- Samanta/ee6de04748b507cdec19cb7af66cfde0870a5b0d; https://doi.org/10.31031/TTEFT.2018.04.000590 Hossain, A., Samanta, A. K., Bhaumik, N. S., Vankar, P. S., & Shukla, D. (2018). 1 \| P a g e Md. Anowar Hossain, “Unethical implementation of power is a global challenge, actually nobody should have power in terms of presidentship/professorship/directorship/chairmanship but everyone has ethical power to serve the society all over the world.” 2 \| P a g e Md. Anowar Hossain, “Unethical implementation of power is a global challenge, actually nobody should have power in terms of presidentship/professorship/directorship/chairmanship but everyone has ethical power to serve the society all over the world.” Research and publication output of Md. Anowar Hossain on Textile Engineering under affiliation of City University, Dhaka, Bangladesh, Year 2006-2010 (academic), 2017-2019 (professional) Md. Anowar Hossain Department of Textile Engineering, City University, Savar, Dhaka, Bangladesh (engr. anowar@yahoo.com) Organic Colouration and Antimicrobial Finishing of Organic Cotton Fabric by Exploiting Distillated Organic Extraction of Organic Tectona grandis and Azardirachta indica with Organic Mordanting Compare to Conventional Inorganic Mordants. International Journal of Textile Science and Engineering, 2018(1), 1-12. https://doi.org/10.5281/zenodo.8245102 Hossain, A., Samanta, A. K., Bhaumik, N. S., Vankar, P. S., & Shukla, D. (2018). Organic Colouration and Antimicrobial Finishing of Organic Cotton Fabric by Exploiting Distillated Organic Extraction of Organic Tectona grandis and Azardirachta indica with Organic Mordanting Compare to Conventional Inorganic Mordants. International Journal of Textile Science and Engineering, 2018(1), 1-12. https://doi.org/10.5281/zenodo.8245102 Hossain, A., Sun, D., & Samanta, A. (2019). Modern Technology versus Rapid Economical Growth in Smart Textiles Incorporated with Encapsulated Phase Change Materials Containing Latent Heat for Special Workers and Extreme Weather Conditions. JResLit Journal of Science and Technology. https://doi.org/10.13140/RG.2.2.15079.62880 Hossain, A., Sun, D., & Samanta, A. (2019). Modern Technology versus Rapid Economical Growth in Smart Textiles Incorporated with Encapsulated Phase Change Materials Containing Latent Heat for Special Workers and Extreme Weather Conditions. JResLit Journal of Science and Technology. https://doi.org/10.13140/RG.2.2.15079.62880 1 \| P a g e r in terms of Hossain, M. A. (17 May 2023). Anowar’s Handbook on Application of Computer in Textiles. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.23199.53923 Hossain, M. A. (17 May 2023). Anowar’s Handbook on Application of Computer in Textiles. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.23199.53923 Hossain, M. A. (2009). Basic Knowledge of Wet Processing Technology, ISBN-978-984-35-2885-8, Issued on 10 August 2022, Department of Archives and Library, Ministry of Cultural affairs, Government of The People’s Republic of Bangladesh (Vol. 1-4). Rupok Publications. https://doi.org/10.5281/zenodo.7844857; Hossain, M. A. (2009). Basic Knowledge of Wet Processing Technology, ISBN-978-984-35-2885-8, Issued on 10 August 2022, Department of Archives and Library, Ministry of Cultural affairs, Government of The People’s Republic of Bangladesh (Vol. 1-4). Rupok Publications. https://doi.org/10.5281/zenodo.7844857; Hossain, M. A. (2010a). Anowar’s Handbook on Textile Raw Materials II. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.29667.94247; https://doi.org/10.5281/zenodo.8241586 Hossain, M. A. (2010a). Anowar’s Handbook on Textile Raw Materials II. https://doi.org/10.5281/zenodo.8240631 Hossain, M. A. (17 May 2023). Anowar’s Handbook on Application of Computer in Textiles. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.23199.53923 3 \| P a g e Md. Anowar Hossain, “Unethical implementation of power is a global challenge, actually nobody should have power in terms of presidentship/professorship/directorship/chairmanship but everyone has ethical power to serve the society all over the world.” Research and publication output of Md. Anowar Hossain on Textile Engineering under affiliation of City University, Dhaka, Bangladesh, Year 2006-2010 (academic), 2017-2019 (professional) Md. Anowar Hossain Department of Textile Engineering, City University, Savar, Dhaka, Bangladesh (engr. anowar@yahoo.com) School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.29667.94247; https://doi.org/10.5281/zenodo.8241586 Hossain, M. A. (2010b). Engineering of Textile Coloration City University, Permanent Campus: Khagan, Birulia, Savar, Dhaka-1216, Bangladesh]. https://doi.org/10.5281/zenodo.7854575; http://dx.doi.org/10.13140/RG.2.2.11281.40801 Hossain, M. A. (2010c). Garments Technology for Merchandiser and Fashion Designer, ISBN- 978-984-35- 2883-4, Issued on 10 August 2022, Department of Archives and Library, Ministry of Cultural affairs, Government of The People’s Republic of Bangladesh. Rupok Publications, 140, Islamia market, Nilkhet, Dhaka, 2010. https://doi.org/10.5281/zenodo.7844933 Hossain, M. A. (2010d). Principle of garments production, ISBN-978-984-35-2884-1, Issued on 10 August 2022, Department of Archives and Library, Ministry of Cultural affairs, Government of The People’s Republic of Bangladesh. Rupok Publications. https://doi.org/10.5281/zenodo.7844918 Hossain, M. A. (2010e). Production monitoring techniques of a knit composite industry. https://doi.org/10.5281/zenodo.7854541 Hossain, M. A. (2019a). Cyclodextrin for Aroma Finishing on Textile Substrate-A Review Article. International Journal of Science and Engineering Investigations, 8(89). http://dx.doi.org/10.13140/RG.2.2.11724.18561; http://www.ijsei.com/archive-88919.htm; https://doi.org/10.5281/zenodo.8242756 Hossain, M. A. (2019a). Cyclodextrin for Aroma Finishing on Textile Substrate-A Review Article. International Journal of Science and Engineering Investigations, 8(89). http://dx.doi.org/10.13140/RG.2.2.11724.18561; http://www.ijsei.com/archive-88919.htm; https://doi.org/10.5281/zenodo.8242756 Hossain, M. A. (2019b). Uster analysis of cotton/polyester blended spun yarns with different counts. Journal of Textile Engineering & Fashion Technology, 5(4). https://doi.org/10.15406/jteft.2019.05.00204 Hossain, M. A. (2019c). Uster Imperfections of 35% Cotton and 65% Polyester Blended Yarn for 40Ne, 50Ne and 60Ne Ring Spun Yarn. South Asian Research Journal of Engineering and Technology, 1(2). https://www.sciencegate.app/document/10.36346/sarjet.2019.v01i02.002 Hossain, M. A. (2023a). Anowar's Handbook on Color Engineering for Textile Engineers (Part-2). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.29805.15844 Hossain, M. A. (2023b). Anowar’s Handbook International Trade and Marketing Management. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.25788.21120; Hossain, M. A. (2023b). Anowar’s Handbook International Trade and Marketing Management. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.25788.21120; Hossain, M. A. (2023c). Anowar’s Handbook of Electrical and Electronics Engineering. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. Research and publication output of Md. Anowar Hossain on Textile Engineering under affiliation of City University, Dhaka, Bangladesh, Year 2006-2010 (academic), 2017-2019 (professional) Md. Anowar Hossain Department of Textile Engineering, City University, Savar, Dhaka, Bangladesh (engr. anowar@yahoo.com) http://dx.doi.org/10.13140/RG.2.2.13624.72966; h //d / / d Hossain, M. A. (2023d). Anowar’s Handbook on Chemistry (Part-1). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.12923.49448 Hossain, M. A. (2023d). Anowar’s Handbook on Chemistry (Part-1). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.12923.49448 Hossain, M. A. (2023e). Anowar’s Handbook on Chemistry (Part-2). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.15879.16801 Hossain, M. A. (2023f). Anowar’s Handbook on Color Engineering for Textile Engineers (Part-01). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.19654.04160 Hossain, M. A. (2023g). Anowar’s Handbook on Color Engineering for Textile Engineers (Part-4). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.16822.88641 Hossain, M. A. (2023h). Anowar’s Handbook on Elements of Mechanical Engineering. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.25368.78089 Hossain, M. A. (2023i). Anowar’s Handbook on Elements of Theory of Machine and Machine Design. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.28724.22405 Hossain, M. A. (2023j). Anowar’s Handbook on Environment and Safety Management. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.36273.97126 Hossain, M. A. (2023k). Anowar’s Handbook on Fabric Structure and Design. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.30821.37606; https://doi.org/10.5281/zenodo.8240534 Hossain, M. A. (2023l). Anowar’s Handbook on Garments Manufacturing Technology. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.27465.93280; 4 \| P a g e Md. Anowar Hossain, “Unethical implementation of power is a global challenge, actually nobody should have power in terms of presidentship/professorship/directorship/chairmanship but everyone has ethical power to serve the society all over the world.” Engineering, Hossain, M. A. (2023n). Anowar’s Handbook on Machine Technology & Maintenance of Wet Processing Machineries. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.22432.76802; https://doi.org/10.5281/zenodo.8240660 Hossain, M. A. (2023o). Anowar’s Handbook on Materials Engineering and Practices. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.14883.02082 Hossain, M. A. (2023p). Anowar’s Handbook on Microprocessor, Robotics and Control Engineering. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.26627.07204 Hossain, M. A. (2023q). Anowar’s Handbook on Statistics for Engineer. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.17714.17602 Hossain, M. A. (2023r). Anowar’s Handbook on Technical Textiles. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.19391.89763; https://doi.org/10.5281/zenodo.8241043 Hossain, M. A. (2023s). Anowar’s Handbook on Textile Physics (Part-1). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.17294.74561; https://doi.org/10.5281/zenodo.8241105 Hossain, M. A. (2023t). Anowar’s Handbook on Textile Project Management. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.35461.32480 Hossain, M. A. (2023u). Anowar’s Handbook on Textile Raw Materials I. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.22537.62568 Hossain, M. A. (2023u). Anowar’s Handbook on Textile Raw Materials I. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.22537.62568 Hossain, M. A. (2023v). Anowar’s Handbook on Textile Testing & Quality Control (Part-3). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.23166.77121; https://doi.org/10.5281/zenodo.8241394 Hossain, M. A. (2023v). Anowar’s Handbook on Textile Testing & Quality Control (Part-3). https://doi.org/10.5281/zenodo.8240547 https://doi.org/10.5281/zenodo.8240547 Hossain, M. A. (2023m). Anowar’s Handbook on Industrial Economics. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Hossain, M. A. (2023m). Anowar’s Handbook on Industrial Economics. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.34176.81925; https://doi.org/10.5281/zenodo.8240589 5 \| P a g e Md. Anowar Hossain, “Unethical implementation of power is a global challenge, actually nobody should have power in terms of presidentship/professorship/directorship/chairmanship but everyone has ethical power to serve the society all over the world.” Engineering, School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.23166.77121; h //d / / d //doi.org/10.5281/zenodo.8241394 Hossain, M. A. (2023w). Anowar’s Handbook on Textile Testing and Quality Control (Part-1). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.14778.16327; 4 \| P a g e i f Hossain, M. A. (2023x). Anowar’s Handbook on Textile Testing and Quality Control (Part-2). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.34071.96169; h //d i /10 5281/ d 8241183 Hossain, M. A. (2023x). Anowar’s Handbook on Textile Testing and Quality Control (Part-2). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. http://dx.doi.org/10.13140/RG.2.2.34071.96169; Hossain, M. A. (2023y). Anowar’s Handbook on Yarn Manufacturing Technology (Part-01). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.19896.52484 Hossain, M. A. (2023y). Anowar’s Handbook on Yarn Manufacturing Technology (Part-01). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.19896.52484 Hossain, M. A. (2023z). Anowar’s Handbook on Yarn Manufacturing Technology (Part-2). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.29857.99683 Hossain, M. A. (2023z). Anowar’s Handbook on Yarn Manufacturing Technology (Part-2). School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka-1216, Bangladesh. https://doi.org/10.13140/RG.2.2.29857.99683 Hossain, M. A. (2023aa). Md. Anowar Hossain, Anowar’s Handbook on Polymer Science & Engineering. School of Fashion and Textiles, RMIT University, 25 Dawson Street, Brunswick, Melbourne, VIC 3056, Australia; Department of Textile Engineering, City University, Khagan, Birulia, Savar, Dhaka- 1216, Bangladesh. https://doi.org/10.13140/RG.2.2.31974.80969 Hossain, M. A., & Samanta, A. (2018). Green Dyeing On Cotton Fabric Demodulated From Diospyros Malabarica and Camellia Sinensis with Green Mordanting Agent. Latest Trends in Textile and Fashion Designing, 2(2), 1-8. http://dx.doi.org/10.32474/LTTFD.2018.02.000132 Hossain, M. 5 \| P a g e Md. Anowar Hossain, “Unethical implementation of power is a global challenge, actually nobody should have power in terms of presidentship/professorship/directorship/chairmanship but everyone has ethical power to serve the society all over the world.” Engineering, A., & Samanta, A. (2018). Green Dyeing On Cotton Fabric Demodulated From Diospyros Malabarica and Camellia Sinensis with Green Mordanting Agent. Latest Trends in Textile and Fashion Designing, 2(2), 1-8. http://dx.doi.org/10.32474/LTTFD.2018.02.000132 Hossain, M. A., Samanta, A., Abser, M. N., & Dilruba, F. A. (2019). A Review on Technological and Natural Dyeing Concepts for Natural Dyeing along with Natural Finishing on Natural Fibre. International Journal of Textile Science and Engineering, 3(1), 1-3. https://doi.org/10.13140/RG.2.2.36811.36648 Hossain, M. A., Samanta, A., Abser, M. N., & Dilruba, F. A. (2019). A Review on Technological and Natural Dyeing Concepts for Natural Dyeing along with Natural Finishing on Natural Fibre. International Journal of Textile Science and Engineering, 3(1), 1-3. https://doi.org/10.13140/RG.2.2.36811.36648 Hossain, M. A., Samanta, A. K., NS, B., PS, V., & Shukla. (2018). Non-toxic Coloration of Cotton Fabric using Non-toxic Colorant and Nontoxic Crosslinker. Journal of Textile Science & Engineering, 8(5), 1-5. https://doi.org/10.4172/2165-8064.1000374 Md. Anowar Hossain, A. K. S. (2019). A cost minimization process of heat and energy consumption for direct dyeing of cotton fabric coloration with triethanolamine. Journal of Textile Engineering & Fashion Technology, 5(5), 235-240. https://doi.org/10.15406/jteft.2019.05.00207 5 \| P a g e Md. Anowar Hossain, “Unethical implementation of power is a global challenge, actually nobody should have power in terms of presidentship/professorship/directorship/chairmanship but everyone has ethical power to serve the society all over the world.”
https://openalex.org/W4389155184	https://link.springer.com/content/pdf/10.1007/JHEP11(2023)211.pdf	English	null	Neutrinos, dark matter and Higgs vacua in parity solutions of the strong CP problem	The Journal of high energy physics/The journal of high energy physics	2,023	cc-by	15,868	Published for SISSA by Springer Received: July 23, 2023 Revised: October 20, 2023 Accepted: November 17, 2023 Published: November 29, 2023 Received: July 23, 2023 Revised: October 20, 2023 Accepted: November 17, 2023 Published: November 29, 2023 Received: July 23, 2023 Revised: October 20, 2023 Accepted: November 17, 2023 Published: November 29, 2023 Keywords: Cosmology of Theories BSM, Early Universe Particle Physics, Particle Nature of Dark Matter, Sterile or Heavy Neutrinos Open Access, c⃝The Authors. Article funded by SCOAP3. Published for SISSA by Springer Published for SISSA by Springer Neutrinos, dark matter and Higgs vacua in parity solutions of the strong CP problem https://doi.org/10.1007/JHEP11(2023)211 Contents 1 Introduction 1 2 Solving the strong CP problem with P 3 2.1 Spontaneous breaking of parity 5 2.2 Higgs effective potential at 2 loops: a second minimum before MPl 6 2.3 Generalizations 9 3 Neutrinos from the mirror world 11 3.1 Mirror Majorana neutrinos 11 3.2 Mirror Dirac neutrinos 15 3.3 Leptogenesis 15 4 Cosmology with a massless mirror photon 17 4.1 Relativistic degrees of freedom 17 4.2 Thermal dark matter: mirror electrons (and mirror up quark) 18 5 Phenomenology of massive mirror photon 22 5.1 Cosmological history 24 6 Conclusions 24 Contents 1 Introduction 1 2 Solving the strong CP problem with P 3 2.1 Spontaneous breaking of parity 5 2.2 Higgs effective potential at 2 loops: a second minimum before MPl 6 2.3 Generalizations 9 3 Neutrinos from the mirror world 11 3.1 Mirror Majorana neutrinos 11 3.2 Mirror Dirac neutrinos 15 3.3 Leptogenesis 15 4 Cosmology with a massless mirror photon 17 4.1 Relativistic degrees of freedom 17 4.2 Thermal dark matter: mirror electrons (and mirror up quark) 18 5 Phenomenology of massive mirror photon 22 5.1 Cosmological history 24 6 Conclusions 24 1 Introduction 2.1 Spontaneous breaking of parityf JHEP11(2023)211 4 Cosmology with a massless mirror photon 4.1 Relativistic degrees of freedom 4.2 Thermal dark matter: mirror electrons (and mirror up quark) 5 Phenomenology of massive mirror photon 5.1 Cosmological history 5.1 Cosmological history 6 Conclusions Neutrinos, dark matter and Higgs vacua in parity solutions of the strong CP problem JHEP11(2023)211 Michele Redi and Andrea Tesi INFN Sezione di Firenze, Via G. Sansone 1, I-50019 Sesto Fiorentino, Italy Department of Physics and Astronomy, University of Florence, Via G. Sansone 1, I-50019 Sesto Fiorentino, Italy E-mail: michele.redi@fi.infn.it, andrea.tesi@fi.infn.it Michele Redi and Andrea Tesi INFN Sezione di Firenze, Via G. Sansone 1, I-50019 Sesto Fiorentino, Italy Department of Physics and Astronomy, University of Florence, Via G. Sansone 1, I-50019 Sesto Fiorentino, Italy E-mail: michele.redi@fi.infn.it, andrea.tesi@fi.infn.it Michele Redi and Andrea Tesi INFN Sezione di Firenze, Via G. Sansone 1, I-50019 Sesto Fiorentino, Italy Department of Physics and Astronomy, University of Florence, Via G. Sansone 1, I-50019 Sesto Fiorentino, Italy E-mail: michele.redi@fi.infn.it, andrea.tesi@fi.infn.it E-mail: michele.redi@fi.infn.it, andrea.tesi@fi.infn.it Abstract: The strong CP problem can be solved if the laws of nature are invariant under a space-time parity exchanging the Standard Model with its mirror copy. We review and extend different realizations of this idea with the aim of discussing Dark Matter, neutrino physics, leptogenesis and collider physics within the same context. In the minimal realization of ref. [1] the mirror world contains a massless dark photon, which leads to a rather interesting cosmology. Mirror electrons reproduce the dark matter abundance for masses between 500–1000 GeV with traces of strongly interacting dark matter. This scenario also predicts deviations from cold dark matter, sizable ∆Neff and colored states in the TeV range that will be tested in a variety of upcoming experiments. We also explore scenarios where the mirror photon is massive and the mirror particles are charged under ordinary electromagnetism with very different phenomenology. We also show that, for the measured values of the SM parameters, the Higgs effective potential can give rise to a second minimum at large field value as required to break spontaneously the parity symmetry. Keywords: Cosmology of Theories BSM, Early Universe Particle Physics, Particle Nature of Dark Matter, Sterile or Heavy Neutrinos ArXiv ePrint: 2307.03161 ArXiv ePrint: 2307.03161 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. 1 Introduction Discrete space-time symmetries, parity P, and time-reversal, T, are not fundamental sym- metries of nature as we know it. Parity is maximally violated by the chiral electro-weak interactions, and the presence of a complex phase in the Cabibbo-Kobayashi-Maskawa (CKM) matrix breaks T perturbatively. In light of the CPT theorem, T violation is equiv- alent to CP violation and indeed all the observed CP violation observed in experiments is compatible with the one described by CKM matrix. This leads to the so called strong CP problem because one can add the topological term to the QCD lagrangian θ αs 16πϵµνρσGµνGρσ , (1.1) (1.1) that breaks P and CP. Such term induces new CP violating effects in the Standard Model (SM), predicting in particular an electric dipole moment for the neutron dn ∼10−15θ e cm. The experimental constraint dn < 10−26 e cm then implies that θ < 10−10, a value that appears inexplicable within the SM. Essentially all the solutions of the strong CP problem rely on the existence of new symmetries. In the axion solution (see [2] for a review) a U(1) global symmetry, known – 1 – as Peccei-Quinn symmetry, is introduced that is anomalous under QCD but otherwise exact. Once the symmetry is spontaneously broken θ becomes a dynamical variable, the axion, whose potential is minimized at the CP preserving point, thus solving dynamically the strong CP problem. In the Nelson-Barr solution [3, 4] CP is assumed to be an exact symmetry of nature. If the symmetry is spontaneously broken in a favourable way an order one CKM phase can be obtained with suppressed θ−term. Similarly to Nelson-Barr models one might attempt to solve the strong CP problem imposing P as a fundamental symmetry that would immediately imply θ = 0. At first sight this possibility is excluded because the SM is a chiral gauge theory that maximally violates P. Nevertheless it was realized long ago that P could be a spontaneously broken symmetry in extensions of the SM. In [5] a solution of the strong CP problem was proposed extending the electro-weak gauge sector. In [6] it was further shown that a full mirror electro-weak sector with common color interactions could give sufficient suppression to the θ−term. 1Similar ideas appeared in the literature in the context of Higgs parity theories [11–14]. 1 Introduction The crucial observation was that the mirror sector should have mirror electro-weak interactions where SM fermions are also doubled but with opposite chirality as required by parity, see also [7–9] for recent works. JHEP11(2023)211 Recently an even simpler realization appeared in ref. [1]. Here P is used in connection with a Z2 symmetry that exchanges the SM with a mirror copy that is parity related, P[SM] = g SM † , P ≡P × Z2 . (1.2) (1.2) Due to P the mirror sector g SM has the same group structure of the SM and same matter content but representation are conjugated. In particular P enforces the condition Due to P the mirror sector g SM has the same group structure of the SM and same matter content but representation are conjugated. In particular P enforces the condition θs ≡θ + ˜θ = 0 . (1.3) (1.3) A simple solution of the strong CP problem then emerges where SU(3)c of the strong interactions is identified with the diagonal subgroup of SU(3)× f SU(3) so that the coefficient of the topological term is θs. Crucially this solution is robust against spontaneous breaking of parity. The safest option that does not introduce new CP violating sources is to break P spontaneously. This can be done if the Higgs potential has a large field minimum so that in the mirror sector it has a large expectation value. Remarkably we find that with the addition of the new colored states a consistent minimum below the Planck scale emerges precisely around the observed values of the top quark and Higgs boson masses, see [10] for related work. In this work we study cosmology and particle physics implications of the parity solution of strong CP. An important ingredient turns out to be neutrinos. Neutrino masses can be generated through the see-saw mechanism that must be mirrored for consistency with P. The symmetry allows to mix SM with mirror neutrinos that thus act as sterile neutrinos from the point of view of the SM. The mirror neutrinos are often long lived and lead to entropy injection in the SM thermal bath. 2 Solving the strong CP problem with P We start reviewing the main aspects of ref. [1]. The model has two exact copies of the SM related by the action of P. In particular, if ψ is a left-handed Weyl fermion in a representation r of SM, its P conjugate would be a (left-handed fermion) ˜ψ P[ψ] = ˜ψ† . (2.1) (2.1) Since our definition of P does not act on the representation of the gauge group, ψ is in the representation ¯r of g SM.i Since our definition of P does not act on the representation of the gauge group, ψ is in the representation ¯r of g SM.i If P is a fundamental symmetry, the matter content of the model is completely fixed, as in table 1. In light of P the lagrangian of the mirror sector is determined by the one of the SM as, Lf SM = LSM(P[ψ, A], Yu,d,e →Y ∗ u,d,e, θ →−θ) . (2.2) (2.2) From the behavior of the Yukawa matrices it is manifest that also the physical ¯θ angles of the two sectors are equal and opposite om the behavior of the Yukawa matrices it is manifest that also the physical ¯θ angles of e two sectors are equal and opposite ¯θ = θ + arg det YuYd, ¯˜θ = ˜θ + arg det Y ∗ u Y ∗ d = −¯θ (2.3) (2.3) In absence of right-handed neutrinos (that we discuss in detail in section 3), the two sectors can communicate at the renormalizable level through the Higgs portal and kinetic mixing of the two hyper-charges, these two terms are invariant under P.2 As a boundary In absence of right-handed neutrinos (that we discuss in detail in section 3), the two sectors can communicate at the renormalizable level through the Higgs portal and kinetic mixing of the two hyper-charges, these two terms are invariant under P.2 As a boundary mixing of the two hyper-charges, these two terms are invariant under P. As a boundary 2The presence of a kinetic mixing would have important implication for the scenario in [1] with a massless mirror photon. Upon diagonalizing the kinetic terms the low energy lagrangian can be cast in the form [16], 2The presence of a kinetic mixing would have important implication for the scenario in [1] with a massless mirror photon. 1 Introduction In the minimal scenario with massless mirror photon the mirror electron is stable an neutral under the SM and can be the Dark Matter (DM).1 If the abundance is determined by thermal freeze-out with the mirror photon we – 2 – predict the DM mass in the range 500–1000 GeV. In this scenario we also predict a small fraction of color dark matter and contribution to ∆Neff. Moreover DM is coupled to a long range force producing deviations from cold dark matter. We also explore scenarios where the mirror photon acquires mass. If the hypercharge is broken to the diagonal all the mirror states have identical electric charge as the SM partners. This forbids a large reheating temperature and can produce deviation from the SM parametrized by the Y parameter [15]. The paper is organized as follows. In section 2 we review the scenario of ref. [1] and discuss generalizations to different symmetry breaking patterns. A study of the Higgs effec- tive potential is presented showing that for the observed top quark mass consistent minima for the mirror world exist. We include right-handed neutrinos in section 3, discussing their lifetime and thermal leptogenesis. We study the cosmology with a massless dark photon in section 4 showing that the scenario is compatible with large reheating temperature and that DM can be reproduced by the mirror electron. Section 5 is devoted to the scenario with where hypercharge is broken to the diagonal. We summarize our findings in 6. JHEP11(2023)211 2 Solving the strong CP problem with P Upon diagonalizing the kinetic terms the low energy lagrangian can be cast in the form [16], LU(1) = −1 4F 2 µν −1 4 ˜F 2 µν −eAµ Jµ √ 1 −ϵ2 − ϵ ˜Jµ √ 1 −ϵ2 −e ˜Aµ ˜Jµ . (2.4) (2.4) As a consequence the mirror matter acquires a charge ϵ under ordinary electromagnetism. Very strong bounds apply on this scenario especially if states of the dark sector are the DM. We will mostly focus on ϵ ≈0 in what follows. As a consequence the mirror matter acquires a charge ϵ under ordinary electromagnetism. Very strong bounds apply on this scenario especially if states of the dark sector are the DM. We will mostly focus on ϵ ≈0 in what follows. – 3 – – 3 – SM SU(3) SU(2) U(1) Q 3 2 1/6 U ¯3 2 −2/3 D ¯3 2 1/3 L 1 1 −1/2 E 1 1 1 g SM f SU(3) f SU(2) ˜U(1) ˜Q ¯3 2 −1/6 ˜U 3 1 2/3 ˜D 3 1 −1/3 ˜L 1 2 1/2 ˜E 1 1 −1 Table 1. Quantum number of left-handed Weyl fermions of SM and mirror SM. Table 1. Quantum number of left-handed Weyl fermions of SM and mirror SM. JHEP11(2023)211 conditions at very high energies the couplings of the two sectors must be equal, but running to low energy can be different when P is spontaneously broken, as we will discuss. conditions at very high energies the couplings of the two sectors must be equal, but running to low energy can be different when P is spontaneously broken, as we will discuss. A necessary ingredient to solve the strong CP is to identify our QCD as the diagonal combination of the two SU(3)’s of the visible and mirror sector. This can be achieved introducing a scalar field Σ, transforming as a bi-fundamental (3, ¯3) or (3, 3) of the prod- uct group, L = LSM + Lf SM −ϵ 2Bµν ˜Bµν −λ12\|H\|2\| ˜H\|2 + Tr[(DµΣ)†DµΣ] −V (Σ) . (2.5) (2.5) e the covariant derivative acts on Σ as DµΣ = ∂µΣ −ig3GµΣ + i˜g3Σ ˜Gµ. 2 Solving the strong CP problem with P where the covariant derivative acts on Σ as DµΣ = ∂µΣ −ig3GµΣ + i˜g3Σ ˜Gµ For a generic choice of the potential the minimum is realized for For a generic choice of the potential the minimum is realized for ⟨Σij⟩= Σ0δij −→SU(3) × f SU(3) SU(3)c , (2.6) (2.6) so that color groups are spontaneously broken to the diagonal that can be identified with the low energy QCD interactions. At tree level the couplings satisfy the boundary condition 1 g2s(MΣ) = 1 g2 3(MΣ) + 1 ˜g2 3(MΣ) , (2.7) (2.7) where gs(µ) is the low energy QCD coupling and where we have loosely defined MΣ as the energy scale where SU(3) × f SU(3) is broken. While other mechanisms of symmetry break- ing exist, see [1] for examples, many conclusions only depend on the symmetry breaking pattern. We will focus on the breaking with scalar bi-fundamental in the rest of the paper. The spontaneous breaking of SU(3) has two remarkable consequences. First, it solves the strong CP problem in the following way. The combination (g3Gµ ± ˜g3 ˜Gµ)/ q g2 3 + ˜g2 3 acquires mass mG = q g2 3 + ˜g2 3Σ0. Being an octet under SU(3)c the phenomenology reduces to the one of colorons [18, 19]. The orthogonal combination corresponding to g3Gµ = ∓˜g3 ˜Gµ is massless and can be identified with the gluons at low energies. Due to P one finds Lθs = θ α3 16πϵµνρσGµνGρσ −˜α3 16πϵµνρσ ˜Gµν ˜Gρσ g3Gµ=∓˜g3 ˜Gµ = 0 . (2.8) (2.8) Crucially the vanishing of the low energy θ angle holds even for g3 ̸= ˜g3 so it holds even after the spontaneous breaking of P. Second, due to eq. (2.6), the mirror quarks are colored – 4 – under ordinary QCD at low energy. This implies that P must be broken because otherwise the new colored states would have identical mass as the SM quarks. Experimentally the lightest mirror quark (the mirror up) must be heavier that 1.3 TeV [1], and given the observed value of the up mass, this requires ˜v/v ≳106 so that ˜v ≳2×108 GeV. Concretely this is obtained when the mirror Higgs ˜H gets a VEV ˜v larger than v = 246 GeV. All the mirror fermion and gauge boson masses will be then lifted as compared to the SM by a factor of ˜v/v. 2 Solving the strong CP problem with P The mirror masses are thus m ˜f = mf ˜v v, m ˜ W, ˜Z = mW,Z ˜v v . (2.9) (2.9) JHEP11(2023)211 The lightest states of the mirror world beside neutrinos are the dark electron and dark up quark with masses. According to lattice estimates the up quark mass is different from zero and in the MS scheme is found mMS u (2 GeV) ≈2.3 ± 0.1 MeV [20], four times the electron mass. Taking in account running in a world where the electro-weak VEV is 108 GeV the ratio changes roughly by factor 2 so that, The lightest states of the mirror world beside neutrinos are the dark electron and dark up quark with masses. According to lattice estimates the up quark mass is different from zero and in the MS scheme is found mMS u (2 GeV) ≈2.3 ± 0.1 MeV [20], four times the electron mass. Taking in account running in a world where the electro-weak VEV is 108 GeV the ratio changes roughly by factor 2 so that, m˜u ≈2m˜e = 400 GeV ˜v 108 GeV . (2.10) (2.10) We can distinguish two different scenarios, We can distinguish two different scenarios, We can distinguish two different scenarios, 1. Σ0 > ˜v At energies E ≲MΣ there are two copies of colored fermions of the SM. This scenario is closely related to the one of [6] where a single gauge group SU(3) was considered. At energies E > mΣ P demands g3 = ˜g3. 2. Σ0 < ˜v For Σ0 ≲10−5˜v the lightest color states are colorons. At energies greater than mΣ the symmetry is restored but the couplings of the two sectors run differently due to breaking fo P. For 10−5˜v ≲Σ0 ≲˜v some fermions are lighter mΣ. P demands that g3 = ˜g3 at energies E > ˜v. 2.1 Spontaneous breaking of parity As we discussed P must be broken so that ˜v ≫v. The breaking can either be spontaneous or soft compatibly with the solution of the strong CP problem. We find it remarkable that the SM has the necessary ingredients to generate spontaneous breaking of P because the Higgs potential develops a new minimum at large field values where the electro-weak symmetry is broken at a high scale. For the observed value of SM parameters however in the SM the second minimum is typically trans-Planckian so it is likely beyond the regime of validity of the SM effective field theory. In the present setup new colored states exist so this conclusion must be reconsidered. Indeed we will find consistent vacua below the Planck scale. Therefore the only assumption required for the parity solution to work is that the SM sits in the standard electro-weak minimum while the mirror world lives in the large field minimum. This also guarantees that the breaking does not introduce new potentially dangerous phases. – 5 – The second minimum is modified by two effects compared to the SM. First the new colored states modify the evolution of the couplings and the effective potential above Min[m˜u , mΣ]. Moreover the Higgs portal coupling λ12 modifies the potential at tree level. We can always tune the electro-weak minimum to the observed value but then the second minimum will be predicted in terms of the SM couplings and it will be different from the one computed in the SM. Note that even if λ12 is set to zero it will be generated by the running so that in general the potential is V (H, ˜H). λ12 is however induced only at high loop order so that λ12 = 0 is in practise consistent. The spontaneous breaking of a discrete symmetry leads to existence of topologically stable domain walls that would be disastrous if ever produced [21] (see also [22] for a recent discussion). For λ12 = 0 the situation in our scenario is different. The domain walls interpolate between (v, ˜v) and (˜v , v) that are exactly degenerate under exact P symmetry. As long as V = V (\|H\|2)+V (\| ˜H\|2) the domain walls factorize into two independent profiles for H and ˜H that are individually unstable so that no domain wall problem arises. 2.1 Spontaneous breaking of parity The reason why this happens is that there exists a family of unstable solutions that interpolate between different minima. The presence of the Higgs portal is however expected to change this conclusion so that a single stable domain wall survives. The domain wall problem could be eliminated if the P symmetry is softly broken. Alternatively the domain wall problem is solved if P is broken during inflation, HI < ˜v. In this case any abundance of domain walls is inflated away. JHEP11(2023)211 3We neglect the 2-loop modification of top yukawa coupling and use the tree level condition (2.7). 2.2 Higgs effective potential at 2 loops: a second minimum before MPl In this section we discuss under what circumstances the SM Higgs effective potential can develop a sub-Planckian minimum compatible with the P solution to the strong CP problem (see also [10] for a different realization). We work in the limit of vanishing quartic portal λ12. In this approximation and at the perturbative order of interest, the appearance of a mimimum at large Higgs vev for the mirror world can be studied by inspecting the SM effective potential, modified by the presence of new matter charged under the SM gauge symmetry as well as new dynamics.i The RGE running of the SM parameters is modified in several ways. First, the P symmetry enforces equality of couplings at and above ˜v. Second, importantly, above MΣ the strong coupling gs of our color SU(3)c is matched to the fundamental couplings of the two SU(3) factors as per eq. (2.7), and this may happen before or after ˜v. For example if MΣ ≫˜v the model becomes the one of ref. [6]. While in the opposite regime above MΣ the running of g3 is modified by the presence of new colored matter. We here explore the case where MΣ < m˜u so that below the SU(3) × SU(3) breaking there is just the SM. The running of the SM parameter can be computed with great precision and we use [25] to run the MS parameters up to the scale ¯µ = MΣ. Above this scale the running of g3 is modified by the presence of the bifundamental complex scalar Σ. The modification of the β-function of g3 at 2-loops reads,3 ∆βg3 = g3 3 (4π)2 1 2 + g5 3 (4π)4 11 (2.11) (2.11) 3We neglect the 2-loop modification of top yukawa coupling and use the tree level condition (2.7). – 6 – The matching condition (2.7) has the implication that the low-energy value of g3 be limited to the range gs(MΣ) ≤g3(MΣ) ≤ √ 2gs(MΣ), since ˜g3 will be inevitably larger due to the faster running in the infra-red. We then study the SM effective potential in this context, by fixing Mt, Mh and αs(MZ) to their standard values, and inspecting for which values of MΣ a new sub-Planckian minimum for h appears. 2.2 Higgs effective potential at 2 loops: a second minimum before MPl The new physics acts as a stabilization for the Higgs quartic coupling: the matching condition will generate an effective larger g3 coupling, that it is known to stabilize the SM potential, on top of the already smaller running due to the presence of Σ. Therefore, by fixing Mt in the experimentally allowed range [23, 24], MΣ should be large enough to allow for g3(MΣ) to be such that a minimum right before MPl appear. Intuitively, very large values of g3(MΣ) will make the second mimimum disappear. JHEP11(2023)211 The solution ˜v(MΣ) will be then very precisely determined upon a judicious choice of g3(MΣ) and ˜g3(MΣ). This has to be determined iteratively satisfying both eq. (2.7) and the P symmetric boundary conditions at ˜v, g3(˜v) = ˜g3(˜v). This is done by considering the running of ˜g3 from ˜v to MΣ including all the thresholds of mirror quarks (below ˜v the mirror sector is simply SU(3)×U(1), we neglect the running of the mirror electromagnetism) up to three loops. We notice that the running of ˜g3 is much faster and can result quite easily in confinement of f SU(3), the running is faster since MΣ ≪m˜u. This poses a tight constraint to identify solutions with ˜g3(MΣ) in the perturbative realm.f The SM beta functions and effective potentials are taken from refs. [25, 26]. New physics only enters through the boundary conditions at MΣ and the new contributions to the β-functions. Here we compute the effective potential for the Higgs as Veff(h) = λeff(h) 4 h4 , λeff(µ = h) = e4Γ(h)[λ(h) + λ(1)(h) + λ(2)(h) + · · · ] . (2.12) (2.12) In order to compute this we use the available β-functions and the expression of the Higgs anomalous dimension, constructing a set of differential equations for (g2 i , y2 i , λ, Γ). The effective potential then reads Veff(h) = 1 4λeff(h)h4. The position of the second SM minimum is found by solving 4λeff(h) + λ′ eff(h)h = 0 (2.13) (2.13) By applying the algorithm discussed above we have found solutions with ˜g3(MΣ) in the per- turbative regime, satisfying to great accuracy (sub-per-mille level) the boundary conditions and having a minimum h < MPl. Results are reported in figures 1 and 2. Notably consis- tent solutions are precisely found for the observed values of the top quark and Higgs mass. 2.2 Higgs effective potential at 2 loops: a second minimum before MPl By applying the algorithm discussed above we have found solutions with ˜g3(MΣ) in the per- turbative regime, satisfying to great accuracy (sub-per-mille level) the boundary conditions and having a minimum h < MPl. Results are reported in figures 1 and 2. Notably consis- tent solutions are precisely found for the observed values of the top quark and Higgs mass. In the scenario MΣ > ˜v below the SU(3) breaking scale there are two copies of colored fermions. The running of gs is thus modified compared to the SM and is larger at high scales. This in turn reduces yt compared to the SM and increases the instability scale until it disappears. In this case we have not found consistent minima with VEV smaller than the Planck scale and vanishing λ12. Many other possibilities exist where only a subset of mirror fermions are lighter than MΣ. A more detailed analysis will appear elsewhere. – 7 – 105 106 107 108 109 1010 1011 1015 1016 1017 1018 MΣ[GeV] v[GeV] g 3(MΣ)>2 Mh=125.15 GeV αs(MZ)=0.1184 mu ~<MΣ v>MPl Mt=172.5 GeV Mt=172.25 GeV Mt=172 GeV Mt=172.75 GeV Mt=173 GeV Figure 1. Parameter space of [1] with spontaneous P breaking. For the experimental value of the top quark mass, mt = 172.5 ± 0.5 GeV [23, 24] a perturbative minimum below the Planck scale emerges for 106GeV < MΣ < 1010GeV. We assume λ12 = 0 and MΣ < m˜u. 105 106 107 108 109 1010 1011 1015 1016 1017 1018 MΣ[GeV] v[GeV] g 3(MΣ)>2 Mh=125.15 GeV αs(MZ)=0.1184 mu ~<MΣ v>MPl Mt=172.5 GeV Mt=172.25 GeV Mt=172 GeV Mt=172.75 GeV Mt=173 GeV JHEP11(2023)211 Figure 1. Parameter space of [1] with spontaneous P breaking. For the experimental value of the top quark mass, mt = 172.5 ± 0.5 GeV [23, 24] a perturbative minimum below the Planck scale emerges for 106GeV < MΣ < 1010GeV. We assume λ12 = 0 and MΣ < m˜u. 172.0 172.5 173.0 1015 1016 1017 1018 1019 Mt[GeV] v[GeV] Mh=125.15 GeV αs(MZ)=0.1184 g3/gs=1.15 g3/gs=1.2 g3/gs=1.25 g3/gs=1.3 Figure 2. Correlation between the large field minimum of the Higgs and the top quark mass in the scenario [1]. Isolines are labelled by their corresponding value of g3(Mσ)/gs(MΣ). We assume λ12 = 0 and MΣ < m˜u. 172.0 172.5 173.0 1015 1016 1017 1018 1019 Mt[GeV] v[GeV] Mh=125.15 GeV αs(MZ)=0.1184 g3/gs=1.15 g3/gs=1.2 g3/gs=1.25 g3/gs=1.3 Figure 2. 2.3 Generalizations The breaking of the two fundamental SU(3)’s to the diagonal is a structural part of the scenario. One might wonder if more general patterns of symmetry breaking can be con- sidered. Focusing on two SM copies one can consider in general the pattern of symmetry breaking, The breaking of the two fundamental SU(3)’s to the diagonal is a structural part of the scenario. One might wonder if more general patterns of symmetry breaking can be con- sidered. Focusing on two SM copies one can consider in general the pattern of symmetry breaking, [SU(3) × SU(2) × U(1)] × [ f SU(3) × f SU(2) × ] U(1)] SU(3)c × H Σ0 + [ f SU(2)× eU(1) →eU(1)em]˜v (2.14) The model of ref. [1] corresponds to the case H = SU(2) × U(1) × f SU(2) × ] U(1), where the electroweak interactions (visible and mirror) are unbroken by Σ.i f [SU(3) × SU(2) × U(1)] × [SU(3) × SU(2) × U(1)] SU(3)c × H Σ0 + [SU(2)×U(1) →U(1)em]˜v (2.14) The model of ref. [1] corresponds to the case H = SU(2) × U(1) × f SU(2) × ] U(1), where the electroweak interactions (visible and mirror) are unbroken by Σ.i The model of ref. [1] corresponds to the case H = SU(2) × U(1) × f SU(2) × ] U(1), where the electroweak interactions (visible and mirror) are unbroken by Σ.i JHEP11(2023)211 Let us first consider the breaking SU(2) × f SU(2) down to the diagonal. In the regime ˜v ≫Σ0 mirror SU(2) is broken and one finds v2 SM = v2 + Σ2 0. The small VEV of Σ0 implies the existence of light vector bosons charged under SU(2) with mass in the 100 GeV range or below that is excluded. The opposite regime ˜v ≪Σ0 is also not viable because v2 SM = v2 + ˜v2 so implying new light colored states. Therefore SU(2) × f SU(2) cannot be broken to the diagonal. This also forbids the possibility of unified breaking pattern of the form SU(5) × f SU(5) →SU(5)d.f A different conclusion holds for hypercharge that can be broken to the diagonal combi- nation without introducing new states with SM charges. We discuss this possibility below. Alternatively eU(1) could be broken while preserving U(1), namely H = SU(2) × U(1) × f SU(2). 2.2 Higgs effective potential at 2 loops: a second minimum before MPl Correlation between the large field minimum of the Higgs and the top quark mass in the scenario [1]. Isolines are labelled by their corresponding value of g3(Mσ)/gs(MΣ). We assume λ12 = 0 and MΣ < m˜u. – 8 – 2 3 Generalizations 4Stability requires λΣ + ˜λΣ > 0. Possible unbroken subgroups compatible with stability are U(3) a U(2)2 U(1). The former is the one discussed in the main text and it is realized for λΣ ≥0, ˜λΣ > −3λΣ λΣ > 0, ˜λΣ > 0. The latter is the solution with Σij = µΣ/ p λΣ + ˜λΣδi3δj3 for λΣ > 0, −λΣ < ˜λΣ < 0. 2.3 Generalizations In order for this to be consistent with P symmetry, one can for example add extra scalars charged under U(1)’s in both sectors or SU(2) triplets that spontaneously break only eU(1). Eventually, upon P breaking, the only unbroken symmetries are the electroweak interactions of the SM. In this case the mirror states have the same quantum numbers of the model of ref. [1] but without massless mirror photon. Massive mirror photon. Breaking U(1) × eU(1) →U(1)Y corresponds to H = SU(2) × U(1)Y × f SU(2). This can be done effectively promoting SU(3) →U(3) and considering the collective breaking U(3) × eU(3) U(3)d . (2.15) (2.15) We notice in fact that the scenario of [1] already contains the necessary ingredients and it is sufficient to give Σ a U(1) charge (q , ±q). The presence of the additional U(1) constrains the potential of Σ of eq. (2.5) to be of the following form We notice in fact that the scenario of [1] already contains the necessary ingredients and it is sufficient to give Σ a U(1) charge (q , ±q). The presence of the additional U(1) constrains the potential of Σ of eq. (2.5) to be of the following form V (Σ) = −µ2 Σtr[ΣΣ†] + λΣtr[ΣΣ†]2 + ˜λΣtr[ΣΣ†ΣΣ†], (2.16) (2.16) where we have absorbed the contribution from the Higgs vevs v and ˜v in the effective mass term µ2 Σ ≥0. If both quartics are positive the only minimum is the one leaving a U(3) invariant, Σ ∝I where Σij = µΣ/ q 3λΣ + ˜λΣδij.4 In the unitary gauge the model of where we have absorbed the contribution from the Higgs vevs v and ˜v in the effective mass term µ2 Σ ≥0. If both quartics are positive the only minimum is the one leaving a U(3) invariant, Σ ∝I where Σij = µΣ/ q 3λΣ + ˜λΣδij.4 In the unitary gauge the model of 4Stability requires λΣ + ˜λΣ > 0. Possible unbroken subgroups compatible with stability are U(3) and U(2)2 U(1). The former is the one discussed in the main text and it is realized for λΣ ≥0, ˜λΣ > −3λΣ or λΣ > 0, ˜λΣ > 0. The latter is the solution with Σij = µΣ/ p λΣ + ˜λΣδi3δj3 for λΣ > 0, −λΣ < ˜λΣ < 0. 2.3 Generalizations – 9 – q = 0 SU(3)C SU(2)L U(1)Y ˜Qu ¯3 1 0 ˜U 3 1 0 ˜Qd ¯3 1 0 ˜D 3 1 0 ˜Le 1 1 0 ˜E 1 1 0 ˜ν 1 1 0 q ̸= 0 SU(3)C SU(2)L U(1)Y ˜Qu ¯3 1 −2/3 ˜U 3 1 2/3 ˜Qd ¯3 1 1/3 ˜D 3 1 −1/3 ˜Le 1 1 −1 ˜E 1 1 1 ˜ν 1 1 0 T bl 2 Mi t t t b b l th t b ki l i th d l q = 0 SU(3)C SU(2)L U(1)Y ˜Qu ¯3 1 0 ˜U 3 1 0 ˜Qd ¯3 1 0 ˜D 3 1 0 ˜Le 1 1 0 ˜E 1 1 0 ˜ν 1 1 0 q ̸= 0 SU(3)C SU(2)L U(1)Y ˜Qu ¯3 1 −2/3 ˜U 3 1 2/3 ˜Qd ¯3 1 1/3 ˜D 3 1 −1/3 ˜Le 1 1 −1 ˜E 1 1 1 ˜ν 1 1 0 JHEP11(2023)211 Table 2. Mirror states quantum numbers below the symmetry breaking scale mΣ in the model with massless dark photon (q = 0) and with hypercharge broken to the diagonal (q ̸= 0). Table 2. Mirror states quantum numbers below the symmetry breaking scale mΣ in the model with massless dark photon (q = 0) and with hypercharge broken to the diagonal (q ̸= 0). eq. (2.5) produces and effective mass term LM = g2 1q2Σ2 0(B −˜B)2 . (2.17) (2.17) Alternatively we could also give a Stueckelberg mass to the diagonal combination. Due to the breaking to the diagonal the mirror states are charged under the SM hypercharge and thus also carry electric charge. In particular the mirror states have Q = Y = ˜T3 + ˜Y = ˜Q . (2.18) (2.18) Therefore all the mirror sector states have electric charge identical to the SM particles but with different masses controlled by ˜v. The SM charges of mirror states are reported in table 2. Note that instead the hyper-charge assignments of mirror matter (which is a good quantum number when ⟨H⟩= 0) are different from the corresponding SM states. In this context the left-handed mirror neutrinos have vanishing hyper-charge (as well as, crucially, the VEV of the mirror Higgs field). Therefore all the mirror sector states have electric charge identical to the SM particles but with different masses controlled by ˜v. f . (2.5) produces and effective mass term Table 2. Mirror states quantum numbers below the symmetry breaking scale mΣ in the model with massless dark photon (q = 0) and with hypercharge broken to the diagonal (q ̸= 0). 3 Neutrinos from the mirror world The generation of SM neutrino masses introduces new structure in the model that must be compatible with P invariance. In this section we consider a type-I see-saw with right- handed neutrinos. To give masses to all neutrinos one needs to add 3 copies (Ni , ˜Ni) of singlet Weyl fermions transforming under P as Ni →˜N† i . We can define the parity states, Ni ± ≡Ni ± ˜Ni →±(Ni ±)† . (3.1) (3.1) We consider the lagrangian invariant under P (see also [27] for a particular choice),5 We consider the lagrangian invariant under P (see also [27] for a particular choice),5 g g ( [ ] p ), Lν = αijLiHN j ++βijLiHN j −+α∗ ij ˜Li ˜HN j +−β∗ ij ˜Li ˜HN j −−M+,i 2 Ni +Ni +−M−,i 2 Ni −Ni −+h.c. , (3.2) JHEP11(2023)211 ν = αijLiHN j ++βijLiHN j −+α∗ ij ˜Li ˜HN j +−β∗ ij ˜Li ˜HN j −−M+,i 2 Ni +Ni +−M−,i 2 Ni −Ni −+h.c. , (3.2) (3.2) where M+ and M−are real and diagonal, while α and β are generic complex matrices in flavor space. In the limit α = β and M+ = M−two independent fermion number symmetries emerge for the visible and the mirror sector that guarantee that the lightest fermion of each sector is stable. In a more symmetric notation, the above lagrangian can be recast as YijLiHFj + eYij ˜Li ˜HFj −Mi 2 FiFi + h.c., Fi ≡(Ni +, N i −) . (3.3) (3.3) The Y, eY yukawa matrices are 3 × 6, and they are parametrized as Y = (α, β) and ˜Y = (α∗, −β∗). In this notation P enforces the following relation The Y, Y yukawa matrices are 3 × 6, and they are parametrized as Y = (α, β) and Y = (α∗, −β∗). In this notation P enforces the following relation (α∗, −β∗). In this notation P enforces the following relation eY = Y ∗I, I = diag[1, 1, 1, −1, −1, −1] . (3.4) (3.4) This parametrization will be useful discussing leptogenesis. This parametrization will be useful discussing leptogenesis. We now discuss the possible structures of the neutrino mass spectrum. Since we consider the limit where max[α, β]v ≪min[M±, max[α, β]˜] the SM neutrinos are always majorana. We can distinguish two cases: 2.3 Generalizations The SM charges of mirror states are reported in table 2. Note that instead the hyper-charge assignments of mirror matter (which is a good quantum number when ⟨H⟩= 0) are different from the corresponding SM states. In this context the left-handed mirror neutrinos have vanishing hyper-charge (as well as, crucially, the VEV of the mirror Higgs field). For Σ0 →∞the dark photon can be integrated out. In this limit B1 = B2, G1 = G2 so that the gauge structure is, SU(3) × SU(2)L × SU(2)R × U(1) (2.19) (2.19) where SU(2)L,R act on SM and mirror fermions respectively. The same scenario was also considered in [6]. More than one mirror. The crucial ingredient of the strong CP solution is the con- volution of space-time symmetry with mirror symmetry. This can be generalized to 2N copies of the SM where, P[SMi] = SMi+1 (2.20) (2.20) Breaking color to the diagonal subgroup, U(3)i × U(3)i+1 U(3) (2.21) (2.21) gives rise at low energy QCD + a tower of states analogous to extra-dimensions. Indeed one could realize such scenario in extra-dimensions with replicas of the SM related by chirality. – 10 – 3.1 Mirror Majorana neutrinos We start with the limit where M± are the heaviest masses, so that N± can be integrated out. They act as see-saw for both the visible and the mirror neutrinos, and they yield the following lagrangian L = i¯L /DL + i¯˜L /D˜L + 1 2AijLiHLjH + 1 2A∗ ij ˜Li ˜H ˜Lj ˜H + BijLiH ˜Lj ˜H + h.c., (3.5) (3.5) where Aij is a symmetric matrix and Bij is an hermitian matrix in flavor space. In terms of the fundamental parameters they are given by where Aij is a symmetric matrix and Bij is an hermitian matrix in flavor space. In terms of the fundamental parameters they are given by Aij = α· 1 M+ ·αT ij + β · 1 M− ·βT ij , Bij = α· 1 M+ ·α† ij − β · 1 M− ·β† ij . (3.6) 5We are here neglecting off-diagonal terms of the form ∆ijNi ˜ Nj with ∆an hermitian matrix. – 11 – A unitary rotation U and a diagonal rephasing P make the first two terms diagonal and real (and therefore equal). This amounts to the transformations L →UPL and ˜L →U∗P ∗˜L, that take the lagrangian into the form A unitary rotation U and a diagonal rephasing P make the first two terms diagonal and real (and therefore equal). This amounts to the transformations L →UPL and ˜L →U∗P ∗˜L, that take the lagrangian into the form L = i¯L /DL + i¯˜L /D˜L + ˜mi ˜v2 δijLiHLjH + ˜mi ˜v2 δij ˜Li ˜H ˜Lj ˜H + ∆ij ˜mj ˜v2 LiH ˜Lj ˜H + h.c. (3.7) where ˜mi is real 2 ˜mi/˜v2δij = [P · UT · A · U · P]ij and ∆ij ˜mj/˜v2 = [PUT BU∗P ∗]ij. The presence of six right-handed neutrinos guarantees that all the Majorana neutrinos (visible and mirror) are massive. L = i¯L /DL + i¯˜L /D˜L + ˜mi ˜v2 δijLiHLjH + ˜mi ˜v2 δij ˜Li ˜H ˜Lj ˜H + ∆ij ˜mj ˜v2 LiH ˜Lj ˜H + h.c. (3 L = i¯L /DL + i¯˜L /D˜L + ˜mi ˜v2 δijLiHLjH + ˜mi ˜v2 δij ˜Li ˜H ˜Lj ˜H + ∆ij ˜mj ˜v2 LiH ˜Lj ˜H + h.c. (3.7) where ˜mi is real 2 ˜mi/˜v2δij = [P · UT · A · U · P]ij and ∆ij ˜mj/˜v2 = [PUT BU∗P ∗]ij. 3.1 Mirror Majorana neutrinos The decay rate of ˜ν is important for the cosmological history. Upon breaking of the mirror weak interactions, ˜L gets a majorana mass from the mirror Weinberg operator. In general, compatibly with P a mixing exists between SM and mirror neutrinos, see eq. (3.7). Such a term allows ˜ν to decay even when electro-weak symmetry is unbroken. Clearly ˜νi acts as three right-handed neutrinos for the SM neutrinos. The tree-level decay rate of the mirror neutrinos into LH + L∗H∗is given by Γ(˜νi →all) ∼(∆†∆)ii 16π ˜m3 i ˜v2 (3.13) (3.13) JHEP11(2023)211 Such a rate becomes faster than Hubble at a temperature Such a rate becomes faster than Hubble at a temperature T˜ν−dec ≲103 GeV m˜u TeV 2 mi 0.1 eV 3/2 \|∆\|2 . (3.14) (3.14) Mirror neutrinos decay to SM final states from the LH˜ν term directly, or through off- shell W/Z and Higgs. The decay width depends on the available decay channels. We here consider the case where the lightest mirror neutrino is below the W mass, so that it decays to three fermion final states as well as lepton (charged or neutrino) plus meson final states below the QCD scale. The phenomenology here is totally analogous to a majorana ‘heavy neutral lepton’ (HNL), so that we can use results from the corresponding literature [27]. We notice that the decay to the lightest SM leptons and neutrino are of the form Γ˜ν→SM = CF G2 F θ2 192π3 ˜m5 (3.15) (3.15) Denoting with mν,light. the mass of the lightest SM neutrino, the decay width is numerically given by 4 Denoting with mν,light. the mass of the lightest SM neutrino, the decay width is numerically given by Γ˜ν→SM ≈2.8 × 104 s−1 CF mν,light. 0.008 eV m˜ν 10 GeV 4 × ∆2 , (3.16) (3.16) where we assumed ∆ij = ∆δij. To determine whether ˜ν is long-lived we can compare it to Hubble at T ≈m˜ν, their ratio reads Γ˜ν→SM H(m˜ν) ≈1.4 × 10−4 CF 90 g∗(m˜ν) 1 2 mν,light. 0.008 eV m˜ν 10 GeV 2 × ∆2 . (3.17) (3.17) [27] we find CF ∼16 for m˜ν ∼10 GeV. From [27] we find CF ∼16 for m˜ν ∼10 GeV. For Γ/H < 1 the neutrinos are long lived. Their energy density for T < mν redshifts non-relativistically until they decay to SM neutrinos when Γ ∼H. 3.1 Mirror Majorana neutrinos The presence of six right-handed neutrinos guarantees that all the Majorana neutrinos (visible and mirror) are massive. (3.7) For vanishing ∆the mass spectrum of mirror neutrinos is determined by the SM neutrinos, JHEP11(2023)211 m˜νi = mνi ˜v2 v2 . (3.8) (3.8) Therefore the mirror neutrinos are determined by the same neutrino mass ordering of the SM. The lightest neutrino mass is bounded by m ≪ p ∆m2sun ≈0.008 eV in the normal ordering (NO), by m ≪ q ∆m2 atm. ≈0.05 eV for the inverted ordering (IO) and m ≫ q ∆m2 atm. ≈0.05 eV quasi-degenerate ordering (QD). One finds, ˜mnormal light. = h 1.9 NO, 12 IO i GeV m˜u TeV 2 ˜minverted light. = h 12 NO, 12 IO i GeV m˜u TeV 2 (3.9) (3.9) Importantly the masses are quadratic in the VEV while the masses of other particles grow linearly. As a consequence for m˜ν > 10 TeV the mirror neutrinos are not the lightest states in the mirror world. For ∆̸= 0 visible and mirror neutrino mix. After ˜H gets a vev, the lagrangian can be written as L = i¯L /DL+i¯˜ν /∂˜ν+ ˜mi ˜v2 δijLiHLjH+ ˜mi 2 ˜νi˜νi+ ∆ij ˜mj √ 2˜v LiH˜νj+h.c.+ 1 ˜v2 [¯˜νγµν ¯ψγµψ] (3.10) and the mixing scales as θij ∼∆ij v ˜v = 2 × 10−6 ∆ij TeV m˜u . (3.11) (3.11) Integrating out the mirror neutrinos we get the final Weinberg operator for the SM neu- trinos L = i¯L /DL + ˜mi ˜v2 " δij + (∆· ∆T )ij 4 # LiHLjH + h.c. (3.12) (3.12) For ∆ij ≪1, the neutrino masses are controlled by the high-scale see-saw, and their masses are strongly correlated with the mirror neutrino masses as in eq. (3.8). Correlation is lost for ∆ij ∼O(1) when the mass of observable neutrinos becomes dominated by the see-saw with the mirror neutrinos. For ∆ij ≪1, the neutrino masses are controlled by the high-scale see-saw, and their masses are strongly correlated with the mirror neutrino masses as in eq. (3.8). Correlation is lost for ∆ij ∼O(1) when the mass of observable neutrinos becomes dominated by the see-saw with the mirror neutrinos. – 12 – – 12 – Mirror neutrinos decay. Due to the spontaneous breaking of parity the mirror neu- trinos ˜ν are unstable towards decay to SM even if they are the lightest mirror fermions. 3.1 Mirror Majorana neutrinos If the decay is sufficiently slow the mirror neutrinos temporarily dominate the energy density of the universe injecting entropy in the SM thermal bath. This process dilutes all the abundance in the dark sector as well as the baryonic asymmetry. In order to avoid constraints from BBN the reheating temperature should be somewhat larger than 5 MeV. This translates into Γ > 25/s. The regions where neutrinos are long lived leading to entropy injection and are shown in blue in figure 3 where the red region is excluded by BBN bounds. – 13 – 10-1 1 101 102 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 1 101 lightest mirror neutrino mass, m[GeV] Γdecay/H(m) mν, light=0.008eV Δ=1 Δ=0.1 Δ=0.01 η=0.03 η=0.1 η=0.3 10-3 10-2 10-1 100 10-1 100 101 102 \|Δ\| lightest mirror neutrino mass [GeV] TR<5 MeV mu'<1.5 TeV mν,light. = 0.008 eV Figure 3. On the left, lifetime of mirror neutrinos for the reference choice of SM neutrino mν = 0.008 eV. On the right, isolines of dilution (3.21) are shown and bounds from BBN and mass mirror up quark. 10-1 1 101 102 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 1 101 lightest mirror neutrino mass, m[GeV] Γdecay/H(m) mν, light=0.008eV Δ=1 Δ=0.1 Δ=0.01 η=0.03 η=0.1 η=0.3 10-3 10-2 10-1 100 10-1 100 101 102 \|Δ\| lightest mirror neutrino mass [GeV] TR<5 MeV mu'<1.5 TeV mν,light. = 0.008 eV lightest mirror neutrino mass, m[GeV] JHEP11(2023)211 Figure 3. On the left, lifetime of mirror neutrinos for the reference choice of SM neutrino mν = 0.008 eV. On the right, isolines of dilution (3.21) are shown and bounds from BBN and mass mirror up quark. Mirror neutrinos decouple after EWSB, at a temperature that can be computed by evaluating rates induced by Fermi operators with leptons (and light quarks). We call T˜ν,dec such a temperature, and mirror neutrinos decouples when relativistic. Their energy density at the time after they have become non-relativistic is ρ˜ν(T) = ρrad(T) g∗(T ˜m) g∗(T˜ν,dec) 1 3 7 8 × 2 × 3 g∗(Tν,dec) T ˜m T (3.18) (3.18) where T ˜m ≈˜m is the temperature when they are non-relativistic. If Γ ≪H(T ˜m), mirror neutrinos are long lived and they reheat the SM, thanks to an entropy injection. 3.1 Mirror Majorana neutrinos Most of the decay happens at a time around Γ ≈H, which might be already in a phase of matter domination driven by the mirror neutrino. At that time the corresponding SM temperature is TΓ ≈2.1 g∗(T ˜m) g∗(T˜ν,dec) 1 9 g∗(TΓ)7 8 × 2 × 3 g∗(Tν,dec) ! 1 3 Γ2M2 Pl T ˜m ! 1 3 . (3.19) (3.19) The maximal effect of entropy injection is when the neutrino decays while dominating the energy density of the universe. This gives the maximal amount of dilution, and correspond to the maximal reheating temperature TR ≈200 MeV∆ p CF r mν,light. 0.008 eV m˜ν GeV 2 . (3.20) (3.20) The dilution is computed as the ratio of the SM entropy before and after the decay of the mirror neutrino. Using the simplified formulas in [28] the dilution factor reads, The dilution is computed as the ratio of the SM entropy before and after the decay of the mirror neutrino. Using the simplified formulas in [28] the dilution factor reads, ηdil ≡s sΓ = Min " 1 , 1.3 g1/4 ∗ Y˜ν s MPlΓ˜ν→SM m2 ˜ν # ≈Min 1 , 0.01 ∆ p CF rmν,light. 10−2 eV m˜ν GeV (3.21) Note that the heaviest neutrinos whose mass should be larger than 10 GeV in light of the constraint on the mirror up quark does not produce significant dilution. The dilution is thus – 14 – dominated by the lightest neutrino. BBN constraints require that the reheating tempera- ture should be larger than 5 MeV or equivalently that Γ ≳25 s−1. As a consequence only in the region around the physical values of neutrino masses the dilution is relevant. Dilution and BBN constrains are shown in figure 3 right for the reference value mµ = 0.008 eV. Scattering of mirror neutrinos. The same couplings that control the decay of mirror neutrino are also relevant for scattering at energies above their mass. Roughly the rate is given by the decay rate replacing m˜ν with the temperature so that the rate goes as T 5. As a consequence the mirror neutrinos decouple when the temperature drops below the mass. JHEP11(2023)211 3.2 Mirror Dirac neutrinos Now we investigate the opposite regime where the scale of the right-handed neutrino is below or comparable to the P breaking scale, namely where ˜v ≫M± finding two different behaviors depending on the relative size of the two contributions. The discussion in this case will lead us to the vanilla see-saw for the SM neutrinos. In this limit, the mirror neutrinos gets a Dirac mass term, ˜Li ˜H(α∗ ijNj + −β∗ ijNj −), that we can read out from eq. (3.2). Below the scale of the Dirac mirror neutrinos, the effective lagrangian involving SM neutrinos and other states is found along the solution α∗ ijNj + = β∗ ijNj −. Neglecting the flavor structure one finds, Lν = 2Re[α∗β] p \|α\|2 + \|β\|2 LHN −(M+ + M−)(β∗)2 2(\|α\|2 + \|β\|2) N2 + h.c. (3.22) (3.22) Therefore, below the P breaking scale, we are left with the standard scenario for the neutrinos where the right-handed neutrino is a combination of N+ and N−. SM neutrinos are set by the see-saw scale, and we lose the correlation with the mirror majorana case. In the case of 3 generations, the Yukawa couplings are complex allowing for CP violation in the neutrino sector as required by leptogenesis. 3.3 Leptogenesis (3.25) (3.25) Given that P sends NL →−N˜L such number is odd under P, so that until P is exact, the decay of N± will not generate any net Ltot number. Essentially, the second Sakharov con- dition is not satisfied for Ltot. This can be checked explicitly by computing the asymmetry in the decay of N in each sector, Given that P sends NL →−N˜L such number is odd under P, so that until P is exact, the decay of N± will not generate any net Ltot number. Essentially, the second Sakharov con- dition is not satisfied for Ltot. This can be checked explicitly by computing the asymmetry in the decay of N in each sector, ε ≡Γ(N →LH) −Γ(N →L∗H∗) Γ(N →LH) + Γ(N →L∗H∗) , ˜ε ≡Γ(N →˜L ˜H) −Γ(N →˜L∗˜H∗) Γ(N →˜L ˜H) + Γ(N →˜L∗˜H∗) , (3.26) (3.26) where N is the heaviest neutrino. In order to compute the above asymmetries it is con- venient to use the symmetric notation of eq. (3.3). From an explicit computation we find (assuming N = F1), ε = 1 8π X Nj̸=N Im[(Y †Y )2 j1] (Y †Y )11 f M2 j M2 N ! ˜ε = 1 8π X Nj̸=N Im[( eY † eY )2 j1] ( eY † eY )11 f M2 j M2 N ! , (3.27) (3.27) where f(x) = √x[ x−2 x−1 −(1 + x) log 1+x x ] (see [29] for a review). We notice that the loop function is symmetric under exchange of the two sectors, as it should. Moreover eY † eY = I(Y †Y )∗I. Notice also that the combination that enters the above expression is the square of (Y †Y )ij. We then have ( eY † eY )2 ij = (Y †Y )2∗ ij , and also noticing that (Y †Y )kk = ( eY † eY )kk, yielding a relative sign between ε and ˜ε upon taking the imaginary part we have explicitly ε = −˜ε, which supports the claim in eq. (3.25). After N decays and before mirror electroweak symmetry breaking an equal and oppo- site amount of lepton asymmetries is present in each sector, which can be transferred to baryon number through the respective sphaleron transitions, leading to equal and opposite baryon and lepton asymmetries in each sector. We notice that at this epoch both B −L and ˜B −˜L are individually conserved. 3.3 Leptogenesis The structure of right-handed neutrinos N± discussed above allows us to discuss baryo- genesis via leptogenesis, exploiting the asymmetric out-of-equilibrium decay of Majorana neutrinos as in standard leptogenesis (see [29] and [30] for reviews). In our context, we identify two different scenarios, that here we briefly sketch leaving further developments for future work. P-broken leptogenesis. Below the scale ˜v a combination of N± acts as Majorana right- handed neutrino for the SM neutrinos (mirror neutrinos are instead Dirac). This falls into the category of standard leptogenesis. In this scenario the leptonic mirror sector plays no particular role since they are heavier than a Majorana combination of N±. Therefore we can map this to the commonly discussed thermal leptogenesis. In particular, the interactions of the Majorana N can be matched to the following (schematic) effective lagrangian αLHN + MNN + αye f M2 H ˜LN(˜L ˜E) + αyd f M2 H ˜LN( ˜Q ˜D) + αyu f M2 H ˜LN( ˜Q ˜U)∗+ (α →β) (3.23) αLHN + MNN + αye f M2 H ˜LN(˜L ˜E) + αyd f M2 H ˜LN( ˜Q ˜D) + αyu f M2 H ˜LN( ˜Q ˜U)∗+ (α →β) (3.23) (3.23) – 15 – The branching fraction of N decaying to mirror states of is suppressed by factors of order y2 SM/4π2 × M4 N/ f M4 H, leading to ∆L + ∆˜L ̸= 0 and \|∆L\| ≫\|∆˜L\|. We predict [B0, L0] = [−28 79 , 51 79]∆L, [ ˜B0, ˜L0] ≈0 , P −broken N −decay . (3.24) (3.24) P-symmetric leptogenesis. In this case the scale MN is the highest in the system, higher than ˜v. This suggests that the decay of N respects P. In this case both SM and mirror neutrinos are inevitably Majorana. Since by symmetry, CP violation occurs equally in each sector, if the decay of N happens when the mirror weak interactions are unbroken (therefore P is not spontaneously broken) a net amount of lepton asymmetry in each sector will be produced. We refer to the lagrangian in eq. (3.2) in the phase where P is unbroken. The decays of N± → LH, L∗H∗, ˜L ˜H, ˜L∗˜H∗are such that the produced number of L is equal to the number of ˜L∗. In light of this it is convenient to define the total generalized lepton number, JHEP11(2023)211 NLtot = NL + N˜L . 6We assume the kinetic mixing ϵ to vanish and the mixed quartic to be irrelevant. 4 Cosmology with a massless mirror photon The mirror sector contains colored states and a massless dark photon that seem at odds with standard cosmology, if the mirror sector is not empty. As we will show, quite surprisingly the presence of a massless dark photon does not exclude the scenario and moreover the mirror electron elegantly produces the DM abundance from thermal freeze-out from mirror photons. The region of parameters selected by DM in particular is close to the experimental bounds. JHEP11(2023)211 The only link between mirror world and the SM is provided by colored states and neutrinos.6 For large reheating temperature the two sectors are in thermal equilibrium. Specifically if TR > ˜v the whole dark sector including neutrinos thermalizes with the SM at a common temperature. Neglecting for the moment the role of right-handed neutrinos, the first important event is the decoupling of mirror neutrinos, when electro-weak interactions of the type ˜ν˜e ↔˜ν˜e go out of equilibrium. Mirror neutrinos, in absence of other dynamics, decouple relativistically at a temperature T˜ν ≈ ˜v v 4 3 Tν,SM ≈5 × 104 GeV m˜u TeV 4/3 (4.1) (4.1) If they are stable, they would overclose the universe given the masses (3.9). The only viable option in this case would be that the reheating temperature is below the mass lightest colored state, m˜u or mΣ, so that the dark sector never reaches thermal equilibrium with the SM. A much more interesting possibility is that the mirror neutrinos decay to the SM, ∆̸= 0. In this case the reheating temperature can be large and the two sectors are in thermal equilibrium initially. Even if TR < T˜ν so that the neutrinos are not in equilibrium due to the mirror weak interactions, the neutrinos portal will thermalize them. After the mirror neutrinos decay the dark sector contains massless mirror photon that contributes the dark radiation and massive stable states, mirror electron and hadrons made of mirror up quark contributing to DM. 3.3 Leptogenesis When the mirror Higgs develops a vev, mirror sphalerons shut down and ˜B becomes conserved. However ˜L violations become active again since the mirror neutrinos gets a – 16 – majorana mass. Moreover, if ∆is sizable this can recouple L and ˜L. In turn, this can potentially lead to a washout of the SM baryon asymmetry produced thus far since both B + L (SM sphalerons) and L violating processes can now be active. This has the effect of (partially) washing out B generated before. We leave this interesting scenario open to future investigations. 7In the limit ˜v →∞mirror weak interactions decouple and individual fermion number of leptons and quarks is conserved. In the relevant region of parameters the decay rate of heavier species, Γ ∼M 5/˜v4 is fast enough to maintain thermal equilibrium with the plasma so the approximate stability does not play a role for the abundances. 4.1 Relativistic degrees of freedom SM and mirror sector maintain equilibrium until T∗≈Min[m˜u , mΣ]/25 through ordinary color interactions. Below T∗the mirror sector contains mirror electrons and mirror pho- tons, while at the same temperature the SM has its full thermal degrees of freedom. (Af- ter the mirror electrons decouple the mirror photon temperature gets reheated compared to the SM). – 17 – Assuming that the two sectors decouple at temperature T∗the evolution of the tem- perature is given in general by, ξ(T) ≡ ˜T(T) T = ˜g∗(T∗) g∗(T∗) 1 3 g∗(T) ˜g∗( ˜T(T)) ! 1 3 (4.2) (4.2) Assuming the mirror up quark to be the lightest colored state we estimate T∗∼m˜u/25. At that temperature the only relativistic degrees of freedom in the mirror sector is the photon while mirror neutrinos typically remain into thermal contact with the SM. We find, Assuming the mirror up quark to be the lightest colored state we estimate T∗∼m˜u/25. At that temperature the only relativistic degrees of freedom in the mirror sector is the photon while mirror neutrinos typically remain into thermal contact with the SM. We find, ξ(T ≤T∗) = 0.34 100 g∗(T∗) 1 3 (4.3) JHEP11(2023)211 (4.3) where is the effective number of degrees of freedom of the SM plus mirror neutrinos. This allows us to compute ∆Neff from the presence of the light mirror photon, as ∆Neff 4 CMB = 8 7 11 4 4 3 ξ 4 CMB ≈0.06 100 g∗(T∗) 4 3 (4.4) (4.4) While this contribution is currently consistent with experimental bounds it can be tested in future experiments.ii This result is modified if neutrinos are long lived. The entropy injection (3.21) modifies the previous results as, ∆Neff ≈0.06η4/3 dil (4.5) rror electrons (and mirror up quark) ∆Neff ≈0.06η4/3 dil (4.5) (4.5) 8We have checked that in the relevant regions of parameters freeze-out continues to take place in the relativistic regime. 4.2 Thermal dark matter: mirror electrons (and mirror up quark) Mirror worlds contain automatically stable particles as a consequence of their accidental symmetries. By construction mirror baryon number and electric charge are accidental symmetries of the mirror sector. It follows that the lightest state carrying baryon number and the mirror electron are stable.7 For the latter, since the U(1) symmetry is gauged and unbroken, stability is exact while for baryon number conservation holds up to dimension 6 operators as in the SM. As we will show the mirror electron is a good DM candidate being neutral under the SM. Its mass is predicted by the DM abundance and it turns out to be borderline with current constraints. The lightest state with baryon number is the mirror up quark. This state is charged under ordinary QCD at low energy so naively it is not a viable DM candi- date. The thermal abundance turns out to be suppressed compared to the mirror electron. Moreover as shown in [31] heavy colored states mostly form deep Coulombian bound states that are SM singlets and are thus good DM components. The residual small fraction of mirror up quark forms QCD-size exotic hadrons binding with ordinary matter. These states feature large hadronic cross-sections and they are very constrained experimentally, – 18 – their fraction should be smaller than O(10−4). Remarkably due to the double suppression of the abundance of hybrid QCD states the scenario appear to be viable. At the same time deviations from CDM are predicted that might be observable in future experiments. Mirror electrons as DM. The lightest charged states in the mirror world are electrons ˜e that are coupled to massless dark photons. The system at energies below m˜u is just dark QED, 1 L = ¯˜ei /D˜e −m˜e¯˜e˜e −1 4 ˜F 2 µν (4.6) (4.6) Mirror electrons and up quark follow an equilibrium distribution due to annihilation into mirror photons. Allowing for a different temperature between visible and mirror sector at decoupling one finds [28], Mirror electrons and up quark follow an equilibrium distribution due to annihilation into mirror photons. Allowing for a different temperature between visible and mirror sector at decoupling one finds [28], JHEP11(2023)211 Ωh2 0.12 = ξ 106.75 g∗(T∗) s g∗(T∗) + ˜g∗(T∗)ξ4 106.75 1 ⟨σeffv⟩(23 TeV)2 (4.7) (4.7) where g∗is the effective number of degrees of freedom of visible and mirror sector at freeze-out and ξ the ratio of temperatures. 4.2 Thermal dark matter: mirror electrons (and mirror up quark) The tree level annihilation cross-sections of electrons is given by, ⟨σe¯ev⟩= πα2 EM m2e (4.8) (4.8) This should be corrected by two related effects: and Sommerfeld enhancement and bound state formation that adds an annihilation channel [32]. For αEM ∼10−2 however the modification to the tree level cross-section is very small and be neglected. At freeze-out ξ ≈1 the temperature is between 10 and 50 GeV so that the pre-factor in eq. (4.7) can be neglected in what follows. Computing the abundance with the tree level cross-section with αEM = 1/128 one finds that mirror electrons reproduce the DM abundance for m˜e ≈300 GeV. Using the fact that m˜u ∼2m˜e we would thus predict m˜u ∼600 GeV that is excluded experimentally. This estimate however neglects the slow decay of mirror neutrinos. If this happens for T ≲m˜e/25 (the temperature where mirror electrons decouple from the thermal bath) it leads to entropy injection in the SM thermal bath that the depletes the DM abundance according to the factor (3.21).8 This effect is generic in most regions of parameter space. We show the DM abundance accounting for the entropy dilution in figures 4 and 5. The critical abundance is reproduced for, MDM = [300, 1000] GeV (4.9) (4.9) MDM = [300, 1000] GeV allowing to evade possible collider bounds on colored states. A precise determination of the DM depends on the details of the neutrino sector and requires the solution of the coupled Boltzmann equations that we leave to future work. There are two potential constraints on DM charged under a long-range interaction: i) the delay of kinetic decoupling (suppressing power on small scales); ii) the modification 8We have checked that in the relevant regions of parameters freeze-out continues to take place in the relativistic regime. – 19 – 10-3 10-2 10-1 1 200 400 600 800 1000 1200 1400 1 10 \|Δ\| mirror electron mass [GeV] no dilution TR<5 MeV mu'< TeV ΩDM for mν=0.008eV 10-3 10-2 10-1 1 200 400 600 800 1000 1200 1400 1 10 \|Δ\| mirror electron mass [GeV] lightest mirror neutrino mass [GeV] no dilution TR<5 MeV mu'< TeV ΩDM for mν=0.008eV 10-3 10-2 10-1 1 200 400 600 800 1000 1200 1400 10 102 \|Δ\| mirror electron mass [GeV] lightest mirror neutrino mass [GeV] no dilution TR<5 MeV mu'<TeV ΩDM for mν=0.05eV Figure 4. 4.2 Thermal dark matter: mirror electrons (and mirror up quark) Mirror electron DM mass as function of mixing ∆accounting for entropy injection (solid black line). On the left the abundance assuming that the relevant entropy injection is associated to the SM neutrino with mass mν = 0.008 eV (normal hierarchy). On the right mν = 0.05 eV (inverted hierarchy). Light red region is excluded by BBN constraints. 10-3 10-2 10-1 1 200 400 600 800 1000 1200 1400 10 102 \|Δ\| mirror electron mass [GeV] lightest mirror neutrino mass [GeV] no dilution TR<5 MeV mu'<TeV ΩDM for mν=0.05eV lightest mirror neutrino mass [GeV] \|Δ\| \|Δ\| Figure 4. Mirror electron DM mass as function of mixing ∆accounting for entropy injection (solid black line). On the left the abundance assuming that the relevant entropy injection is associated to the SM neutrino with mass mν = 0.008 eV (normal hierarchy). On the right mν = 0.05 eV (inverted hierarchy). Light red region is excluded by BBN constraints. JHEP11(2023)211 10-4 10-3 10-2 10-1 200 400 600 800 1000 1200 1400 lightest SM neutrino mass [eV] mirror electron mass [GeV] no dilution TR<5 MeV Δ>1 mu'<TeV mν > 50 meV Δ=0.1 ΩDM ΩDM Δ=0.01 Figure 5. Prediction for the SM lightest neutrino mass in connection with the DM relic abundance, for different values of ∆. Light red region is excluded by BBN. 10-4 10-3 10-2 10-1 200 400 600 800 1000 1200 1400 lightest SM neutrino mass [eV] mirror electron mass [GeV] no dilution TR<5 MeV Δ>1 mu'<TeV mν > 50 meV Δ=0.1 ΩDM ΩDM Δ=0.01 Figure 5. Prediction for the SM lightest neutrino mass in connection with the DM relic abundance, for different values of ∆. Light red region is excluded by BBN. to DM properties in clusters and galaxies. The first problem is not very relevant for heavy DM. The kinetic decoupling can be estimated to happen Tk ≈MeV(M/TeV)3/2/ξ2 × (0.01/α) [33], safely before BBN for heavy masses, while the second constraint extends to even larger DM masses [34]. The strongest constraints arise from the structure of galaxy halos as for the observed ellipticity of the NGC720 halo [35]. Demanding that the interaction are sufficiently weak not to deform the DM velocity distribution over the lifespan of a galaxy one can estimate the bound [34] to DM properties in clusters and galaxies. The first problem is not very relevant for heavy DM. 4.2 Thermal dark matter: mirror electrons (and mirror up quark) The kinetic decoupling can be estimated to happen Tk ≈MeV(M/TeV)3/2/ξ2 × (0.01/α) [33], safely before BBN for heavy masses, while the second constraint extends to even larger DM masses [34]. The strongest constraints arise from the structure of galaxy halos as for the observed ellipticity of the NGC720 halo [35]. Demanding that the interaction are sufficiently weak not to deform the DM velocity distribution over the lifespan of a galaxy one can estimate the bound [34] α ≲0.2 MDM TeV 2 (4.10) (4.10) so that mirror electrons in the TeV range can be good DM candidates. so that mirror electrons in the TeV range can be good DM candidates. – 20 – Another constraint on this scenario arise from underground direct detection exper- iments. As discussed in [11, 12] even if absent at tree level a kinetic mixing between hyper-charges arises at loop level. For MΣ > m˜u running effects at 4-loop order generate ϵ ∼10−8 that is already in tension with Xenon 1T. This conclusion assumes that the local DM density is not modified by the supernova shock waves that tend to expel it from the galaxy [36], see however [37]. The effect is suppressed if MΣ ∼m˜u as in that case the run- ning only arises at higher loop level. This would however require a coincidence of scales. In any case direct detection experiments appear very relevant for this scenario and might already exclude this scenario. JHEP11(2023)211 Colored DM fraction. The abundance of color triplets with no visible electric charge leads to a component made of colored DM [38]. Naively DM made of strongly interacting particles is not viable because for example it would form exotic nuclei where one of the ordinary quarks is replaced by the exotic quark. These nuclei have QCD like cross-sections and lead to very strong constraint on their abundance that can be at best a small fraction of DM. However for heavy DM, colored particles can form deep Coulombian bound states with binding energy EB ∼α2 3MQ much larger than the confinement energy. These bound states are small color singlets that behave as collision-less DM for practical purposes. The fraction of DM that ends up into exotic nuclei with QCD cross-sections and into Coulombian bound states is a dynamical question that requires to solve the coupled Boltzmann equations. 4.2 Thermal dark matter: mirror electrons (and mirror up quark) As it turns out most of the DM actually forms deep bound states that are energetically favoured leaving just a small fraction of strongly interacting heavy quarks that hadronize with ordinary matter. This general mechanism was explored in details in [38] for a scenario with a Dirac color octet. The QCD-like stable bound states have in this case zero electric charge leading to weaker constraints. It was in particular shown that the abundance can be reproduced for a mass M ˜Q = 12.5 TeV. In the current scenario the heavy quark is a color triplet with zero charge leading to exotic nuclei with fractional electric charges. The experimental constraints are in this case stronger and it is unclear whether the scenario would be allowed if this made 100% of DM. In our case however only a fraction of DM is in the form of colored particles leading to weaker constraints. Extremely strong bounds were however derived in [36] re-interpreting searches of magnetic monopoles assuming that they are accelerated by supernova shock-waves and arrive to the detector with sufficient energy. y pfi gy The abundance from freeze-out can be simply estimated using the ⟨σu¯uv⟩= 2 43S4/3 + 41 43S−1/6 × 43 54 πα2 3 M2u , Sλ = 2πλα3/vrel 1 −e2πλα3/vrel (4.11) ⟨σu¯uv⟩= 2 43S4/3 + 41 43S−1/6 × 43 54 πα2 3 M2u , Sλ = 2πλα3/vrel 1 −e2πλα3/vrel (4.11) (4.11) where S is the Sommerfeld enhancement (computed in the massless limit) and we neglected the subleading decay to mirror photons. Using vrel ∼0.3 at decoupling and α3(Mu) = 0.1 one finds an abundance of colored states Ωu¯u/Ωe¯e ∼0.1. We note that this estimate is conservative. As discussed in [38] bound state formation enhances the effective annihilation cross-section reducing the abundance. Moreover after QCD confinement about half of the particles bind into unstable bound states leaving half of colored particles in states with net baryonic charge. Therefore the fraction of strongly interacting DM is very suppressed. – 21 – In summary the scenario of [1] leads to sharp predictions for DM made of mirror electrons and up quarks with mass in the TeV range. The scenario is currently close to experimental limit on DM charged under a long range force predicting new colored states just above the collider constraints that give rise to a small component of strongly interacting DM. 4.2 Thermal dark matter: mirror electrons (and mirror up quark) Future experiments can thus test this scenario. 5 Phenomenology of massive mirror photon Let us now turn to the scenario where hypercharge as color is broken to the diagonal combination. In this case a combination of U(1) gauge bosons has mass and the mirror states acquire SM quantum numbers as in the table 2 right. In particular the states have identical electric charge as the SM partners.9 JHEP11(2023)211 To understand the phenomenology of the mirror photon in this context, it is useful to focus on the effective lagrangian of the neutral gauge bosons, Lneutral = −1 4(W 2 µν + ˜W 2 µν + B2 µν + ˜B2 µν + 2ϵBµν ˜Bµν) +v2 8 (g2Wµ −g1Bµ)2 + ˜v2 8 (g2 ˜Wµ −g1 ˜Bµ)2 + g2 1q2Σ2 0(Bµ −˜Bµ)2 (5.1) (5.1) where we neglect the difference of mirror couplings due to different running below ˜v and we also added a kinetic mixing for hypercharge as allowed by the symmetries. Given that for phenomenological reasons ˜v ≫v, it is a good approximation to work in the limit ˜v →∞. To leading order this corresponds to replace ˜W = g1/g2 × ˜B in the above lagrangian so that, ˜v≫v neutral = −1 4(W 2 µν + B2 µν) −1 4 1 + g2 1 g2 2 ! ˜B2 µν −ϵ 2Bµν ˜Bµν + v2 8 (g2Wµ −g1Bµ)2 + g2 1q2Σ2 0(Bµ −˜Bµ)2 (5 (5.2) The 3×3 mass matrix can be exactly diagonalized since has a zero eigenvalue corresponding to the SM photon. However there are two distinct regimes depending on the relative size of v and qΣ0: Light dark photon, MA ≪MZ. This corresponds to qΣ0 ≪v so we can further expand around v →∞. Integrating out Wµ at tree level from (5.2) one finds, L MA≪MZ eff = −1 4 1 + g2 1 g2 2 ! (B2 µν + ˜B2 µν)−ϵ 2Bµν ˜Bµν +q2Σ2 0(Bµ−˜Bµ)2−g1BµJµ EM−g1 ˜Bµ ˜Jµ EM (5.3) (5.3) 9As discussed in section 2.3 adding extra-dynamics such as electro-weak scalar triplets, the photon could acquire mass in the mirror sector without breaking hypercharges to the diagonal. In this case the mirror sector remains electrically neutral under the SM and the phenomenology can be similar to the one of [1] avoiding however possible constraints of DM charged under long interactions. 9As discussed in section 2.3 adding extra-dynamics such as electro-weak scalar triplets, the photon could acquire mass in the mirror sector without breaking hypercharges to the diagonal. 5 Phenomenology of massive mirror photon In this case the mirror sector remains electrically neutral under the SM and the phenomenology can be similar to the one of [1] avoiding however possible constraints of DM charged under long interactions. – 22 – where we have added the coupling to electro-magnetic currents. In terms of the mass eigenstates γ = 1/ √ 2(B + ˜B) (massless photon) and A = 1/ √ 2(B −˜B) (the dark photon) the lagrangian takes the form L MA≪MZ eff = −1 4 1 + g2 1 g2 2 + ϵ ! γ2 µν −1 4 1 + g2 1 g2 2 −ϵ ! A2 µν + 2g2 1q2Σ2 0A2 µ −g1 √ 2γµ(Jµ EM + ˜Jµ EM) −g1 √ 2Aµ(Jµ EM −˜Jµ EM) (5.4) For E < mA the SM and mirror electric currents couple identically to the massless dark photon: the mirror states have same electric charge independently of ϵ. Note however that the coupling is reduced by 1/ √ 2 below and above mA so that QED is completely modified at low energies.f JHEP11(2023)211 In order to connect with the dark photon literature it is useful a different basis. We define γ = B and B −˜B = √ 2A so that L MA≪MZ eff = −1 2 1 + g2 1 g2 2 + ϵ ! γ2 µν −1 2 1 + g2 1 g2 2 ! A2 µν + 1 √ 2 1 + g2 1 g2 2 + ϵ ! γµνAµν +2q2Σ2A2 µ −g1γµ(Jµ EM + ˜Jµ EM) + √ 2g1Aµ ˜Jµ EM (5.5) (5.5) In the language of dark photon this corresponds to an order 1 mixing. Constraints on CMB µ−distoritions require MA ≲10−15 eV and even stronger bounds follow from Jupiter magnetic field, see [39] for a review. For such small masses the phenomenology reduces to the one of ref. [1]. Heavy dark photon, MA ≫MZ. In this regime from eq. (5.2) the mass of the dark photon is m2 A ≈4q2Σ2 0, and it behaves as a perfect copy of the SM hyper-charge, since it couples to the hyper-charge current with coupling g1/ √ 2. Direct searches for this type on gauge bosons has been studied for example in [40, 41] with bounds in the multi-TeV region. A heavy dark photon also leaves its imprints in terms of higher-dimensional operators in the SM effective theory. 5 Phenomenology of massive mirror photon To study the precision physics effects on the SM it is convenient to integrate out ˜B and expand in momenta, LSMEFT = −1 4W 2 µν −1 4 2 + g2 1 g2 2 ! B2 µν −ϵ 2B2 µν + v2 8 (g2Wµ −g1Bµ)2 + (g2 1 + g2 2)2 4g2 2q2Σ2 0 (∂ρBµν)2 (5 6) (5.6) From the kinetic term we extract the SM hypercharge gauge coupling, 1 g2 Y = 2(1 + ϵ) g2 1 + 1 g2 2 (5.7) (5.7) The 4 derivatives term gives a contribution to the precision electro-weak parameter Y parameter [42] Y = 2(1 + ϵ)2m2 W m2 A (g2 2 + g2 Y )2 (g2 2 −g2 Y ) (5.8) (5.8) Current bound require \|Y \| < 0.2 × 10−3 [43] so that mA > 10 TeV that is stronger than the one from direct searches. The bound is also expected to improve with future LHC data [40, 44, 45]. Current bound require \|Y \| < 0.2 × 10−3 [43] so that mA > 10 TeV that is stronger than the one from direct searches. The bound is also expected to improve with future LHC data [40, 44, 45]. – 23 – – 23 – 5.1 Cosmological history Naively since the dark photon is massive one might expect that this scenario would be less constrained. In fact the opposite is true. Due to the spontaneous breaking of hypercharge to the diagonal all the mirror states have the same electric charge as the SM states. Since the mirror electron and up quark are stable in order to escape from bounds their abundance should be negligible. As we have shown if the mirror sector is initially in thermal equilibrium with the SM the abundance of electrons saturates the DM for masses below TeV. The only possibility is thus that the mirror sector is never in thermal equilibrium with the SM. Given that the bridge between the two sectors is given by colored states we estimate, JHEP11(2023)211 TR < 1 25Min[m˜u , mΣ] (5.9) (5.9) When this condition is satisfied the abundance of mirror is negligible. Since the reheating temperature is low the mechanisms thermal leptogenesis cannot be realized in this frame- work. The only states that can be populated are thus neutrinos that are produced through the neutrino portal. When this condition is satisfied the abundance of mirror is negligible. Since the reheating temperature is low the mechanisms thermal leptogenesis cannot be realized in this frame- work. The only states that can be populated are thus neutrinos that are produced through the neutrino portal. Mirror neutrinos as Dark Matter? An interesting possibility for DM is offered by the mirror neutrinos that are the only neutral states when hypercharge is broken to the diagonal, see table 2. Mirror neutrinos with masses in the KeV range can give rise to sterile neutrino DM, see [46, 47] for reviews. Due to the mixing with the SM neutrinos they can be produced via freeze-in from the SM thermal bath. The abundance is roughly reproduced for, θ 10−5 × m˜ν 10 keV ≈1 . (5.10) (5.10) Note that if the mirror neutrinos are Majorana the mixing is given by (3.11) so that it is difficult to obtain the critical abundance from freeze-in given the bound m˜u > 1.5 TeV. Other mechanisms could however be considered. Moreover in the regime where mirror neutrinos are Majorana the mirror neutrinos have masses given by eq. (3.8). This implies that the lightest neutrino must be lighter than 10−9 eV if the lightest mirror neutrino is in the KeV range. 6 Conclusions All known solutions of the strong CP problem rely on the existence of a new symmetry. In the case of the QCD axion the SM should be augmented with a U(1)PQ global symmetry only broken by the QCD anomaly. The existence of an almost exact (apart from anoma- lies) global symmetry raises the question of axion quality problem [48], especially since in quantum gravity no global symmetries are known to exist. In the Nelson-Barr solution CP must be assumed to be an exact symmetry that is spontaneously broken and a non-generic structure of couplings of new fermions must also be enforced.i From this point of view we find it fascinating that the strong CP problem might be solved by a space-time symmetry such as a generalized parity, P. This idea was originally advocated in the context of left-right models [5] and further generalized in [6] but has not – 24 – received as much attention as the QCD axion or even the Nelson-Barr mechanism. Here we have built upon ref. [1] where an even simpler scenario was considered. The SM is extended with a mirror copy related by spacetime parity. The breaking of SU(3) × f SU(3) →SU(3)c then implies a boundary condition θs = θ + ˜θ = 0 solving the strong CP problem. In this work we extended ref. [1] in several ways. First we have identified different patterns of symmetry breaking that allow to solve the strong CP problem but with dif- ferent phenomenological implications. The existence of other minima in the SM Higgs effective potential suggests the possibility to break P spontaneously. We have shown that in the simplest scenario where the breaking of color is realized by a scalar bi-fundamental consistent high scale vacua exist precisely for the measured value of the top quark and Higgs masses. We have then included right-handed neutrinos necessary to give masses to visible and mirror neutrinos. This leads to an interesting structure of mirror neutrinos whose masses scale quadratically with the mirror Higgs VEV. The neutrino dynamics is particularly important for the cosmological history of the model as they are typically long lived. Moreover if the reheating temperature is large thermal leptogenesis can be realized in the SM without producing an asymmetry in the mirror world. JHEP11(2023)211 The phenomenology depends crucially on the fate of the mirror photon. 6 Conclusions If the mir- ror hypercharge remains unbroken as in [1] all the mirror states have no electric charge under the SM. This leads to a very predictive scenario that is determined by the VEV of the mirror Higgs. Assuming a large reheating temperature the dark photon contributes to the effective number of relativistic degrees of freedom, predicting sizable ∆Neff. The mirror electrons reproduce the DM abundance for mass in the range 500–1000 GeV, that is marginally allowed by astrophysical and collider constraints and would produce signals in direct detection experiments. A small fraction of strongly interacting DM is also predicted in the form of bound states of the mirror up quark that however leads to severe experi- mental constraints. The detailed predictions depend on the mirror neutrinos whose decay leads entropy injection in the SM plasma. Overall the DM scenario appears somewhat in tension with experimental constraints motivating extensions of the minimal setup. If the pattern of symmetry breaking also includes hypercharge so that U(1) × eU(1) → U(1)Y the mirror states have the same electric charge as the SM states. This implies that the mirror electron and up quarks that are stable have electric charge. As a consequence this scenario is only viable for a reheating temperature lower than the mirror electron mass. The only viable DM candidate is the lightest mirror neutrino that could realize sterile neutrino DM depending on the mass of the lightest neutrino. Direct and indirect collider constraints require that the dark photon mass is larger than 5-10 TeV. All in all solving the strong CP problem through P appears as an attractive possibility that should be considered seriously. In this work we have just scratched the surface of the phenomenology of mirror P world. A more detailed analysis will be surely needed to study the details of dark matter, leptogenesis and collider physics in this scenario. 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W4388295236.txt	https://bmcpsychiatry.biomedcentral.com/counter/pdf/10.1186/s12888-023-05321-7	en		Emotional control in selected somatic and psychiatric diseases	BMC psychiatry	2,023	cc-by	8,115	Orzechowska et al. BMC Psychiatry (2023) 23:802 https://doi.org/10.1186/s12888-023-05321-7 BMC Psychiatry Open Access RESEARCH Emotional control in selected somatic and psychiatric diseases Agata Orzechowska1, Paulina Maruszewska1, Małgorzata Gałecka2, Philip Hyland3, Daniel Boduszek4,5, Piotr Gałecki1 and Katarzyna Bliźniewska-Kowalska1* Abstract The aim was to assess the level of subjective control of emotional states among patients treated for dermatological and gastrointestinal somatic diseases compared to those with depressive and anxiety disorders. The results were related to the analyzed dimensions of emotion regulation in healthy subjects. Materials and methods The reports of the conducted studies were compiled for a total of 310 people, including 120 patients diagnosed with a somatic disease (psoriasis, rosacea, irritable bowel syndrome, and gastroesophageal reflux), as well as 96 patients diagnosed with depressive disorders and 30 patients with anxiety disorders. The control group consisted of healthy subjects (64 individuals). To assess the psychological variables analyzed, the subjects completed the Emotion Regulation Questionnaire developed by J. Brzeziński. Results The study showed that the patients suffering from a chronic somatic symptom disorder, similarly to those treated for depression and anxiety disorders, differed from the healthy individuals in most aspects of emotional control. The patients with dermatological and gastrointestinal diseases differed statistically significantly from the patients with depression and the patients with anxiety disorders in relation to three dimensions of emotional control. Patients with a somatic disease are characterized by higher emotional and rational motivation, lower emotional resilience and lower emotional arousal. Conclusions A chronic disease co-occurs with the emotional sphere of a person’s daily functioning. Regardless of the diagnosis in terms of somatic disorders and mental illnesses, the way in which emotional states are controlled can be an important factor in the onset of the disease, coping with it as well as the treatment process. Keywords Emotional control, Emotion regulation, Somatic Disease, Mental Illness *Correspondence: Katarzyna Bliźniewska-Kowalska katarzyna.blizniewska-kowalska@umed.lodz.pl 1 Department of Adult Psychiatry, Medical University of Lodz, Lodz 91-229, Poland 2 Department of Psychotherapy, Medical University of Lodz, Lodz 91-229, Poland 3 Department of Psychology, Maynooth University, Maynooth, Co. Kildare, Ireland 4 Faculty of Psychology, SWPS University, Wrocław 53-238, Poland 5 Department of Psychology, University of Huddersfield, Huddersfield HD1 3DH, UK © 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://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. Orzechowska et al. BMC Psychiatry (2023) 23:802 Introduction Each person is characterized by a tendency to react emotionally in a certain way, which is typical and fairly independent of the situation. This tendency involves three components of emotions, i.e., the individual’s subjective feelings, physiological changes, and the expression of emotion as presented in the person’s behavior. The ability to control behavior subject to emotion is inherent in these components. Emotional control is the process by which people can influence what emotions they experience, when the emotions are exacerbated, and how they evaluate and expresses them. Emotional control is an indicator of emotional maturity and the ability to constructively express one’s emotional states depending on the situational context [1, 2]. Emotional and affect control is a component of emotion regulation, which is a broader term—although it is sometimes used interchangeably with emotional control, and includes the processes responsible for initiating, modeling, and maintaining the experience as well as expression of emotions. Emotion regulation refers to the processes of self-control and to correcting the ways in which emotions are expressed, changing their content and intensity, modulating emotion-related behaviors, and serving to undertake adaptive and goal-oriented action strategies. Emotional control is responsible for strengthening, weakening or changing an emotional response. A person with properly developed emotional control is able to inhibit their emotions in some situations and freely reveal them in others [3–6]. Although emotional responses are usually appropriate to the demands of the environment and allow for an effective action, there are times when a person’s experienced emotions lead to behavior that is contrary to their needs and goals. Features of dysfunctional experience and regulation of emotions are usually observed and analyzed in people suffering from mental illnesses, such as affective disorders, anxiety disorders or personality disorders, fitting into the clinical picture of these disorders. It has been shown that such people reveal problems in understanding emotions and their control and, as a result, may have periodic or long-term difficulties in forming close emotional ties, or their ties are characterized by ambivalence, conflicts or overdependence on others [4, 7]. Of the most common and costly brain diseases listed in terms of treatment, more than 60% of the social and economic costs are generated by mental disorders from the group of depressive and anxiety disorders, being one of the most common reasons for seeking help from specialists [8]. The two disorders share similar biological mechanisms that determine their underlying causes, as well as common psychological mechanisms, including emotional and cognitive phenomena such as chronic worry and ruminations, fixed negative thought patterns Page 2 of 11 about oneself, one’s surroundings and the future, resulting in interpersonal problems [9]. Recently, emotion regulation processes have also been considered in the context of chronic somatic diseases, not only as a reflection of the negative consequences of the disease, such as its inconvenience and chronic nature, but also as a risk factor involved in their formation. The emotional sphere of the personality, including mainly tension and negative emotions and difficulties in regulating them properly, in conjunction with difficult and stressful situations, co-occur with a decrease in the body’s immunity [10, 11]. The psychophysiological mechanism of emotion may be an important and common risk factor relevant to the course of both mental and somatic chronic diseases. The prevalence of unpleasant emotions and the inability to discharge negative emotional tension increase vulnerability to stress and adversely affect health. Thus, the control of emotions in difficult situations, which undoubtedly include the struggle with a chronic disease, is important for the process of coping with the course of the disease [12, 13]. The regulation of emotions takes place through three brain regions working together. The brainstem structures are responsible for the most elementary, innate and unconscious drive reactions (arousal or inhibition and autonomic responses). The limbic system, including mainly the amygdala and hippocampus, modifies emotional responses depending on incoming external stimuli—the environment. The prefrontal cortex, on the other hand, is responsible for feelings (conscious emotions) and control over emotions [14, 15]. The formed biological structures of the human brain are subject to various external impacts, which can act supportively or negatively at each developmental stage determining our resilience and coping ability [16]. Thus, fixed abnormal behavioral traits can lead to dysregulation of the functioning of specific brain structures and a constant proinflammatory response by the immune system, triggering a cascade of reciprocal feedback. These changes can consequently lead to mood disorders, including depression, personality disorders, psychoses, anxiety disorders and dementia syndromes [7]. Psychoneuroimmunology is the study of the interactions among behavioral, neural, and endocrine, and immune processes. The brain communicates with the immune system through autonomic nervous system and neuroendocrine activity. Both pathways generate signals that are perceived by the immune system via receptors on the surface of lymphocytes and other immune cells. An activated immune system, in turn, generates cytokines that are perceived by the nervous system. Thus, bidirectional connection between the brain and the immune system reinforces the hypothesis that immune changes Orzechowska et al. BMC Psychiatry (2023) 23:802 Page 3 of 11 Table 1 Characteristics of the study groups part 1- mental problems versus healthy control Variable Depression (N = 96) Gender Age 66 F 30 M 47.07 ± 11.12 (min. 18 / max. 61) Age depression versus anxiety disorders Z = 4.12 p = 0.000 (p < 0.05) Cohen’s d = 2.15 Age depression versus healthy subjects Z = 6.68 p = 0.000 (p < 0.05) Cohen’s d = 3.15 Anxiety disorders (N = 30) 17 F 13 M 36.63 ± 11.25 (min. 19 / max. 60) Age anxiety disorder versus healthy subjects Healthy subjects (N = 64) 38 F 26 M 32.77 ± 10.56 (min. 20 / max. 61) Z = 1.71 p = 0.088 F—female, M—male; N- number; ±—standard deviation; Z—Mann-Whitney U test; p—statistical significance could mediate some of the effects of psychological factors on health and disease [17]. Immunological disorders and chronic inflammation, which can consequently cause similar abnormal psychological reactions and disorders as in mental illnesses, can also occur in the course of some chronic somatic symptom disorders [13, 18]. The aim of the study was to analyze emotional control variables in several somatic diseases (dermatological- psoriasis, rosacea- and gastroenterological—irritable bowel syndrome, gastroesophageal reflux disease) compared to patients with selected mental illnesses (depression and anxiety disorders), against healthy subjects. Materials and methods Surveys among patients with somatic disease and mental illness were conducted in dermatology and gastroenterology departments, as well as in the psychiatric ward of university hospitals in Lodz, Poland. The somatic disease group included patients with psoriasis (30 patients), rosacea (30 patients), irritable bowel syndrome (IBS) (30 patients), and gastroesophageal reflux disease (GERD) (30 patients). The mean age of all individuals surveyed in this group was M = 38.91 years, standard deviation (SD) = 11.74, minimum age = 18 years, maximum = 55 years. The patients with depressive disorders, including diagnoses of depressive episode (F 32 according to ICD-10 criteria) and recurrent depressive disorder (F 33) − 96 patients, and patients with anxiety disorders (30 patients) in the form of phobias (F 40) and other anxiety disorders (F 41), were under hospital treatment. The mean age of all the people surveyed in this group was M = 44.59 years, standard deviation (SD) = 11.97, minimum age = 18 years, maximum = 61 years. Patients with mental comorbidity, somatic disease other than the disease entities selected for the study, including neurological, inflammatory and oncological, were excluded from the study. The authors did not interfere with the diagnosis and treatment process at any stage of the study. The research was performed individually by the authors using the research methods described below. Medical data on the course of the disease were obtained directly from the patients and from attending physicians (with the patients’ consent). The comparison group (64 people) included those who did not meet the criteria for a diagnosis of mental disorders and somatic diseases. The mean age of all the people surveyed in this group was M = 32.77 years, SD = 10.56, minimum age = 20 years, maximum age = 61 years. Detailed sociodemographic data describing all the subjects invited to the study, including patients with somatic disease and mental illness, and the control group of healthy subjects, are provided in Tables 1 and 2. The selection of participants for the study group was random. Each subject gave written informed consent to participate in the study. Each study was conducted in accordance with the rules of the Data Protection Act, and its design was approved by the Bioethics Committee of Table 2 Characteristics of the study groups part 2 – somatic diseases Variable Psoriasis (N = 30) Rosacea (N = 30) Gastroesophageal reflux (GERD) (N = 30) Gender Age 13 F 17 M 38.33 ± 12.81 (min. 18 / max. 55) 18 F 12 M 43.43 ± 9.92 (min. 27 / max. 55) 12 F 18 M 41.43 ± 10.96 (min. 19 / max. 55) Total: Age depression versus somatic diseases Age somatic diseases versus healthy subjects Z = 5.12 p = 0.000 (p < 0.05) Cohen’s d = 3.34 Z = 3.39 p = 0.001 Cohen’s d = 2.89 Age anxiety disorders versus Z = 0.95 somatic diseases p = 0.344 Age mental illnesses versus somatic diseases F—female, M – male; N- number; ±—standard deviation; Z—Mann-Whitney U test; p—statistical significance Z = 3.82 p = 0.0001 Cohen’s d = 3.43 Irritable bowel syndrome (IBS) (N = 30) 22 F 8 M 32.43 ± 10.57 (min. 19 / max. 55) Orzechowska et al. BMC Psychiatry (2023) 23:802 Page 4 of 11 Table 3 Duration of disease Table 6 Emotional control: mental illness vs. healthy subjects Number of patients (somatic disease) over 10 years 6–10 years 3–5 years 1–2 years up to a year TOTAL Number of patients (mental illness) 18 12 38 27 31 126 36 19 31 25 9 120 Table 4 Emotional control: somatic diseases versus mental illness Mean (M) somatic disease KE MER 13.97 16.68 Mean (M) mental illness 14.74 13.50 OE KS PE 13.57 17.65 14.00 14.71 17.66 18.36 Z p Cohen’s d 1.53 0.126 6.40 0.000 3.42 (p < 0.05) 2.52 0.012 3.43 0.12 0.908 7.55 0.000 3.42 (p < 0.05) Z—Mann-Whitney U test; p—statistical significance; Cohen’s d- effect size measure; KE—expressivity control; MER—emotional and rational motivation; OE—emotional resilience; KS—situation control; PE—emotional arousal Table 5 Emotional control: somatic diseases versus healthy people Mean (M) healthy subjects 13.52 15.00 14.55 14.78 Z p KE MER OE KS Mean (M) somatic disease 13.97 16.68 13.57 17.65 0.67 2.89 1.57 5.71 PE 14.00 12.45 2.73 0.503 0.004 0.117 0.000 (p < 0.05) 0.006 Cohen’s d 2.89 2.88 2.89 Z—Mann-Whitney U test; p—statistical significance; Cohen’s d- effect size measure KE—expressivity control; MER—emotional and rational motivation; OE— emotional resilience; KS—situation control; PE—emotional arousal the Medical University of Lodz under the following applications: RNN/882/11/KB and RNN/383/11/KB. Emotion Regulation Questionnaire developer by J. Brzeziński [19] was used for the study. The method contains 45 statements rated on a four-point scale. It is used to measure subjective control of emotions in difficult situations, which consists of five dimensions: (a) expressivity control (KE)—measures an individual’s ability to control the outward manifestations of experienced emotions (body movements, gesticulation, facial expressions). A high score indicates excessive expressivity control, a low score shows underdeveloped expressivity control. Mean (M) healthy subjects Z KE MER OE KS Mean (M) mental illness 14.74 13.50 14.71 17.66 p Cohen’s d 13.52 15.00 14.55 14.78 2.13 2.55 0.51 6.42 0.033 0.011 0.613 0.000 (p < 0,05) 7.62 0.000 (p < 0,05) 2.82 2.82 PE 18.36 12.45 2.82 2.81 Z—Mann-Whitney U test; p—statistical significance; Cohen’s d- effect size measure KE—expressivity control; MER—emotional and rational motivation; OE— emotional resilience; KS—situation control; PE—emotional arousal (b) emotional and rational motivation (MER) - indicates the type of motivation of the individual, i.e., the type of control of one’s own behavior, which on the one hand can be emotional (impulsive) and on the other rational (controlled and thoughtful). A high score indicates controlled and thoughtful behavior, and a low score shows impulsive behavior. (c) emotional resilience (OE)—measures the ability of the subject to prevent behavioral disorganization under the influence of experienced positive and negative emotions, as well as the ability to influence the source of emotions, allowing to suppress the current emotional process. A high score indicates resilience to emotions and excessive control over one’s own behavior; a low score signifies nonresilience to emotions and an inability to control the behavior associated with them. (d) situation control (KS)—indicates the individual’s ability to control emotogenic (emotion-triggering) situations, to perceive and interpret them appropriately, taking into account whether the individual easily enters or avoids emotion-triggering situations. A high score indicates excessive control of the situation manifested mainly in the form of anxiety, and resulting from defensively oriented perception and interpretation. A low score means that the individual enters into various emotiontriggering situations too easily, without thinking about the consequences of doing so. (e) emotional arousal (PE)—measures the threshold of general emotional arousal and illustrates the degree to which it is easy to enter an emotional state after being exposed to emotion-triggering stimuli. The lower the score on this scale, the more difficult it is for the individual to enter an emotional state, i.e., the less emotionally excitable they are [19]. The subjects provided other sociodemographic variables, like gender, age, duration of disease, by filling out a short questionnaire prepared by the study authors. Orzechowska et al. BMC Psychiatry (2023) 23:802 STATISTICA 13.3 PL software was used for statistical analysis of the results obtained. A two-sided critical area was assumed during the statistical verification of the hypotheses. In order to choose the type of measurement, an analysis of the variables under study was conducted, which showed that the hypothesis of conformity to normal distribution should be rejected. In order to demonstrate the statistical significance of the association of the analyzed variables among the patients treated for somatic, affective and anxiety disorders and among the healthy individuals, a statistical analysis was performed based on non-parametric tests, including the MannWhitney U test and the Spearman’s rank correlation coefficient. The significance level of p < 0.05 was adopted in all statistical methods used. Results The study groups of patients with somatic symptom disorders were dominated by those with chronic disease— more than 10 years and 3 to 5 years. Among the patients with mental illness, those affected for 3 to 5 years and patients with illness for 1 to 2 years were the largest group (Table 3). The patients with dermatological and gastrointestinal diseases differed statistically significantly from the patients with depression and the patients with anxiety disorders in relation to three dimensions of emotional control. The people with somatic disease are characterized by higher emotional and rational motivation (MER), lower emotional resilience (OE) and lower emotional arousal (PE) (Table 4). Table 5 shows that—compared to the healthy individuals—the patients treated for dermatological and Page 5 of 11 gastrointestinal diseases have statistically significantly higher levels of emotional and rational motivation (MER), situation control (KS) and emotional arousal (PE). The patients with mental disorders—compared to healthy subjects—recorded the highest number of statistically significant differences in the analyzed dimensions of emotional control. They have higher levels of expressivity control (KE), situation control (KS) and emotional arousal (PE) than the healthy subjects. The dimension of emotional and rational motivation (MER) scored significantly lower compared to the healthy people. The results described are shown in Table 6. A graphical presentation of the obtained results of the differences between the average values in each group is shown in Figs. 1 and 2. All subgroups of somatic symptom disorders and mental illnesses were compared with the healthy subjects. Tables and figures include selected results of the statistical analysis without breakdown by patient group. On the situation control (KS) subscale that measures an individual’s ability to control emotion-triggering situations as well as their correct perception and interpretation, most of the subjects with somatic disease and mental illness scored higher than the healthy subjects. This indicates a defensive attitude and a tendency to control the situation in the form of anxiety. In the group of patients with anxiety disorders (M = 16.43; SD = 2.65) and patients with psoriasis (M = 16.20; SD = 3.00), these scores were slightly lower compared to the others. Those with irritable bowel syndrome (M = 18.23; SD = 3.57) and rosacea (M = 18.90; SD = 3.19) recorded similar results to the patients with depression (M = 18.04; SD = 3.04). Fig. 1 Emotional control scores among all study subjects. KE—expressivity control; MER—emotional and rational motivation; OE—emotional resilience; KS—situation control; PE—emotional arousal Orzechowska et al. BMC Psychiatry (2023) 23:802 Page 6 of 11 Fig. 2 Emotional control scores among all study subjects. KE—expressivity control; MER—emotional and rational motivation; OE—emotional resilience; KS—situation control; PE—emotional arousal The dimension of emotional arousal (PE), which refers to the ease of entering an emotional state under the influence of emotion-triggering stimuli, was dominant in the group of patients with mental illness. On the other hand, expressivity control (KE) (controlling the outward manifestations of experienced emotions) and emotional resilience (OE) (assessing the ease of entering an emotional state under the influence of emotion-triggering stimuli) in terms of the mean scores obtained were most similar in all the patients studied compared to the control subjects. Interestingly, in the emotional and rational motivation (MER) subscale, higher scores—compared to those with depressive and anxiety disorders, and even compared to the healthy people—were obtained by those with somatic diseases, i.e., psoriasis (M = 17.33; SD = 4.44), rosacea (M = 15.60; SD = 3.29), gastroesophageal reflux (M = 17.30; SD = 3.15), irritable bowel syndrome (M = 16.50; SD = 3.56). A higher score on this scale indicates motivation to engage in more rational and less emotional behavior in emotion-triggering situations. Age, duration of Illness and gender of study subjects Subjects with a diagnosis of chronic somatic symptom disorders were statistically significantly different in age from those with mental illness and from those in the control group. After a detailed statistical analysis, the group of patients with anxiety disorders was not statistically significantly different from those with somatic disease (all ‘dermatological’ and ‘gastrointestinal’ patients combined) and from the healthy subjects, as can be seen in Tables 1 and 2, presenting the characteristics of the subjects. This has to do with the nature of the diseases in question— the mean age of onset typical of the disease entity and the availability of the subjects during hospitalization and outpatient visits during the course of the study. Among those with somatic diseases, the age of the subjects co-varied statistically significantly with a small number of the emotional control variables discussed. In the patients with psoriasis, a positive correlation was for emotional and rational motivation (R = 0.38; p = 0.036) and situation control (R = 0.38; p = 0.040). A negative correlation for expressivity control (R= -0.41; p = 0.024) was confirmed among the patients with gastroesophageal reflux. Age did not correlate statistically significantly with the variables analyzed in the group of patients with rosacea and irritable bowel syndrome as well as among patients with anxiety disorders. In contrast, among those with depressive disorders, a significant positive correlation extended only to situation control (R = 0.28; p = 0.005). Age co-occurred statistically significantly with expressivity control (R = 0.29; p = 0.021) and emotional and rational motivation (R = 0.26; p = 0.037) among the healthy subjects. Duration of illness did not correlate statistically significantly in the anxiety disorder, psoriasis and irritable bowel syndrome groups. In the group of patients suffering from depression, a significant negative correlation was for emotional resilience (R= -0.20; p = 0.048). A significant negative correlation was also confirmed for emotional arousal (R= -0.37; p = 0.044) among the patients with rosacea. The patients suffering from gastroesophageal reflux had a significant negative correlation in situation control (R= -0.48; p = 0.007) and emotional arousal (R= -0.37; p = 0.044). In all the cases described, longer duration of illness co-occurred with lower intensity of the listed dimensions of emotional control. Gender differentiated the subjects (patients with somatic disease, mental illness and the control group) in a statistically significant way with reference to only two dimensions concerning emotional control, namely situation control (Z = 2.95; p = 0.003) and emotional arousal Orzechowska et al. BMC Psychiatry (2023) 23:802 (Z = 4.25; p = 0.000), which had higher levels among women. The differences between men and women were greatest among patients with depression. They addressed the following dimensions of emotional control: expressivity control, situation control and emotional arousal, with higher results for women in all the dimensions mentioned. Discussion Measuring emotional control takes on particular importance in the context of scientific reports that prove the existence of a relationship between the way emotions are expressed and the occurrence of mental, somatic and psychosomatic diseases [20]. There are few reports in scientific studies comparing patients with chronic somatic disease to patients with psychiatric disorders in terms of control of emotional states. Most reports in the literature refer to common factors that link somatic diseases to depression or the cooccurrence of psychiatric disorders with chronic physical illness, mainly in a biological context [18, 21, 22]. The association of somatic diseases with depression and anxiety is grounded in the presence of biological factors common to these diseases. Both depression and anxiety disorders, as well as a number of diseases of civilization (hypertension, coronary heart disease, diabetes and even dementia), share a common immunological background. An increase in immune system activity and the adverse metabolic changes described above can be seen in each of the aforementioned diseases [23]. The same brain regions that are particularly sensitive to the consequences of weakened immune defenses are responsible for both emotion dysregulation and cognitive dysfunction, i.e., the anterior and medial cingulate cortex, the dorsolateral and ventromedial prefrontal cortex, the anterior insula, and the amygdala [24, 25]. This would also suggest similar emotional reactions in these diseases. In some situations, the difference between somatic diseases and mental illnesses can blur in terms of emotional reactions. The reason for the co-occurrence of anxiety disorders with somatic diseases as with depression may be related to common biological and behavioral processes involved in the development of these types of diseases. There are also similarities in the pathophysiology of anxiety and selected somatic diseases or somatization tendencies [26, 27]. The accepted models of disease formation primarily take into account biological factors relating to the genetic background, biological dysregulation conditions involving the activities of individual systems and organs of the body, which the authors of the presented paper did not directly address. It is pointed out that psychological and environmental factors—in addition to biological Page 7 of 11 factors—play a significant role in the development of diseases, which in their theoretical assumptions emphasize the importance of personality traits (the totality of the way of thinking, experienced emotions and behavior), early human experience and adaptation to environmental demands, or the way of cognitive elaboration of one’s own experiences and evaluation of oneself. Certain personality traits can promote illness, while at the same time, changes in selected personality traits can be a consequence of the disease process [28]. Deficits in the regulation of emotional control can include and simultaneously co-occur with the phenomenon of alexithymia, which is described as a disorder of cognitive and affective processes that limits a person’s access to his own mental states and awareness of his own emotions. Alexithymia is treated as a stable personality trait of patients, which, along with other personality factors, predisposes to the occurrence of various somatic and mental illnesses. The limited ability of a person with alexithymia to become aware of and process his own emotions using cognitive processes leads to both a focus on somatic sensations accompanying emotional arousal and a compulsive, poorly controlled response to negative stimulation [29]. In the analysis of the study variables presented in this publication, the patients with skin diseases and the patients with gastrointestinal disorders differed statistically significantly from those with psychiatric disorders and from the healthy subjects on three dimensions of emotional control. People with somatic disease are characterized by higher emotional and rational motivation, lower emotional resilience and lower emotional arousal than those with mental illness. Compared to the healthy individuals, the patients treated for dermatological and gastrointestinal diseases have statistically significantly higher levels of emotional and rational motivation, situation control and emotional arousal. The patients with mental disorders—compared to the healthy subjects— recorded the highest number of statistically significant differences in the analyzed dimensions of emotional control. They have higher levels of expressivity control, situation control and emotional arousal than the healthy subjects. They scored lower on the dimension of emotional and rational motivation compared to the healthy people. The results described for the people with mental illness remain consistent with many other studies addressing this topic. In addition, regardless of statistical significance, there was similarity in the individual factors comprising emotional control on the dimensions that differentiate people with somatic disease and mental illness from healthy individuals. The literature on this issue indicates that emotion regulation disorders are important in the development and course of many mental illnesses such as depressive Orzechowska et al. BMC Psychiatry (2023) 23:802 disorders, bipolar affective disorder, borderline personality disorder, generalized anxiety disorder, and eating disorders [30]. Numerous studies on emotion regulation—regardless of the very conceptualization of the construct and the methods used—unequivocally point to the differences that exist between people with a diagnosis of a mental disorder and the population of healthy people [31, 32]. Emotion regulation in patients who reveal mental disorders is usually characterized by multiple deficits. Their emotional reactions are usually excessive, violent, marked by long-lasting negative emotional states; they perform an adaptive function insufficiently, and most often are clearly dysfunctional for a given situational context. The literature provides a big number of reports on the association of maladaptive emotion regulation strategies with the severity of psychopathological symptoms [33, 34]. Empirically, the negative impact of emotion suppression on health—expressed as an increase in the level of psychopathological symptoms—has been established. At the same time, emotion acceptance has been proven to reduce the severity of symptoms. It has been repeatedly shown that emotion suppression is related to the level of depressive symptoms and leads to an increase in negative emotions. Studies evaluating the relationship between the ability to control emotions and the severity of symptoms of depressive and anxiety disorders have shown that the more people control, suppress and devalue their emotions, the higher their levels of anxiety and depression symptoms. Conversely, the more accepting they are of their emotions—both negative and positive—the less severe the symptoms of depression and anxiety [35]. The results in subsequent publications clearly indicate reduced levels of emotion acceptance, increased suppression, more frequent ruminations, and negligible use of positive reformulation strategies in these patients compared to healthy individuals [36–38]. Research also shows that suppression of positive emotions is associated with anhedonia, increased negative emotions and elevated levels of depression [34, 35]. Dermatological and gastroenterological diseases are among those with complex etiologies in which a significant role is played by both biological and psychological factors. In most cases, these diseases cause bothersome symptoms that impede daily functioning and may even result in unsightly changes in appearance, affecting the patient’s body image and self-esteem, and to a large extent determine the patient’s social relations [21, 38–40]. In our own studies from 2008 to 2010 [41, 42] the determinants of emotional control among people treated for gastroesophageal reflux disease and irritable bowel syndrome were assessed in conjunction with levels of experienced stress, coping styles and perceived anxiety. The results of the analysis conducted in 2008 showed that the patients studied did not differ statistically significantly in Page 8 of 11 terms of the psychological factors analyzed, but the variables examined co-occurred with the severity of selected somatic symptoms. The results obtained in 2010, on a slightly larger number of patients, showed the prevalence of unfavorable aspects of emotional control in the group of patients diagnosed with IBS, as well as higher levels of perceived stress and anxiety as an ongoing personality trait and currently experienced condition. Also in reports from 2009 [43], in which 70 people with dermatological diseases were surveyed, the Coping Inventory for Stressful Situations by R.H. Moss and Emotion Regulation Questionnaire by J. Brzeziński were used to assess psychological factors. The type of skin disease only partially differentiated the patients studied in terms of the psychological factors analyzed. They included control of emotion-triggering situations, emotional arousal and cognitive avoidance. The women surveyed differed from the men in terms of control of emotional expressivity and control of emotion-triggering situations, and in terms of overall emotional resilience. Among all the patients studied, positive aspects of emotional control correlated with less frequent use of less favorable stress coping strategies. The results presented suggest that the unfavorable aspects of emotional control may be related not so much to the type of disease entity, but to the disease itself, which may be close to the research results achieved in the current work. Atroszko et al. [44] studied 31 patients aged 18–78 suffering from arterial hypertension. They compared the values obtained on the Emotion Regulation Questionnaire by J. Brzeziński, the Type A-Framingham scale and the Emotionality, Activity and Sociability Temperament Questionnaire with a control group of men aged 18–87. The results gave a consistent picture of the emotional functioning of a man with hypertension as a person with a high tendency to react with dissatisfaction and anger, striving to maintain control in emotion-triggering situations to avoid experiencing emotions, especially avoiding failure and feeling tension, and characterized by a tendency to be competitive and hostile. The authors of the study concluded that the tendency to frequently experience negative emotions may be a mediating factor between personality traits and the development of hypertension. The relationship between emotional functioning and somatic diseases has been a topic of scientific research for several decades. The mechanism of this relationship is explained on the basis of various models that describe the relationship between emotions and physiological responses, mediated by other psychological factors such as stress and personality traits. Individual susceptibility to reacting with negative emotions and emotional control processes is of interest from this perspective [23, 45]. Measuring emotional control takes on particular Orzechowska et al. BMC Psychiatry (2023) 23:802 importance in the context of scientific reports that prove the existence of a relationship between the way emotions are expressed and the occurrence of mental, somatic and psychosomatic diseases. In our study, people with somatic symptom disorders scored higher on the emotional and rational motivation subscale and on most dimensions of emotional control compared to people with depressive and anxiety disorders, and even relative to healthy people. This may be related to the nature and course of chronic diseases. A chronic disease is a stressor of a special nature, requiring specific measures to overcome the symptoms of the disease and cope with chronic difficult emotional states. Coping which involves cognitive and behavioral efforts to transform or eliminate burdens and associated emotional tension may secondarily—in the course of a long-term disease—affect the intensification of the tendency to emotional control observed in the results we obtained [46]. Emotional control versus age, onset of Disease and gender In our study, we showed the existence of heterogeneous relationships between age and various dimensions of emotional control across groups. Age differentiated the subjects, but did not co-occur in a statistically significant way with a large number of emotional control variables. In the vast majority of them, selected dimensions of emotional control increased with age. Other studies addressing this topic also indicate that the ability to control emotions increases with age, as the ability to understand and name one’s own emotions associated with the development of social interactions rises. One study found that factors such as emotional suppression and aggression control, indicating a tendency toward less emotional expression, are highest in the group of people over 55. This phenomenon is explained by the fact that older adults may be better able to understand and accept their inner subjective experiences, condense them, and do not feel such a strong need to express their emotions. In addition, perhaps older people suppress the expression of negative emotions more, or experience fewer negative emotional reactions compared to younger people [47]. The more mature a person is, the more effective they are able to make the use of emotion-focused coping strategies such as distancing, self-control and positive reappraisal [48]. Some researchers conclude that as the subjects get older, they approach a more favorable continuum in terms of the listed psychological factors, which may be related to emotional maturity and a sense of life stability, a formed personality and self-esteem, and acceptance of their own illness [49]. In our own research and considering all the groups studied, we have shown that women enter a state of emotional arousal with greater ease and at the same time show a greater ability to control expressivity. In contrast, Page 9 of 11 in the group of depressive patients, women were characterized by greater emotional arousal, with stronger expressivity control and situation control. In the literature we find data indicating that women score higher on emotional expressivity than men in a group of patients suffering from various types of chronic diseases. The results also support the view that emotions are culturally constructed and context dependent. Cultural changes and social consequences that affect age and gender roles can have a direct impact on the ways in which emotions are experienced and expressed [47]. Duration of disease did not correlate in a statistically significant way in the group of patients with anxiety disorders, psoriasis and irritable bowel syndrome. In the group of patients with depression, rosacea, gastroesophageal reflux, it was shown that longer duration of disease co-occurred with lower intensity of selected dimensions of emotional control. In the case of patients with depression, emotional resilience decreased with longer duration of disease, while in the case of patients with somatic disease, emotional arousal diminished mainly. This may suggest a tendency toward better emotional functioning with the duration of the disease in the case of a somatic condition, but not a mental one, which is understandable given the nature of mental disorders. A study by Kossakowska et al. [50] showed that the duration of a dermatological disease was not correlated with the severity of negative emotional control, such as anger, depressive mood and anxiety, but was strongly correlated with the severity of disease progression as assessed by clinicians [50]. In other studies conducted with patients suffering from psoriasis, it was shown that disease duration was associated with a greater degree of disease acceptance and ability to regulate emotions. With longer duration of disease, the tendency to use denial strategies decreased [51]. In contrast, in a group of people with rheumatoid arthritis, it was shown that with disease duration women behaved more ambiguously—they became emotionally unstable and worried, while at the same time persevering and reconciled to life. A greater number of adaptive behaviors were observed in men [52]. The duration of the disease is indirectly related to the intensity of the disorder’s symptoms. Limitations The heterogeneity of the comparison groups of subjects, in terms of the number of subjects studied and demographic variables, may be considered a limitation of the presented project. They varied significantly in terms of age, which is related to the prevalence of specific disease entities in the age range and the availability of healthy individuals who consented to the study. The study results should be treated with caution. Further research in this Orzechowska et al. BMC Psychiatry (2023) 23:802 area should continue on larger group sizes in specific disease entities. Conclusions A somatic disease and mental disorder of a chronic nature entails a fundamental and usually inauspicious change in a person’s life in the form of persistent ailments that cause suffering and restrictions on the performance of important social roles—both family- and work-related. Both categories of disease entities are closely related to the function of the nervous system and to the human mental sphere. Emotional processes, which have a significant impact on the way of thinking, behavior and physiological reactions, are a factor that determines the overall functioning, as well as the quality of life of an individual. Comparing patients with somatic disease to patients with mental illness in terms of controlling emotional reactions raises new possibilities for covering the described groups with similar therapeutic interventions that pay attention to the patient’s emotional sphere. The importance of various disease factors should be studied and taken into account in the treatment process, with the aim of selecting the most optimal therapeutic tasks. Control of emotional reactions, which is the variable analyzed in the current study, is one of the established personality traits, as we described in the discussion of the work. Other well-established personality traits and their contribution to somatic and psychiatric disorders will become the subject of our upcoming studies. In the given study, the authors also did not address the therapeutic side of the described phenomenon, and due to the significant number of patients affected by this problem, it requires much more research attention in the future. List of abbreviations IBS irritable bowel syndrome GERD gastroesophageal reflux disease Acknowledgements Not applicable. Authors’ contributions Substantial contributions to the conception A.O., P.M. Design of the work A.O., P.M., K. B-K., the acquisition, analysis A.O., K. B-K., M.G. Interpretation of data A.O., P.M., M.G., K. B-K. Resources P.G. Have drafted the work or substantively revised it P.H., D.B, P.G. Funding The paper was financed by the Medical University of Lodz number: 503/1-062-02/503-11-001. Data Availability Upon request from corresponding author. 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https://openalex.org/W4381334874	https://www.frontiersin.org/articles/10.3389/fimmu.2023.1211171/pdf	English	null	Second-line therapy for patients with steroid-refractory aGVHD: systematic review and meta-analysis of randomized controlled trials	Frontiers in immunology	2,023	cc-by	9,375	TYPE Systematic Review PUBLISHED 20 June 2023 DOI 10.3389/fimmu.2023.1211171 TYPE Systematic Review PUBLISHED 20 June 2023 DOI 10.3389/fimmu.2023.1211171 OPEN ACCESS OPEN ACCESS EDITED BY Giuseppe Milone, University Hospital Polyclinic Vittorio Emanuele, Italy REVIEWED BY Xiao-Dong Mo, Peking University People’s Hospital, China Philippe Lewalle, Institut Jules Bordet, Universite´ Libre De Bruxelles, Belgium CORRESPONDENCE Xi Li lixi1988xn@163.com Yunding Zou zouyunding@163.com Shuangnian Xu xushuangnian@tmmu.edu.cn †Th h h ib d ll OPEN ACCESS EDITED BY Giuseppe Milone, University Hospital Polyclinic Vittorio Emanuele, Italy REVIEWED BY Xiao-Dong Mo, Peking University People’s Hospital, China Philippe Lewalle, Institut Jules Bordet, Universite´ Libre De Bruxelles, Belgium CORRESPONDENCE Xi Li lixi1988xn@163.com Yunding Zou zouyunding@163.com Shuangnian Xu xushuangnian@tmmu.edu.cn †These authors have contributed equally to this work and share ﬁrst authorship RECEIVED 25 April 2023 ACCEPTED 08 June 2023 PUBLISHED 20 June 2023 CITATION Luo C, Huang X, Wei L, Wu G, Huang Y, Ding Y, Huang Z, Chen J, Li X, Zou Y and Xu S (2023) Second-line therapy for patients with steroid-refractory aGVHD: systematic review and meta-analysis of randomized controlled trials. Front. Immunol. 14:1211171. doi: 10.3389/fimmu.2023.1211171 Chengxin Luo 1,2†, Xiangtao Huang 1,2†, Ling Wei 1,2†, Guixian Wu 1,2, Yarui Huang 1,2, Yaqun Ding 1,2, Zhen Huang 1,2, Jieping Chen 1,2, Xi Li 3, Yunding Zou 1,2 and Shuangnian Xu 1,2* 1Center for Hematology, Southwest Hospital, Third Military Medical University, Chongqing, China, 2Chongqing Key Laboratory of Tumor Immunotherapy, Chongqing Science & Technology Commission, Chongqing, China, 3Department of Hematology, Daping Hospital, Third Military Medical University, Chongqing, China Objective: Steroids-refractory (SR) acute graft-versus-host disease (aGVHD) is a life-threatening condition in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the optimal second-line therapy still has not been established. We aimed to perform a systematic review and meta-analysis of randomized controlled trials (RCTs) to compare the efﬁcacy and safety of different second-line therapy regimens. Luo C, Huang X, Wei L, Wu G, Huang Y, Ding Y, Huang Z, Chen J, Li X, Zou Y and Xu S (2023) Second-line therapy for patients with steroid-refractory aGVHD: systematic review and meta-analysis of randomized controlled trials. Front. Immunol. 14:1211171. doi: 10.3389/fimmu.2023.1211171 Methods: Literature search in MEDLINE, Embase, Cochrane Library and China Biology Medicine databases were performed to retrieve RCTs comparing the efﬁcacy and safety of different therapy regimens for patients with SR aGVHD. Meta-analysis was conducted with Review Manager version 5.3. The primary outcome is the overall response rate (ORR) at day 28. Pooled relative risk (RR) and 95% conﬁdence interval (CI) were calculated with the Mantel-Haenszel method. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/, identiﬁer CRD42022342487. KEYWORDS acute graft-versus-host disease, steroids-refractory, second-line therapy, mesenchymal stroma cells, ruxolitinib, meta-analysis, randomized controlled trials acute graft-versus-host disease, steroids-refractory, second-line therapy, mesenchymal stroma cells, ruxolitinib, meta-analysis, randomized controlled trials Introduction review and meta-analysis of randomized controlled trials (RCTs) comparing different second-line therapies in patients with steroids- refractory aGVHD following allo-HSCT, aiming to provide evidences for regimens selection in clinical practice and clues for future study design. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a key treatment strategy for patients with high-risk hematological malignancies and severe non-malignant hematological disorders. The applications of allo-HSCT continuously increased over the past several decades (1). Despite substantial advancements in transplantation technologies, acute graft-versus-host disease (aGVHD) remains one of the most common complications and crucial contributing factor for transplant-related mortality (TRM) in patients undergoing allo-HSCT (2). Approximately 40%–60% of patients developed moderate to severe (grade II–IV) aGVHD after allo-HSCT despite standard prophylaxis (3, 4). Severe aGVHD is a life-threatening condition, associating with a dismal long-term overall survival (OS) lower than 30% for grade III disease and lower than 5% in patients with grade IV disease (5, 6). Above all, aGVHD is a major challenge in clinical practice limiting the application of allo-HSCT and compromising its beneﬁts. COPYRIGHT © 2023 Luo, Huang, Wei, Wu, Huang, Ding, Huang, Chen, Li, Zou and Xu. 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. Results: Eight eligible RCTs were included, involving 1127 patients with SR aGVHD and a broad range of second-line therapy regimens. Meta-analysis of 3 trials investigating the effects of adding mesenchymal stroma cells (MSCs) to other second-line therapy regimens suggested that the addition of MSCs is associated with signiﬁcantly improvement in ORR at day 28 (RR = 1.15, 95% CI = 1.01–1.32, P = 0.04), especially in patients with severe (grade III–IV or grade C–D) aGVHD (RR = 1.26, 95% CI = 1.04–1.52, P = 0.02) and patients with multiorgan involved (RR = 1.27, 95% CI = 1.05–1.55, P = 0.01). No signiﬁcant difference was observed betwwen the MSCs group and control group in consideration of overall survival and serious adverse events. Treatment outcomes of the other trials were comprehensively reviewed, ruxolitinib showed signiﬁcantly higher ORR and complete response rate at day 28, higher durable overall response at day 56 and longer failure-free survival in comparison with other regimens; inolimomab shows similar 1-year therapy success rate but superior long-term overall survial in comparison with anti-thymocyte globulin, other comparisons did not show signiﬁcant differences in efﬁcacy. Frontiers in Immunology Frontiers in Immunology 01 frontiersin.org Luo et al. Luo et al. 10.3389/ﬁmmu.2023.1211171 Conclusions: Adding MSCs to other second-line therapy regimens is associated with signiﬁcantly improved ORR, ruxolitinib showed signiﬁcantly better efﬁcacy outcomes in comparison with other regimens in patients with SR aGVHD. Further well-designed RCTs and integrated studies are required to determine the optimal treatment. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/, identiﬁer CRD42022342487. Systematic review registration: https://www.crd.york.ac.uk/PROSPERO/, identiﬁer CRD42022342487. Literature search and study selection This study was reported according to preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement (16). The protocol was registered on the International Prospective Register of Systematic Reviews (PROSPERO) and available online (registration number CRD42022342487). We searched MEDLINE (by Ovid), Embase, Cochrane Library and China Biology Medicine (CBM) databases on April 6, 2022 with no date and language restriction, the search procedure was repeated on April 13, 2023. The main search terms are “graft versus host disease” or “GVHD” in combination with “refractory”, “resistant”, “persistent”, “second- line” or “salvage”. Reference lists of eligible trials and relevant reviews were manually checked for additional trials. Steroids remains the ﬁrst-line therapy for aGVHD, however, only 40%–60% of patients can achieve durable response with initial therapy (7–9). Patients with grade III-IV aGVHD, hyperacute GVHD, older age or multiple organs involvement are associated with high risk of treatment failure (10–12). The prognosis of patients with steroids-refractory aGVHD is extremely poor, with a GVHD-related mortality around 70% at 2 years (10, 13). Currently available second-line therapy for patients with steroids- refractory aGVHD mainly include mycophenolate mofetil (MMF), tacrolimus, anti-thymocyte globulin (ATG), Janus kinase (JAK) 1/2 inhibitor ruxolitinib, interleukin 2 receptor (IL-2R) antibodies (daclizumab, inolimomab, basiliximab), mammalian target of rapamycin (mTOR) inhibitors (sirolimus, everolimus), tumor necrosis factor-alpha (TNF-a) inhibitors (inﬂiximab, etanercept), anti-CD52 antibody alemtuzumab, anti-a4b7 integrin antibody vedolizumab, extracorporal photopheresis (ECP) and mesenchymal stroma cells (MSCs) used alone or in combination (2, 9, 14). However, the optimal second-line therapy still has not been established (2, 15). In this study, we performed a systematic Two investigators (CXL and XTH) independently assessed eligibility of retrieved citations; disagreements were resolved by discussion with a third investigator (SNX). The inclusion criteria are: (і) included patients receiving allo-HSCT as therapy for hematological disorders, and developing steroids-refractory aGVHD of any grade following allo-HSCT; (ii) compared the efﬁcacy and safety of two or more different second-line therapy regimens for steroids-refractory aGVHD; (iii) study design is randomized controlled trial (RCT). According to previously published agreements, steroids-refractory aGVHD is deﬁned as: disease progression after at least 3 days of treatment with methylprednisolone ≥2 mg/kg/day or equivalent; lack of response after at least 7 days of treatment; or failure to taper the methylprednisolone dose to < 0.5 mg/kg/day or the prednisone Frontiers in Immunology 02 frontiersin.org Luo et al. 10.3389/ﬁmmu.2023.1211171 dose to < 0.6 mg/kg/day (17, 18). Data extraction and quality assessment Two investigators (CXL and LW) independently extracted data on trial characteristics (ﬁrst author, publication year, study design), patients’ characteristics (age, gender, underlying disease, donor type, graft source, grade of aGVHD), type and dosage of second-line therapy regimens, median follow-up and treatment outcomes. The primary outcome is the overall response rate (ORR) at day 28, which is deﬁned as the proportion of patients who achieved complete response (CR) or partial response (PR) at day 28. Secondary outcomes include CR rate, OS, and adverse events (AEs). For dichotomous data, number of patients with events and total number of patients analyzed were extracted. For time-to-event data, hazard ratios (HRs) and 95% conﬁdence intervals (CIs) were extracted. The ln(HR) and standard error (SE) were calculated directly, or estimated from the log-rank P value and the number of events with the previously established methods when the HRs and 95% CIs were not reported (19). For trials with multiple publications, survival data were extracted from the report with the longest follow- up. Predesigned forms were used to extracted data and cross-checked to reach a consensus between the two investigators. The characteristics of the 8 included RCTs are summarized in Table 1. The speciﬁc deﬁnition of steroids-refractory aGVHD in each included trail is summarized in Supplementary Table 1. A broad range of second-line therapy strategies for steroids-refractory aGVHD are investigated, the dosage of all regimens are provided in Supplementary Table 2. Three trials investigated the effects of adding MSCs to other second-line therapy regimens (including MMF, ATG, inﬂiximab, etanercept, daclizumab, pentostatin, denileukin diftitox, ECP, alemtuzumab, and basiliximab plus cyclosporine or tacrolimus) (21–23). One trial (the REACH2 trial) compared the efﬁcacy and safety of ruxolitinib with other best available care (investigator’s choice from ATG, ECP, MSCs, LD- MTX, MMF, everolimus, sirolimus, etanercept, or inﬂiximab) (24). Other trials compared high-dose methylprednisolone (HD-MP) plus OKT3 (a murine anti-CD3 monoclonal antibody) or ATG with HD- MP alone, or compared ATG with inolimomab (a monoclonal antibody against IL-2R) or ABX-CBL (a murine monoclonal antibody against CD147) (25–29). The primary endpoints were largely different, including ORR (at day 28, 30 or 100), durable complete response, therapy success rate, and OS at day 180. The Median follow-up ranges from 180 days to 58.4 months (Table 1). Literature search and study selection Retrospective studies, single-arm studies, dose-escalating studies, case reports or case series, and post- hoc analysis were excluded. records based on title and abstract, the remaining 386 records were included for further screening. Subsequently, we excluded 38 studies on GVHD prophylaxis, 23 studies on ﬁrst-line therapy of aGVHD, 11 dose-ﬁnding studies, 6 protocol and 281 non-RCT studies. Ultimately, 27 records (19 conferences abstract and 8 full- text article) for 8 eligible RCTs were included for the systematic review (21–29). The ﬂow chart of study selection is provided in Figure 1. Data extraction and quality assessment Methodological quality of included RCTs were assessed with the Cochrane risk of bias tool based on following aspects: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting and other bias (20). Two investigators independently performed risk of bias assessment; any disagreements were resolved by consensus. A total of 1127 patients were included and most of them were diagnosed with hematological malignancies (Table 1). Patient’s age, gender and grade of aGVHD were well matched between the experimental group and control group in 7 of the included trials that with enough information. As for donor type, 5 trials with relevant information included both related and unrelated donors. Graft sources were reported in 5 trials, which included both bone marrow (BM) and peripheral blood stem cells (PBSCs). Two trials included small proportions of patients (0.6%-12%) receiving cord blood (CB) transplantation (23, 24). Statistical analyses All statistical analyses were performed with Review Manager version 5.3 (Revman, The Cochrane Collaboration, 2014). For dichotomous data, the pooled risk ratios (RRs) and 95% conﬁdence intervals (CIs) were calculated using the Mantel-Haenszel method. For time-to-event data, the pooled HRs and 95% CIs were calculated using the generic inverse-variance method. Heterogeneity was assessed with Chi-square test and I2 statistic. Random-effects model was used when there is signiﬁcant heterogeneity (P ≤0.1, I2 > 50%), otherwise ﬁxed-effects model was used. Subgroup analyses were performed according to the patients’ age (< 18 years and ≥18 years), grade of aGVHD, involved organ (skin, gastrointestinal, and liver), and the number of involved organs (single organ and multiorgan). All P values are two-sided, and a P value of < 0.05 indicated statistical signiﬁcance except that of heterogeneity test. The results of risk of bias assessment suggest that random sequence generation is adequate in 3 trials, allocation sequence concealment is adequate in 2 trials, whereas the other trials did not provide sufﬁcient information to evaluate selection bias (Supplementary Figure 1). As for blinding of participants and personnel, 2 trials are double-blind, 4 trials are open-label. Outcome assessments were performed in blinded manner in 3 trials. All of the included trials are free from attrition bias, reporting bias and any other bias except for one trial that only reported as a conference abstract and did not provide sufﬁcient information to evaluate. Overall response rate The databases search retrieved 5185 potentially relevant records. We removed 783 duplicates and excluded 4016 irrelevant Treatment outcomes including overall response rate (ORR), complete respou rate and overall survival of all included trials are Frontiers in Immunology 03 frontiersin.org Luo et al. 10.3389/ﬁmmu.2023.1211171 FIGURE 1 The ﬂow chart of study selection. CBM, China Biology Medicine; GVHD, graft-versus-host disease; RCT, randomized controlled trial. FIGURE 1 The ﬂow chart of study selection. CBM, China Biology Medicine; GVHD, graft-versus-host disease; RCT, randomized controlled trial. GURE 1 he ﬂow chart of study selection. CBM, China Biology Medicine; GVHD, graft-versus-host disease; RCT, randomized controlled trial. TABLE 1 Characteristics of included trials and patients. Study Study design Treatment group No. of patients Age* Gender (Male%) Underlying disease Donor Graft source aGVHD grade at baseline Median follow- up Zhao 2022 (21) Phase 3 RCT, open-label, multicenter (NCT02241018) MSC + Second-line therapy 99 28 (16– 59) 63% AML: 39.4%; ALL: 45.5%; Others: 15.2% HLA matched: 51.5%; HLA mismatched: 48.5% PBSC: 53.5%; PBSC + BM: 46.5% II: 36.4%; III: 41.4%; IV: 22.2% 19.8 months Second-line therapy 99 29 (16– 57) 57% AML: 49.5%; ALL: 43.4%; Others: 7.1% HLA matched: 51.5%; HLA mismatched: 48.5% PBSC: 57.6%; PBSC + BM: 42.4% II: 37.4%; III: 44.4%; IV: 18.2% 12.3 months Oosten 2022 (22) Phase 3 RCT, double-blind, multicenter (HOVON-113 trial) MSC + Second-line therapy 20 NA NA NA NA NA II: 12%; III: 68%; IV: 20% 24.0 months Second-line therapy 21 NA NA NA NA NA Kebriaei 2020 (23) Phase 3 RCT, double-blind, multicenter (NCT00366145) MSC + Second-line therapy 163 43.8 ± 16.6 56% NA Unrelated: 57%; Related: 43% BM: 12%; PBSC: 78%; CB: 10% B: 23%; C: 50%; D: 27% 180 days Second-line therapy 80 40.0 ± 18.1 59% NA Unrelated: 58%; Related: 42% BM: 17%; PBSC: 70%; CB: 12% B: 26%; C: 57%; D: 17% 180 days (Continued) 04 04 frontiersin.org Frontiers in Immunology 10.3389/ﬁmmu.2023.1211171 Luo et al. TABLE 1 Continued Study Study design Treatment group No. Overall response rate of patients Age* Gender (Male%) Underlying disease Donor Graft source aGVHD grade at baseline Median follow- up Zeiser 2020 (24) Phase 3 RCT, open-label, multicenter (REACH2 trail, NCT02913261) Ruxolitinib 154 53 (12– 73) 60% AML: 37.7%; ALL: 16.2%; CML: 3.9%; MDS: 16.9%; NHL: 5.8%; Others: 19.2% NA BM: 12.3%; PBSC: 87.0%; CB: 0.6% I: 1.3%; II: 32.5%; III: 44.2%; IV: 19.5% 5.04 months Investigator’s choice 155 54 (13– 71) 59% AML: 40.6%; ALL: 10.3%; CML: 1.3%; MDS: 18.7%; NHL: 12.3%; Other: 16.8% NA BM: 19.4%; PBSC: 76.1%; CB: 4.5% I: 0%; II: 34.8%; III: 43.9%; IV: 20.6% 3.58 months Socie 2017, 2019 (25, 26) Phase 3 RCT, open-label, multicenter (EudraCT 2007-005009- 24) Inolimomab 49 46.2 ± 12.6 45% AML: 29%; ALL: 14%; MDS: 8%; CLL: 8%; Lymphoma: 16%; Other: 25% MSD: 31%; MUD: 63%; MMUD: 6% PBSC: 81.6%; BM: 18.4% I: 2%; II: 22.4%; III: 63.3%; IV: 12.2% 58.4 months ATG 51 47.1 ± 12.96 51% AML: 20%; ALL: 12%; MDS:14%; CLL: 12%; Lymphoma: 20%; Other: 22% MSD: 39%; MUD: 59%; MMUD: 2% PBSC: 76.5%; BM: 23.5% I: 6.1%; II: 12.2%; III: 65.3%; IV: 16.3% 58.4 months Knop 2007 (27) RCT, multicenter (by EBMT) HD-MP + OKT3 40 40 (19– 65) 70% AML/MDS: 45%; ALL: 2.5%; CML: 30%; Other: 22.5% MSD: 30%; MUD: 52.5%; MMUD: 17.5% PBSC: 70%; BM: 30% II: 25%; III: 62.5%; IV: 12.5% NA HD-MP 40 39 (19– 56) 65% AML/MDS: 40%; ALL: 12.5%; CML: 32.5%; Other: 10% MSD: 50%; MUD: 35%; MMUD: 15% PBSC: 62.5%; BM: 37.5% II: 25%; III: 62.5%; IV: 12.5% NA MacMillan 2007 (28) Phase 2/3 RCT, multicenter, open-label ABX-CBL 48 38 (2- 65) 65% AML: 29%; ALL: 6%; CML: 15%; MDS: 12%; Lymphoma: 21%; Other: 17% MRD: 40%; MMRD: 2%; MUD: 44%; MMUD: 14% NA B/C: 85%; D: 15% 180 days ATG 47 39 (2- 65) 57% AML: 19%; ALL: 17%; CML: 26%; MDS: 8%; Lymphoma: 17%; Other: 13% MRD: 47%; MMRD: 4%; MUD: 34%; MMUD: 15% NA B/C: 91%; D: 15% 180 days Van Lint 2006 (29) RCT, multicenter (by GITMO) HD-MP + ATG 27 36 (2– 63) 56% AML/ALL: 44%; CML: 26%; Other: 30% HLA- identical sibling: 52%; Unrelated: 48% NA I: 15%; II: 59%; III: 26% 1134 days HD-MP 34 33 (2– 64) 50% AML/ALL: 53%; CML: 35%; Other: 12% HLA- identical sibling: 47%; Unrelated: 53% NA I: 21%; II: 56%; III: 24% 1134 days Age is presented as mean ± standard deviation or median with range. Frontiers in Immunology Overall response rate Subgroup analyses according to patients’ age and involved organ did not show any statistically signiﬁcant results (Supplementary Figures 2, 3). investigator’s choice from other second-line therapy regimens, including ATG, etanercept, ECP, inﬁliximab, MSCs and MMF (24). The ORR at day 28 of these regimens ranges from 30% to 60%, leading to a total ORR of 39% (Table 2). Ruxolitinib is associated with a signiﬁcantly higher ORR at day 28 (62% versus 39%, P < 0.001) and durable overall response at day 56 (40% versus 22%, P < 0.001) in comprison with investigator’s choice. The other 4 trials reported ORR at different time phase or 1-year therapy success rate (Table 2). Two trials investigated the efﬁcacy of HD-MP plus ATG or anti-CD3 antibody OKT3, suggested that HD-MP plus ATG did not signiﬁcantly improve ORR at day 30 over HD-MP alone (55% versus 48%; P = 0.3), and HD-MP plus OKT3 did not signiﬁcantly improve ORR at day 100 over HD-MP alone (53% versus 33%; P = 0.06) (27, 29). One trial compared the efﬁcacy of anti-CD147 antibody ABX-CBL with ATG, reported an ORR of 56% for the ABX-CBL group at a median of 22 days (range 7–72 days) and 57% for the ATG group at a median of 28 days (range 2–50 days), no signiﬁcant difference was observed (28). The other trial compared inolimomab with ATG and reported 1-year therapy success rate as the primary endpoin (deﬁned as the overall Another trial (the REACH2 trial) reporting ORR at day 28 as primary endpoint compared the efﬁcacy of ruxolitinib with TABLE 2 Treatment outcomes of included trials. Overall response rate ABX-CBL, a murine monoclonal antibody against CD147; aGVHD, acute graft-versus-host disease; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ATG, anti-thymocyte globulin; BM, bone marrow; CLL, chronic lymphoblastic leukemia; CML, chronic myeloid leukemia; CR, complete response; DCR, durable complete response; EBMT, European Group for Blood and Marrow Transplantation; ECP, extracorporeal photopheresis; GITMO, Gruppo Italiano Trapianto Midollo Osseo; HD-MP, high-dose methylprednisolone; HL, Hodgkin lymphoma; HLA, Age is presented as mean ± standard deviation or median with range. ABX-CBL, a murine monoclonal antibody against CD147; aGVHD, acute graft-versus-host disease; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ATG, anti-thymocyte globulin; BM, bone marrow; CLL, chronic lymphoblastic leukemia; CML, chronic myeloid leukemia; CR, complete response; DCR, durable complete response; EBMT, European Group for Blood and Marrow Transplantation; ECP, extracorporeal photopheresis; GITMO, Gruppo Italiano Trapianto Midollo Osseo; HD-MP, high-dose methylprednisolone; HL, Hodgkin lymphoma; HLA, human leukocyte antigen; MDS, myelodysplastic syndrome; MMF, mycophenolate mofetil; MMUD, mismatched unrelated donor; MP, methylprednisolone; MRD, matched related donor; MSC, mesenchymal stem cell; MSD, matched sibling donor; MRD, matched unrelated donor; NA, not available; NHL, non-Hodgkin lymphoma; OKT3, a murine monoclonal antibody against CD3; ORR, overall response rate; PBSC, peripheral blood stem cells; RCT, randomized controlled trial; SR, steroid-refractory. 05 Frontiers in Immunology frontiersin.org Luo et al. 10.3389/ﬁmmu.2023.1211171 10.3389/ﬁmmu.2023.1211171 summarized in Table 2. The ORR at day 28 were reported in 4 trials, of which 3 trials investigated the effects of adding MSCs to other second-line therapy regimens (21–23). The ORR at day 28 of those second-line therapy regimens without MSCs ranges from 36.4% to 70.7%, addition of MSCs increased it to 47.6-82.8% (Table 2). The result of meta-analysis suggested that adding MSCs to other second-line therapy regimens is associated with signiﬁcant increase of ORR at day 28 (RR = 1.15, 95% CI = 1.01–1.32, P = 0.04; Figure 2A). Subgroup analyses were performed based on patients’ age, grade of aGVHD, involved organ, and the number of involved organs, suggests that the addition of MSCs is associated with signiﬁcant improve of ORR at day 28 in patients with severe (grade III–IV or grade C–D) aGVHD (RR = 1.26, 95% CI = 1.04– 1.52, P = 0.02) and patients with multiorgan involved (RR = 1.27, 95% CI = 1.05–1.55, P = 0.01) although the subgroup differences were not statistically signiﬁcant (Figures 3A, B). (Continued) Overall response rate Study Treatment group ORR CR rate OS Relapse of underlying disease Common AEs Zhao 2022 (21) MSC + Second-line therapy 82.8% (82/99) (at day 28)† 56.6% (56/99) (at day 28)† 6-month OS: 68.7%; 1- year OS: 67.1%; 3-year OS: 63.4% 3-year CIR: 10.1% Hematologic (grade ≥3): 37.4%†; Infection (grade ≥3): 65.7%† Second-line therapy (Basiliximab + calcineurin inhibitor) 70.7% (70/99) (at day 28) 40.4% (40/99) (at day 28) 6-month OS: 60.6%; 1- year OS: 54.8%; 3-year CIR: 13.5% Hematologic (grade ≥3): 53.5%; Infection (grade ≥3): 78.8% Oosten 2022 (22) MSC + Second-line therapy 60% (12/20) (at day 28) NA 1-year OS: 45% NA Infection grade 3: 22%; Infection grade 4: 39% Second-line therapy (MMF) 38% (8/21) (at day 28) NA 1-year OS: 33% NA Infection grade 3: 25%; Infection grade 4: 45% Kebriaei 2020 (23) MSC + Second-line therapy 58.3% (95/163) (at day 28) 36.8% (60/163) (DCR) 180-day OS: 34% 180-day CIR: 8% All infection: 88.3%; Edema peripheral: 35.6%; Abdominal pain: 22.7%; Thrombocytopenia: 22.1% MSC + ATG 61.8% (21/34) 41.2% (14/34) MSC + MMF 63.0% (17/27) 48.2% (13/27) MSC + Inﬂiximab 62.1% (18/29) 44.8% (13/29) MSC + Etanercept 47.6% (10/21) 33.3% (7/21) MSC + Daclizumab 73.3% (11/15) 33.3% (5/15) Second-line therapy 54.3% (44/81) (at day 28) 32.1% (26/81) (DCR) 180-day OS: 42% 180-day CIR: 9.9% All infection: 81.5%; Edema peripheral: 33.3%; Abdominal pain: 17.3%; Thrombocytopenia: 22.2% ATG 58.8% (10/17) 23.5% (4/17) MMF 50.0% (8/16) 31.3% (5/16) Inﬂiximab 53.9% (7/13) 23.1% (3/13) Etanercept 36.4% (4/11) 45.5% (5/11) Daclizumab 54.6% (6/11) 54.6% (6/11) (Continued) (Continued) 06 Frontiers in Immunology frontiersin.org Luo et al. Overall response rate 10.3389/ﬁmmu.2023.1211171 Study Treatment group ORR CR rate OS Relapse of underlying disease Common AEs Zeiser 2020 (24) Ruxolitinib 62% (96/154) (at day 28)† 34% (53/154) (at day 28)† 6-month OS: 59.5%; 1- year OS: 48.7% 18-month CIR: 13% Thrombocytopenia: 33%†; Anemia: 30%; CMV infection: 26%; Infection (grade ≥ 3): 22% (up to day 28) Investigator’s choice 39% (61/155) (at day 28) 19% (30/155) (at day 28) 6-month OS: 50.4%; 1- year OS: 43.6% 18-month CIR: 19% Thrombocytopenia: 18%; Anemia: 28%; CMV infection: 21%; Infection (grade ≥ 3): 19% (up to day 28) ATG 30% (6/20) 15% (3/20) Etanercept 45.5% (10/22) 27.3% (6/22) ECP 43.9% (18/41) 19.5% (8/41) Inﬂiximab 35.3% (6/17) 11.8% (2/17) MSC 60% (9/15) 20% (3/15) MMF 32% (8/25) 16% (4/25) Socie 2017, 2019 (25, 26) Inolimomab 28.5% (Therapy success rate#) NA 1-year OS: 47%; OS after LTFU: 30.6%† 1-year CIR: 13% Viral, bacterial, fungal infection: 78%, 82%, 35% ATG 21.5% (Therapy success rate#) NA 1-year OS: 40%; OS after LTFU: 19.6% 1-year CIR: 6% Viral, bacterial, fungal infection: 92%, 84%, 37% Knop 2007 (27) HD-MP + OKT3 53% (21/40) (at day 100) NA 1-year OS: 45%; NA CRS: 60%; Hyperglycemia: 43%; Viral, bacterial, fungal infection: 35% †, 10%, 10% HD-MP 33% (13/39) (at day 100) NA 1-year OS: 36%; NA Hyperglycemia: 49%; Viral, bacterial, fungal infection: 72%, 27%, 10% MacMillan 2007 (28) ABX-CBL 56% (27/48) (at a median of 22 days) 29% (14/48) (at a median of 77 days) 180-day OS: 35.4% NA Infection and infestations: 97.8%; Blood culture positive: 56.5%; Hypertension: 30.4%; Pneumonia: 33%† ATG 57% (27/47) (at a median of 28 days) 32% (15/47) (at a median of 78 days) 180-day OS: 44.7% NA Infection and infestations: 100%; Blood culture positive: 45.7%; Hypertension: 28.3%; Pneumonia: 65% Van Lint 2006 (29) HD-MP + ATG 55% (15/27) (at day 30) 33% (9/27) (at day 30) OS at the end of follow-up: 34% NA NA HD-MP 48% (16/34) (at day 30) 24% (8/34) (at day 30) OS at the end of follow-up: 36% NA NA *DCR, durable complete response, deﬁned as CR for at least 28 consecutive days within the ﬁrst 100 days after enrollment. #Therapy success was deﬁned as overall survival at 1 year without replacement of the baseline allocated treatment. †Results with signiﬁcant difference in comparison with control group (P < 0.05). Overall response rate ABX-CBL, a murine monoclonal antibody against CD147; ATG, anti–thymocyte globulin; CIR, cumulative incidence of relapse; CR, complete response; CRS, cytokine release syndrome; DCR, durable complete response; ECP, extracorporeal photopheresis; HD-MP, high-dose methylprednisolone; LTFU, long-term follow up (with a median follow-up of 58.4 months); MMF, mycophenolate mofetil; MP, methylprednisolone; MSCs, mesenchymal stem cells; NA, not available; OKT3, a murine monoclonal antibody against CD3; ORR, overall response rate; OS, overall survival; SAEs, serious adverse events. survival at 1 year without replacement of the baseline allocated treatment), and no signiﬁcant differences was observed (28.5% versus 21.5%; P = 0.188) (25, 26). (DCR, deﬁned as CR for at least 28 consecutive days within the ﬁrst 100 days after enrollment) as primary endpoint and no signiﬁcant difference was observed (35% versus 30%, P = 0.42) (21, 23). The REACH2 trial reported that ruxolitinib led to signiﬁcantly higher CR rate at day 28 in comparison with investigator’s choice from other second-line therapy (34% versus 19%, P = 0.004) (24). As for the trial comparing HD-MP plus ATG with HD-MP alone, CR rate at dat 30 was reported, and no signiﬁcant difference was observed (33% versus 24%, P = 0.40) (29). For ABX-CBL versus ATG, The CR rates are 29% at a median of 77 days (range 14-77 days) in ABX- CBL group versus 32% at a meidan of 78 days (range 21-88 days) in ATG group (28). Frontiers in Immunology Complete response rate The complete response (CR) rate were reported in 5 of the included trials (reviewed in Table 2). One of the 2 trials investigating the effects of adding MSCs with data regarding CR rate showed that the addition of MSCs led to a signiﬁcantly improvement in CR rate at day 28 (56.6% versus 40.4%, P = 0.02), whereas another trial reported durable complete response Frontiers in Immunology frontiersin.org 07 Luo et al. 10.3389/ﬁmmu.2023.1211171 B A FIGURE 2 Forest plot for meta-analysis including MSC-related trials. (A) Overall response rate at day 28. (B) Overall survival. MSC, mesenchymal stem cells. A B alysis including MSC-related trials. (A) Overall response rate at day 28. (B) Overall survival. MSC, mesenchymal stem cells. Overall survival and relapse of underlying disease suggests that there is no signiﬁcant difference in OS (HR = 0.89, 95% CI = 0.70–1.15, P = 0.38; Figure 2B). As for other comparisons, ruxolitinib did not lead to signiﬁcant improvement in median OS (11.1 months versus 6.5 month; HR = 0.83, 95% CI = 0.60–1.15) but signiﬁcantly prolonged median failure-free survival (5 months versus 1 month; HR = 0.46, 95% CI = 0.35–0.60); inolimomab showed more favorable OS over ATG (HR = 0.57, 95% CI = 0.35– 0.95, P = 0.03) after long-term follow-up (up to 104 months, with a After a median follow-up ranging from 180 days to 58.4 months, the 8 included trials reported overall survival (OS) at 180 days, 6 months, 1 year, 18-months, 3 years or the end of follow-up (summarized in Table 2). Meta-analysis of the 3 trials investigating the effects of adding MSCs to other second-line therapy regimens B A FIGURE 3 Forest plot for subgroup analysis on ORR at day 28 according to (A) the grade of aGVHD and (B) the number of involved organs. aGVHD, acute graft-versus-host disease; MSC, mesenchymal stem cells. B A FIGURE 3 Forest plot for subgroup analysis on ORR at day 28 according to (A) the grade of aGVHD and (B) the number of involved organs. aGVHD, acute graft-versus-host disease; MSC, mesenchymal stem cells. A B B FIGURE 3 Forest plot for subgroup analysis on ORR at day 28 according to (A) the grade of aGVHD and (B) the number of involved organs. aGVHD, acute graft-versus-host disease; MSC, mesenchymal stem cells. FIGURE 3 Forest plot for subgroup analysis on ORR at day 28 according to (A) the grade of aGVHD and (B) the number of involved organs. aGVHD, acute graft-versus-host disease; MSC, mesenchymal stem cells. 08 Frontiers in Immunology frontiersin.org Luo et al. 10.3389/ﬁmmu.2023.1211171 10.3389/ﬁmmu.2023.1211171 median 58.4 months) (25). The other trials did not report signiﬁcant differences in OS between experimental group and control group (Table 2). The cumulative incidence of relapse of underlying disease (at 180 days, 1 year, 18-months, or 3 years) are provided in 4 trials, no signiﬁcant difference is reported (Table 2). available treatments; inolimomab was associated with superior long-term OS versus ATG. These evidences suggest that MSCs- contained second-line therapy regimens and ruxolitinib are asscociated with favorable efﬁcacy outcomes in patients with steroid-refractory aGVHD. Safety The most common adverse events (AEs) reported in the 8 included trials are hematologic toxicities and infection (summarized in Table 2). One trial investigating the effects of adding MSCs to other second-line therapy regimens suggested that the MSCs group is associated with lower incidences of grade 3–4 hematologic toxicities (37.4% versus 53.5%, P = 0.022) and grade 3–4 infection (65.7% versus 78.8%, P = 0.039) (21). The REACH2 trial reported that ruxolitinib is associated with higher rates of thrombocytopenia (33% versus 18%, P = 0.003) compared with the investigator’s choice group (24). In comparison with ATG, inolimomab showed slightly lower incidences of viral infection (78% versus 92%, P = 0.05), and ABX-CBL showed lower incidences of pneumonia (33% versus 65%, P = 0.002). In comparison with HD-MP alone, HD-MP plus OKT3 is associated with lower risk of viral infection (35% versus 72%, P = 0.001), but caused cytokine release syndrome (CRS) at a rate around 60%. Other reported data in about AEs did not show any signiﬁcant difference between experimental group and control group. Meta-analyses results show that the MSCs plus other second- line therapy regimens is associated with similar risk of serious AEs (RR = 0.99, 95% CI = 0.88–1.12, P = 0.91), serious AEs with outcome of death (RR = 0.95, 95% CI = 0.56–1.59, P = 0.83), and infection-related death (RR = 1.17, 95% CI = 0.51–2.66, P = 0.71) in comparison with the control group (Supplementary Figure 4). Overall survival and relapse of underlying disease y The beneﬁts from MSCs in the prophylaxis and treatment of immune-related disorders are widely investigated due to its multipotency and immunomodulatory properties (30). Various mechanisms are involved in the immunoregulating activities of MSCs, such as suppressing the proliferation and activation of CD4+ T cells, increasng the percentage of regulatory T cells, and modifying the cytokine secretion proﬁle of different immune cells (30–32). In patients undergoing allo-HSCT, co-transplantation of MSCs can promotes engraftment and reduce the risk of severe aGVHD (33– 35). For the treatment of GVHD, MSCs are often administrated in combination with other regimens, and the efﬁcacy varies greatly due to the differences in MSCs source and dosage, disease characteristics and the combined regimens (35–38). Commercial MSCs product Remestemcel-L (Prochymal) was approved pediatric patients in Canada and New Zealand, and Temcell was approved in Japan, but the role of MSCs in the treatment of steroid-refractory aGVHD still require to be established (38). A meta-analysis of 13 non-randomised studies obtained an ORR of 72% and a 6-month survival of 63% for MSCs treatment in patients with steroid-refractory aGVHD (37). Afterwards, three RCTs were published and our meta-analyses results suggested that the addition of MSCs to other second-line therapy is associated with signiﬁcant improvement in ORR, especially in patients with severe disease and multiorgan involvement (21–23). An ongoing phase 3 RCT (NCT04629833) comparing MSCs with best available therapy (BAT) in patients with steroid-refractory aGVHD will be helpful to further establish the beneﬁts of MSCs (39). However, the source and dosage of MSCs, and the choice of combined regimens vary across these 3 published RCTs. Considering the combined regimens, the highest ORR (82.8%) and CR rate (56.6%) at day 28 were reported when MSCs are administrated in combination with basiliximab and calcineurin inhibitor (reviewed in Table 2), further studies are required to determine the optimal treatment strategy of MSCs in patients with different characteristics. Discussion Steroid-refractory aGVHD is a life-threatening conditioning resulting in mortality rates higher than 90%. Treatment of steroid- refractory aGVHD remains a great challenge in clinical practice, the standard strategy still has not been established. This study performed a systematic review and meta-analysis of RCTs comparing the efﬁcacy and safety of different second-line therapy regimens in patients with steroid-refractory aGVHD undergoing allo-HSCT. The reported treatment outcomes (including overall response rate, complete response rate, overall survival and main safety outcomes) of all second-line therapy in 8 eligible RCTs were comprehensively reviewed, and meta-analysis of 3 trials about MSCs was performed. We ﬁnd that the addition of MSCs to other second-line therapy regimens are associated with signiﬁcantly improved ORR at day 28, especially in patients with severe (grade III–IV or grade C–D) aGVHD and patients with multiorgan involved. The overall survival, risk of serious AE and serious AE-related death are similar between the MSCs and non-MSCs group. As for other comparisons, ruxolitinib showed signiﬁcantly higher ORR and CR rate than other best Ruxolitinib, and selective inhibitor of Janus kinase (JAK) 1 and JAK2, is the only approved drugs for the treatment of steroid-refractory aGVHD by US Food and Drug Administration (FDA). The JAK1/2 signaling plays an important role in the mechasims of GVHD through mediating the activation and proinﬂammatoy cytokines release of T cell, neutrophils and dendritic cells (DCs) (40, 41). Preclinical studies suggested that ruxolitinib can effectively inhibit JAK1/2 signaling, and ameliorate both acute and chronic GVHD while preserving graft- versus-tumor activity (42, 43). For patients with steroid-refractory aGVHD, ruxolitinib orally given at a dose of 5–10 mg achieved a ORR at day 28 of 55–82% with acceptable toxicity (24, 44, 45). The multicenter RCT (REACH2 trial) reported that compared with a control group incorporting a series of seond-line therapy regimens, ruxolitinib led to signiﬁcantly higher ORR at day 28, higher durable overall response at day 56 and superior failure-free survival (24). A recently published meta-analysis including both prospective and retrospective studies reported an ORR of 77% and a 6-month survival of 63.9% for ruxolitinib in patients with steroid-refractory Frontiers in Immunology 09 frontiersin.org Luo et al. 10.3389/ﬁmmu.2023.1211171 follow-up data, which we think are very important for patients undergoing allo-HSCT, are absent in several included studies. aGVHD, seem to be comparable to that of MSCs- or basiliximab-based second-line therapy regimens (46). 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 authors. Discussion RCT directly comparing the efﬁcacy and safety of ruxolitinib with speciﬁc second-line regimens such as MSCs- and basiliximab-based regimen is unavailable, further trials and integrated studies are required to validate the advantages of ruxolitinib over other srategies. In conclusion, this systematic review and meta-analysis of RCTs suggests that MSCs-contained second-line therapy regimens and ruxolitinib are associated with favorable efﬁcacy outcomes in patients with steroids-refractory aGVHD, but further well-designed RCTs and integrated studies are still required to determine the optimal treatment. Antagonists of IL-2R represent another attractive option for the therapy of steroid-refractory aGVHD. Blocking IL-2 signaling with IL-2R antagonists such as antibodies against IL-2R alpha chain (CD25) (basiliximab, daclizumab and inolimomab) and denileukin diﬁtitox (a recombinant fusion protein composed of IL-2 fragment and diphtheria toxin) show promising activity in steroid-refractory aGVHD (47–50). The effects of combining IL-2R antagonists with inhibitors of TNF-a signaling (etanercept and inﬂiximab) were also investigated (51–54). According to the 2 RCTs included in our review, basiliximab achieved an ORR at day 28 of 70.7% and 82.8% with or without MSCs respectively, inolimomab achieved a 1-year therapy success rate of 28.5% and an advantage in long-term survival over ATG (21, 25). No RCT comparing the efﬁcacy of these four IL- 2R antagonists is available, a meta-analysis pooling data from both prospective and retrospective studies suggested that basiliximab- based therapy is associated the highest ORR of 81%, followed by 71% for daclizumab, 56% for denileukin diﬁtitox and 54% for inolimomab (55). Well-designed prospective studies will be helpful to validate the advantages of basiliximab over other IL-2R antagonists or other regimens in the therapy of steroid-refractory aGVHD. 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. Funding There are several regimens of other mechanisms that were not involved in included trials, such as the anti-a4b7 integrins monoclonal antibody vedolizumab and the anti-interleukin-6 receptor (IL-6R) monoclonal antibody tocilizumab. A systematic review and meta-analysis have evaluated the efﬁcacy of vedolizumab in the treatment of gastrointestinal aGVHD and obtained a pooled long-term ORR higher than 70% (56). As for steroid-refractory gastrointestinal aGVHD, several retrospective studies of small sample size were published and the reported ORR ranges from 45% to 79% (57–59). The efﬁcacy of tocilizumab in steroid- refractory aGVHD was also only evaluated in a few retrospective studies with small sample size, reporting CR rate of 40%-60% (60, 61). Further studies are warranted to determine the efﬁcacy and safety of these regimens with different mechanisms. This study was partly supported by the National Natural Science Foundation of China (NSFC82000169), Natural Science Foundation of Chongqing (cstc2020jcyj-msxmX1058), Project from Southwest Hospital (2022DZXDX004), Project from Third Military Medical University (2021XJS15), and the Logistics Project (AWS17J007). The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. Author contributions SX, YZ, XL and JC contributed to the study conception and design, supervised the study and made critical revision in the manuscript. CL, XH and LW performed database searching, study selection, data extraction, statistical analyses and manuscript writing. GW, YH, YD, and ZH helped with data extraction, data checking and data interpretation. All authors contributed to the article and approved the submitted version. 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. There are several limitations in this study. First of all, the number of published RCTs regarding therapy of steroid- refractory aGVHD is limited. Secondly, the comparative statistical analysis failed to include all investigated regimens since the regimens applied in the control groups varied largely across included tirals, including HD-MP, ATG and an incoporation of a series of best available choice, making it impractical to integrated evidences with traditional or network-meta-analysis. In future research, uniﬁcation of control regimen will be helpful to increase the application of analyzed results for guiding clinical practice. Thirdly, the reported efﬁcacy outcomes also varied across included tirals, making it impractical to integrate data from different trials. 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https://openalex.org/W2963173807	https://repository.arizona.edu/bitstream/10150/627632/1/019001.pdf	English	null	Ground-based adaptive optics coronagraphic performance under closed-loop predictive control	Journal of astronomical telescopes, instruments, and systems	2,018	cc-by	17,412	Ground-based adaptive optics coronagraphic performance under closed-loop predictive control Item Type Article Authors Males, Jared R.; Guyon, Olivier Citation Jared R. Males, Olivier Guyon, "Ground-based adaptive optics coronagraphic performance under closed-loop predictive control," Journal of Astronomical Telescopes, Instruments, and Systems 4(1), 019001 (6 February 2018). https:// doi.org/10.1117/1.JATIS.4.1.019001 Submission: Received 1 June 2017; Accepted 21 December 2017 DOI 10.1117/1.JATIS.4.1.019001 Publisher SPIE-SOC PHOTO-OPTICAL INSTRUMENTATION ENGINEERS Journal JOURNAL OF ASTRONOMICAL TELESCOPES INSTRUMENTS AND SYSTEMS Rights © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Download date 24/10/2024 04:04:00 Item License https://creativecommons.org/licenses/by/3.0/ Version Final published version Link to Item http://hdl.handle.net/10150/627632 Ground-based adaptive optics coronagraphic performance under closed-loop predictive control Item Type Article Authors Males, Jared R.; Guyon, Olivier Citation Jared R. Males, Olivier Guyon, "Ground-based adaptive optics coronagraphic performance under closed-loop predictive control," Journal of Astronomical Telescopes, Instruments, and Systems 4(1), 019001 (6 February 2018). https:// doi.org/10.1117/1.JATIS.4.1.019001 Submission: Received 1 June 2017; Accepted 21 December 2017 DOI 10.1117/1.JATIS.4.1.019001 Publisher SPIE-SOC PHOTO-OPTICAL INSTRUMENTATION ENGINEERS Journal JOURNAL OF ASTRONOMICAL TELESCOPES INSTRUMENTS AND SYSTEMS Rights © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Download date 24/10/2024 04:04:00 Item License https://creativecommons.org/licenses/by/3.0/ Version Final published version Link to Item http://hdl.handle.net/10150/627632 Ground-based adaptive optics coronagraphic performance under closed-loop predictive control Jared R. Males Olivier Guyon Jared R. Males, Olivier Guyon, “Ground-based adaptive optics coronagraphic performance under closed-loop predictive control,” J. Astron. Telesc. Instrum. Syst. 4(1), 019001 (2018), doi: 10.1117/1.JATIS.4.1.019001. 1 Introduction in a significant improvement in contrast between the planet and the star, but also results in needing large diameter telescopes to resolve planets at such small separations.4 This places such observations in the realm of ground-based telescopes equipped with adaptive optics (AO) and coronagraphs. The quest to characterize Earth-like planets was brought into sharp focus by the discovery of a terrestrial exoplanet orbiting the nearest star, Proxima Centauri.1 With a maximum projected separation of around 36 mas, Proxima b could in principle be characterized with current generation instruments,2 and will be readily studied with next-generation giant segmented mirror tele- scopes (GSMTs) such as the 25-m Giant Magelllan Telescope (GMT), 30-m Thirty Meter Telescope, and 39-m Extremely Large Telescope. Indeed, such observations are recognized as one of the key science goals of the GSMTs,3,4 and Proxima b will not be the only target. Results from the Kepler mission5 have made it clear that small planets occur frequently around low-mass stars.6,7 In addition to Proxima b, the recent discov- eries of Ross 128 b,8 LHS 1140 b,9 and the seven terrestrial plan- ets orbiting TRAPPIST-110 likewise point to a high frequency of terrestrial planets in low-mass star systems near the Earth. The analysis presented here is motivated by the question: what are the fundamental limits of ground-based high-contrast coronagraphic imaging of terrestrial exoplanets? Here we attack one part of this complicated issue: finding the fundamental limit imposed on coronagraphic contrast by residual atmospheric turbulence behind a closed-loop AO control system. Much previous work has been done modeling AO perfor- mance in the spatial-frequency domain.11–14 This technique treats the AO system as a spatial filter, casting time and tempo- ral-frequency domain processes (such as measurement noise and control filtering) into the the spatial domain as filters, and analyzing the effect of the combined AO system on the input turbulence power spectral density (PSD). While some nearby planets, such as those orbiting TRAPPIST-1 and LHS 1140 b, will be amenable to atmospheric characterization with the transit spectroscopy technique, the low a priori transit probability means that the majority of nearby planets will have to be characterized with resolved direct imaging. Low-mass stars present unique challenges and oppor- tunities. With lower stellar luminosity, planets with the same equilibrium temperature occur closer to the star. Ground-based adaptive optics coronagraphic performance under closed-loop predictive control Jared R. Malesa,* and Olivier Guyona,b,c,d aUniversity of Arizona, Tucson Steward Observatory, Tucson, Arizona, United States bNational Institutes of Natural Sciences, Astrobiology Center, Mitaka, Tokyo, Japan cUniversity of Arizona, College of Optical Science, Tucson, Arizona, United States dNational Institutes of Natural Sciences, National Astronomical Observatory of Japan, Subaru Telescope, Hilo, Hawaii, United States Jared R. Malesa,* and Olivier Guyona,b,c,d aUniversity of Arizona, Tucson Steward Observatory, Tucson, Arizona, United States bNational Institutes of Natural Sciences, Astrobiology Center, Mitaka, Tokyo, Japan cUniversity of Arizona, College of Optical Science, Tucson, Arizona, United States dNational Institutes of Natural Sciences, National Astronomical Observatory of Japan, S Abstract. The discovery of the exoplanet Proxima b highlights the potential for the coming generation of giant segmented mirror telescopes (GSMTs) to characterize terrestrial—potentially habitable—planets orbiting nearby stars with direct imaging. This will require continued development and implementation of optimized adaptive optics systems feeding coronagraphs on the GSMTs. Such development should proceed with an understanding of the fundamental limits imposed by atmospheric turbulence. Here, we seek to address this question with a semianalytic framework for calculating the postcoronagraph contrast in a closed-loop adaptive optics system. We do this starting with the temporal power spectra of the Fourier basis calculated assuming frozen flow tur- bulence, and then apply closed-loop transfer functions. We include the benefits of a simple predictive controller, which we show could provide over a factor of 1400 gain in raw point spread function contrast at 1 λ∕D on bright stars, and more than a factor of 30 gain on an I ¼ 7.5 mag star such as Proxima. More sophisticated predictive control can be expected to improve this even further. Assuming a photon-noise limited observing technique such as high-dispersion coronagraphy, these gains in raw contrast will decrease integration times by the same large factors. Predictive control of atmospheric turbulence should therefore be seen as one of the key technologies that will enable ground-based telescopes to characterize terrestrial planets. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. [DOI: 10.1117/1.JATIS.4.1.019001] Keywords: coronagraph; adaptive optics; exoplanets; high-contrast imaging. Paper 17024 received Jun. 1, 2017; accepted for publication Dec. 21, 2017; published online Feb. 6, 2018. Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary. Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems o Journal of Astronomical Telescopes, Instruments, and Systems 4(1), 019001 (Jan–Mar 2018) *Address all correspondence to: Jared R. Males, E-mail: jrmales@email. arizona.edu Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.or 1 Introduction This results The previous analytic treatment of AO performance most closely related to our treatment here is Guyon.15 Using the cor- respondence between Fourier modes and speckle amplitude, coupled with straightforward scaling from the input PSD, a der- ivation of the postcoronagraph contrast and its dependence on various AO system parameters was developed. The resulting scaling laws are readily applicable to system design and analysis and have proven to be useful in placing results from RV and transit surveys in context.16 The goal of this study is to present a way to analyze the achievable contrast in a closed-loop AO system, including Journal of Astronomical Telescopes, Instruments, and Systems 019001-1 Jan–Mar 2018 • Vol. 4(1) Jan–Mar 2018 • Vol. 4(1) nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems o Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . 5. We will ignore all non-Kolmogorov wavefront error sources. This means we do not consider static or non- common path errors within the coronagraph nor ana- lyze telescope error sources (e.g., vibrations). We also consider only free atmosphere turbulence, that is, we do not consider dome seeing. Such error sources are ultimately important in real systems; however, free atmosphere turbulence is the dominant term which must be dealt with first. both current generation telescopes and future GSMTs. To do this we develop a semianalytic framework in the temporal frequency domain. That is, we explicitly calculate temporal PSDs rather than using the spatial-filtering approach. At least in the frozen- flow regime, these should produce nearly identical results. Our exploration of this method was motivated by the fact that actual AO systems must be optimized and controlled in the time and temporal-frequency domains. The paper is organized as follows. In Sec. 2, we state our entering assumptions. In Sec. 3, we lay out our notation and physical description of the problem, including the statistics of the Fourier basis. In Sec. 4, we derive a coronagraph model and deal with a subtle detail depending on how residual variance is calculated. In Sec. 5, we calculate the temporal PSDs of Fourier modes assuming von Kármán turbulence in frozen flow. In Sec. 6, we review the standard model of AO control with optimal gains and present a predictive controller based on the linear prediction formalism. Then, in Sec. 3.1 Coordinate System We will describe the incoming wavefront at the entrance pupil of the telescope. The position vector ~q in this plane is defined by the unit vectors ð^u; ^vÞ, such that (1) EQ-TARGET;temp:intralink-;e001;326;431~q ¼ u^u þ v^v: (1) EQ-TARGET;temp:intralink-;e001;326;431~q ¼ u^u þ v^v: We will restrict our analysis to the unobstructed circular aperture of diameter D defined by We do not seek to describe any particular ExAO system, nor address the details of design. Rather, our goal is to assess what can be achieved with an ideal system limited only by the reality of atmospheric turbulence. As such, our main assumptions are as follows: EQ-TARGET;temp:intralink-;e002;326;378Að~qÞ ¼ 4 πD2 ; if q ≤D 2 0; if q > D 2 : (2) (2) Spatial-frequency is a vector quantity Spatial-frequency is a vector quantity 1. We consider only on-axis guide stars and neglect anisoplanatism. This is of minor concern at the small angular separations we are concerned with for exopla- net imaging. EQ-TARGET;temp:intralink-;e003;326;321~k ¼ ku ^u þ kv ^v: (3) EQ-TARGET;temp:intralink-;e003;326;321~k ¼ ku ^u þ kv ^v: (3) Wavefront control is inherently a discrete problem, and the aperture defines the discrete spatial-frequency sampling 2. Our analysis will be monochromatic. We do not con- sider the effects of atmospheric dispersion or chroma- ticity of the air index of refraction or scintillation within the wavefront sensor (WFS) band. We also assume that the WFS and science wavelengths are the same, ignoring differential chromatic effects. EQ-TARGET;temp:intralink-;e004;326;267Δk ¼ 1 D : (4) (4) Hence, we will often discretize spatial-frequency as Hence, we will often discretize spatial-frequency as EQ-TARGET;temp:intralink-;e005;326;217~kmn ¼ m D ^u þ n D ^v; (5) (5) 3. We ignore WFS aliasing. This is an approximation, but with justification: we analyze a pyramid WFS (PyWFS), which is less susceptible to aliasing than the Shack–Hartmann WFS.17 Furthermore, a spatial filter18 could be incorporated in a PyWFS essentially eliminating aliasing from concern. where m and n are integer indices. 2 Assumptions This analysis is concerned with AO in the “extreme” regime: high actuator counts (∼2000 on a 6.5-m aperture, ∼21;000 on a 25.4 m) and high loop speeds (∼2000 Hz or more). Furthermore, we are mainly interested in the coronagraphic raw contrast, that is the ratio of the intensity of the point spread function (PSF) at some separation from the star, to the intensity at the peak of the star’s image or the PSF. Such extreme-AO (ExAO) systems are typically constructed for the purpose of high-contrast imaging of exoplanets and other narrow-angle circumstellar targets. 1 Introduction 7, we use the previous results to predict the postcoronagraph contrasts for 6.5- and 25.4-m telescopes for a range of guide star magnitudes and compare optimal integrator and linear predictive controllers. We discuss our results in Sec. 8 and conclude in Sec. 9. 6. We will not consider changes in turbulence, e.g., changes in the wind velocity or changes in seeing. 7. We will not consider details of coronagraphic architec- ture. However, we do show that our idealized model is representative of real-world coronagraphs. Thus, what follows can be taken as goal setting: we desire to know how well a coronagraph could perform if limited only by atmospheric turbulence. 3 Incoming Wavefront We begin by introducing our notation and physical description of the problem. We summarize our notation in Table 1. 3.2 Spatial Power Spectral Densities The spatial PSD of the phase at the observation wavelength λ in the von Kármán model19 is 4. We assume perfect knowledge of the AO system com- ponents. This means we neglect errors in calibration (e.g., WFS gain) and assume that we know the spatial and temporal transfer functions of all components. 4. We assume perfect knowledge of the AO system com- ponents. This means we neglect errors in calibration (e.g., WFS gain) and assume that we know the spatial and temporal transfer functions of all components. EQ-TARGET;temp:intralink-;e006;326;115 PvKð~k;λÞ¼0.0218 λ0 λ 2 1 r5∕3 0 1 ðk2 þk2 0Þ11∕6 ðrad2∕m−2Þ; (6) (6) Jan–Mar 2018 • Vol. 4(1) Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Table 1 Summary of notation. Table 1 Summary of notation. where r0 is Fried’s parameter20 and λ0 is the wavelength at which r0 is reported. The outer scale, L0, is included in the parameter Symbol Units Parameter ~q m Position vector in the pupil plane u; v m Position coordinates in the pupil plane u ^; ^v Coordinate unit vectors in the pupil plane ~k m−1 Spatial frequency vector in the pupil plane Δk m−1 Discrete spatial frequency sampling ku; kv m−1 Spatial frequency components in the pupil plane m; n Integer indices of discrete spatial frequencies D m Telescope diameter Að~qÞ Aperture function λ m Wavelength of observation and wavefront sensing P rad2 m2 Spatial power spectrum of the wavefront λ0 m Reference wavelength for turbulence parameters r 0 m Fried parameter for atmospheric turbulence strength Q Denotes the Fourier transform of a mode z m Height above the observatory C2nðziÞ Normalized turbulence strength for layer i Mmn Denotes a Fourier mode hmn m Amplitude of a mode describing the wavefront phase amn Amplitude of a mode describing the wavefront amplitude Φð~q; tÞ rad The phase of the wavefront Að~q; tÞ The amplitude of the wavefront σ2mn rad2 The variance of the amplitude of a mode V m∕s Wind speed Θ rad Wind direction τ s Denotes a time delay ~r λ∕D Position vector in the image plane I photons∕s∕ðλ∕DÞ2 Intensity in the image plane PSF photons∕s∕ðλ∕DÞ2 Point-spread function S Strehl ratio C Contrast ratio f Hz Temporal frequency T mn rad2 ∕Hz Temporal power spectrum of a single mode HðsÞ Denotes a transfer function Journal of Astronomical Telescopes, Instruments, and Systems 0190 EQ-TARGET;temp:intralink-;e007;326;719k0 ¼ 1 L0 : (7) (7) The mean value over the aperture, or the piston term, has no effect on our problem. However, the amplitude of the piston mode is nonzero in the above PSD, so we should subtract the power in piston from the PSD. The spatial PSD of a mode on the aperture is given as21,22 EQ-TARGET;temp:intralink-;e008;326;621Pmodeð~kÞ ¼ Poð~kÞjQmodeð~kÞj2; (8) (8) where Poð~kÞ is the input PSD ignoring the aperture, and Qmode denotes the Fourier transform of the mode on the aperture. Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . As a Zernike polynomial, piston is simply Zpiston ¼ 1, and its Fourier transform on Að~qÞ is23 EQ-TARGET;temp:intralink-;e009;326;539Qpiston ¼ 2 J1ðπDkÞ πDk : (9) (9) So the piston subtracted phase PSD is So the piston subtracted phase PSD is EQ-TARGET;temp:intralink-;e010;326;486PvK;subð~k; λÞ ¼ PvKð~k; λÞf1 −½2JiðπDkÞ2g; (10) (10) where the Jinc function is where the Jinc function is EQ-TARGET;temp:intralink-;e011;326;438JiðxÞ ¼ J1ðxÞ x (11) (11) and J1 is the cylindrical Bessel function of the first kind. We model atmospheric turbulence as occurring in discrete layers. To account for the Fresnel propagation among these layers, we use the functions15 EQ-TARGET;temp:intralink-;e012;326;356 Xðk; λÞ ¼ X i C2nðziÞcos2ðπzik2λÞ; Yðk; λÞ ¼ X i C2nðziÞsin2ðπzik2λÞ; (12) (12) where C2nðzÞ is the normalized turbulence strength profile as a function of altitude z. With these corrections, we have the PSD of the phase EQ-TARGET;temp:intralink-;e013;326;255PΦð~kÞ ¼ PvK;subð~k; λÞXðk; λÞ (13) (13) and the PSD of the amplitude EQ-TARGET;temp:intralink-;e014;326;208PAð~kÞ ¼ PvK;subð~k; λÞYðk; λÞ (14) (14) at the aperture of the telescope. at the aperture of the telescope. at the aperture of the telescope. Journal of Astronomical Telescopes, Instruments, and Systems 019001-2 Journal of Astronomical Telescopes, Instruments, and Systems 019001-2 Jan–Mar 2018 • Vol. 4(1) ed From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms 3.4.1 Ignoring the aperture (17) In Eq. (25), the Fourier transform Q describes how the PSD is sampled by the modes. If we were to ignore the spatial effect of the aperture, we would have where p ¼ 1 (in the superscript we will use þ and −). These modes are neither odd nor even and are normalized EQ-TARGET;temp:intralink-;e018;63;588 Z Að~qÞ½Mp mnð~qÞ2d~q ¼ 1; (18) EQ-TARGET;temp:intralink-;e026;326;584Qmn ∼δðj~k −~kmnjÞΔk; (26) (18) (26) where δðxÞ is the Dirac delta distribution, which is the Fourier transform of the sine and cosine on an infinite domain. With this approximation, we have the variance of a mode where δðxÞ is the Dirac delta distribution, which is the Fourier transform of the sine and cosine on an infinite domain. With this approximation, we have the variance of a mode although not orthogonal, on the aperture. For completeness, we show how the normalization is derived in Appendix A.1. The Fourier transforms on the aperture are (see Appendix A.2) although not orthogonal, on the aperture. For completeness, we show how the normalization is derived in Appendix A.1. The Fourier transforms on the aperture are (see Appendix A.2) EQ-TARGET;temp:intralink-;e027;326;517σ2mn ¼ PΦð~kmn; λÞðΔkÞ2 ¼ PΦð~kmn; λÞ D2 ðradÞ: (27) EQ-TARGET;temp:intralink-;e019;63;516Qp mnð~kÞ ¼ ð1 þ ipÞJiðπDkþmnÞ þ ð1 −ipÞJiðπDk−mnÞ; (19) (19) (27) where for notational simplicity, we have defined 3.4 Statistics of the Fourier Basis where we have included a complex-valued coefficient h to illustrate the form of this function. Decomposing this into sines and cosines, we have Following the recipe of Noll,23 we can write the covariance of any two modes over the aperture as EQ-TARGET;temp:intralink-;e016;63;719 Mmn ¼ Refhg½cosð2π~kmn · ~qÞ þ i sinð2π~kmn · ~qÞ þ Imfhg½i cosð2π~kmn · ~qÞ −sinð2π~kmn · ~qÞ; (16) EQ-TARGET;temp:intralink-;e025;326;712hhp mnhp 0 m 0n 0i ¼ Z ∞ 0 Z 2π 0 Qp mnQp 0 m 0n 0PðkÞdθk dk: (25) (25) We now make use of this expression to analyze the statistics of the Fourier basis under von Kármán turbulence. We now make use of this expression to analyze the statistics of the Fourier basis under von Kármán turbulence. where Ref·g and Imf·g denote the real and imaginary parts, respectively. This suggests a real-valued Fourier basis defined as EQ-TARGET;temp:intralink-;e017;63;644Mp mnð~qÞ ¼ cosð2π~kmn · ~qÞ þ p sinð2π~kmn · ~qÞ; (17) 3.3.1 Fourier basis Here, we develop a real-valued form of the Fourier basis. Consider the complex exponential form of the basis24 EQ-TARGET;temp:intralink-;e015;326;93Mmn ¼ heið2π~kmn·~qÞ; (15) 1-3 Jan–Mar 2018 • Vol. 4(1) EQ-TARGET;temp:intralink-;e015;326;93Mmn ¼ heið2π~kmn·~qÞ; (15) (15) Journal of Astronomical Telescopes, Instruments, and Systems 019001-3 Journal of Astronomical Telescopes, Instruments, and Systems 019001-3 Jan–Mar 2018 • Vol. 4(1) Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . 3.4.2 Modal variance EQ-TARGET;temp:intralink-;e020;63;471 kþmn ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ku þ m D 2 þ kv þ n D 2 r ; k−mn ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ku −m D 2 þ kv −n D 2 r : (20) Including the aperture, the variance of a mode in radians is given as (20) EQ-TARGET;temp:intralink-;e028;326;430σ2mn ¼ 2π λ 2 hjhp mnj2i ¼ Z ∞ 0 Z 2π 0 jQp mnj2PðkÞdθk dk: ( We have chosen this form of the Fourier basis primarily because it yields the useful result (28) EQ-TARGET;temp:intralink-;e021;63;380jQþmnð~kÞj2 ¼ jQ−mnð~kÞj2: (21) Now since (21) EQ-TARGET;temp:intralink-;e029;326;361jQp mnj2 ¼ Qp mnQp mn ¼ 2½Ji2ðπDkþmnÞ þ Ji2ðπDk−mnÞ; (29) (29) This means that each spatial frequency can be described by a single spatial PSD, and, as we will show a single one-sided (positive frequencies only) temporal PSD. independent of p, it follows that hjhþmnj2i ¼ hjh−mnj2i. From now on, we will drop the p superscript except where it is explicitly needed. In Fig. 1, we show the modal variance versus spatial fre- quency. This was calculated using numerical integration in double precision with the GNU Scientific Library (GSL26) routines gsl_integration_qagiu (for the radial integral) and gsl_integration_qag (for the azimuthal integral). We found that an absolute tolerance of 10−10 and a relative tolerance of 10−4 gave good results. 3.3.2 The Wavefront in the Fourier basis We denote the wavefront phase as Φð~q; tÞ, which has units of radians. The coefficient of a mode is calculated as EQ-TARGET;temp:intralink-;e022;63;265hp mnðtÞ ¼ λ 2π Z Að~qÞMp mnð~qÞΦð~q; tÞd~q: (22) (22) Figure 1 also shows the naive result using Eq. (27). The sim- ple estimate based on the PSD is significantly below the true variance. These coefficients are real-valued. Poyneer and Veran25 showed that the Fourier basis can be used for analysis and syn- thesis, even though it is not orthogonal on an aperture. Since our slightly modified basis is simply a linear combination of the cosine and sine, their results apply to it as well. This means that we can write One might guess that AO correction will reduce or eliminate this discrepancy. A simple way to estimate the effect of AO con- trol on these statistics is to assume frozen flow and apply a scal- ing for the time-delay error. Following Guyon15 (as modified in Appendix B), the corrected PSD is approximated as EQ-TARGET;temp:intralink-;e023;63;156Φð~q; tÞ ¼ 2π λ X mn ½hþmnðtÞMþmnð~qÞ þ h−mnðtÞM−mnð~qÞ: (23) (23) EQ-TARGET;temp:intralink-;e030;326;131Pcorð~k; λÞ ≈ PΦð~kÞð2πVkÞ2τ2 tl; k ≤D 2d PΦð~kÞ; k > D 2d ; (30) (30) Similarly, we represent the wavefront amplitude aberrations as Similarly, we represent the wavefront amplitude aberrations as Similarly, we represent the wavefront amplitude aberrations as X EQ-TARGET;temp:intralink-;e024;63;103Að~q; tÞ ¼ 1 þ X mn ½aþmnðtÞMþmnð~qÞ þ a−mnðtÞM−mnð~qÞ: (24) where V is the wind speed, τtl is the total latency in the control system, and d is the actuator spacing projected on the aperture. pes, Instruments, and Systems 019001-4 Jan–Mar 2018 • Vol. 4(1) The intensity in an image plane is given as Fig. 1 Modal variance versus spatial frequency. We compare the result of Eq. (25) (solid lines) with the simple estimate of PΦ∕D2 (dashed lines). The black curves are for uncorrected turbulence, and the red curves are for a simple treatment of AO correction. The discrepancy between the solid and dashed curves is due to the convolution on the aperture, which is neglected in the simple estimate. EQ-TARGET;temp:intralink-;e031;326;600Ið~r; tÞ ¼ jFfAð~qÞAð~q; tÞeiΦð~q;tÞgj2: (31) (31) The operator Ff·g describes the propagation of the wave- front from pupil to final image plane. From here on we assume this is the Fourier transform defined as in Appendix A.2, though it could be a series of transforms corresponding to various coronagraph components.27 We define the system PSF as the result of the above with no aberrations, that is hmn ¼ 0 and amn ¼ 0 for all m; n, which is We next recalculated the modal variances with the corrected PSD, using V ¼ 10 m∕s, τtl ¼ 1.25 ms, and d ¼ 13.5 cm on a D ¼ 6.5 m aperture. EQ-TARGET;temp:intralink-;e032;326;490PSFð~rÞ ¼ jFfAð~qÞgj2: (32) The variance with and without the aperture is also shown in Fig. 1. Though the discrepancy is reduced, the simple estimate based on the PSD is still below the true variance. This means that the variance of a Fourier mode is not simply proportional to the PSD when it is sampled by the aperture. EQ-TARGET;temp:intralink-;e032;326;490PSFð~rÞ ¼ jFfAð~qÞgj2: (32) In the case of the circular unobstructed aperture [Eq. (2)], this is the airy pattern The discrepancy is due to the aperture: there is a convolution inherent in Eq. (25) not addressed by Eq. (27). We show how to address this and discuss its implications for various coronagraph models, in Sec. 4. EQ-TARGET;temp:intralink-;e033;326;437PSFð~rÞ ¼ ½2JiðπDr∕λÞ2; (33) (33) where we have used the image plane coordinate ~r ¼ λ~k. Telescope apertures are typically more complicated, consisting of secondary mirror obscuration and support structures, possibly segments, and can be noncircular. Furthermore, it could be posi- tion dependent due to the coronagraph. The result will be more difficult to analyze, usually requiring numerical calculation. Journal of Astronomical Telescopes, Instruments, and Systems 019001-4 Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Fig. 1 Modal variance versus spatial frequency. We compare the result of Eq. (25) (solid lines) with the simple estimate of PΦ∕D2 (dashed lines). The black curves are for uncorrected turbulence, and the red curves are for a simple treatment of AO correction. The discrepancy between the solid and dashed curves is due to the convolution on the aperture, which is neglected in the simple estimate. case. In the figure, we show the covariances normalized by the modal variances, that is the correlation. There is significant correlation between various modes in the uncorrected case, but an AO system acts to suppress this. In the following section we show that this correlation between Fourier modes can be accounted for by convolving the PSF with the PSD. 4 Postcoronagraph Contrast We next derive a PSD-based model of a coronagraph fed by an AO system in the long-exposure limit and assess its validity. Journal of Astronomical Telescopes, Instruments, and Systems 019001-4 Journal of Astronomical Telescopes, Instruments, and Systems 019001-4 01-4 Jan–Mar 2018 • Vol. 4(1) Journal of Astronomical Telescopes, Instruments, and Systems 019001-4 Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/t Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems o We next recalculated the modal PSD, using V ¼ 10 m∕s, τtl ¼ 1 a D ¼ 6.5 m aperture. The variance with and without Fig. 1. Though the discrepancy is based on the PSD is still below t that the variance of a Fourier mo to the PSD when it is sampled by The discrepancy is due to the a inherent in Eq. (25) not addressed address this and discuss its implica models, in Sec. 4. 3.4.3 Correlation We next calculated the covarianc according to Eq. (25). The results rected von Kármán turbulence, an Fig. 1 Modal variance versus spati result of Eq. (25) (solid lines) with (dashed lines). The black curves a and the red curves are for a simp The discrepancy between the solid the convolution on the aperture, wh estimate. Fig. 2 Correlatio p 0 ¼ p and (b) m which is the va the modal varian Journal of Astronomical Telescopes Males We next recalculated the modal variances PSD, using V ¼ 10 m∕s, τtl ¼ 1.25 ms, an a D ¼ 6.5 m aperture. The variance with and without the apertu Fig. 1. Though the discrepancy is reduced, based on the PSD is still below the true va that the variance of a Fourier mode is not to the PSD when it is sampled by the aper The discrepancy is due to the aperture: th inherent in Eq. (25) not addressed by Eq. (2 address this and discuss its implications for v models, in Sec. 4. 3.4.3 Correlation We next calculated the covariance of pair according to Eq. (25). The results are shown rected von Kármán turbulence, and in Fig. Fig. 1 Modal variance versus spatial frequen result of Eq. (25) (solid lines) with the simple (dashed lines). The black curves are for unc and the red curves are for a simple treatme The discrepancy between the solid and dash the convolution on the aperture, which is neg estimate. Fig. 2 Correlations betwee p 0 ¼ p and (b) modes with which is the variance of e the modal variances. Males and Guy Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . 4.1 Coronagraph Model The intensity in an image plane is given as Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Fig. 3 Same as Fig. 2, but for AO corrected von Kármán turbulence. After AO correction removes low-spatial-frequency power, the modal correlations are much smaller but not zero. Fig. 3 Same as Fig. 2, but for AO corrected von Kármán turbulence. After AO correction removes low-spatial-frequency power, the modal correlations are much smaller but not zero. coronagraph, we can use a second-order Taylor expansion28,29 to approximate the postcoronagraph PSF as30 coronagraph, we can use a second-order Taylor expansion28,29 to approximate the postcoronagraph PSF as30 covariances of the form hhp mnhp 0 m 0n 0i. So, to proceed we make the ansatz that we can temporarily ignore the aperture as in Sec. 3.4.1. The effect of this assumption is that all covariances are 0, which leads directly to to EQ-TARGET;temp:intralink-;e034;63;505 Ið~r; tÞ ≈ F Að~qÞ X mnAmnð~q; tÞ 2 þ jFfAð~qÞΦð~q; tÞgj2: (34) EQ-TARGET;temp:intralink-;e038;326;479hIΦ;mnð~rÞi ¼ PΦð~kmn; λÞ D2 ½PSFð~r −~kmnλÞ þ PSFð~r þ ~kmnλÞ (38) EQ-TARGET;temp:intralink-;e038;326;479hIΦ;mnð~rÞi ¼ PΦð~kmn; λÞ D2 ½PSFð~r −~kmnλÞ þ PSFð~r þ ~kmnλÞ (34) Macintosh et al.30 only addressed phase, but the extension to amplitude is straightforward from their arguments. Dealing with the phase component first, we have (38) for a single term in the sum on the first line of Eq. (37). This is the classic result of a Fourier aberration producing a pair of symmetric speckles.31 We illustrate this in Fig. 4. EQ-TARGET;temp:intralink-;e035;63;412IΦð~r; tÞ ≈ X mnFfAð~qÞΦmnð~q; tÞg 2 : (35) (35) Finally, we perform the sum over all spatial frequencies. The complete intensity due to Φ at ~r is Evaluating a single term gives Evaluating a single term gives EQ-TARGET;temp:intralink-;e039;326;366hIΦð~rÞi ¼ X mn hIΦ;mnð~rÞi: (39) (39) EQ-TARGET;temp:intralink-;e036;63;356FfAð~qÞΦmnð~q; tÞg ¼ 2π λ ½hþmnðtÞQþmnð~kÞ þ h−mnðtÞQ−mnð~kÞ: (36) This summation is an unnormalized convolution with the PSF and can be thought of as adding photons from nearby speckles. This step accounts for the effect of the aperture. In the AO corrected regime, it increases the intensity by as much as 50%, explaining the discrepancies in Fig. 1. Then taking the modulus squared and pulling out the terms with mn ¼ m 0n 0 gives (we are suppressing the t dependence of h) Now we apply the lesson of Sec. 3.4.2 above. If we instead use the variance calculated with Eq. 3.4.3 Correlation We next calculated the covariance of pairs of Fourier modes according to Eq. (25). The results are shown in Fig. 2 for uncor- rected von Kármán turbulence, and in Fig. 3 for the corrected Equation (31) can not be evaluated in closed form in the gen- eral case. However, by assuming small aberrations and an ideal Fig. 2 Correlations between the Fourier modes in uncorrected von Kármán turbulence. (a) Modes with p 0 ¼ p and (b) modes with p 0 ≠p. Each covariance point is normalized to the associated diagonal in (a), which is the variance of each mode. In uncorrected turbulence, the correlations are high relative to the modal variances. Fig. 2 Correlations between the Fourier modes in uncorrected von Kármán turbulence. (a) Modes with p 0 ¼ p and (b) modes with p 0 ≠p. Each covariance point is normalized to the associated diagonal in (a), which is the variance of each mode. In uncorrected turbulence, the correlations are high relative to the modal variances. Journal of Astronomical Telescopes, Instruments, and Systems 019001-5 Journal of Astronomical Telescopes, Instruments, and Systems 019001-5 Jan–Mar 2018 • Vol. 4(1) ded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Journal of Astronomical Telescopes, Instruments, and Systems 019001-6 org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibra Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . (28), then we can skip the convolution and arrive directly at EQ-TARGET;temp:intralink-;e037;63;268 IΦð~r;tÞ≈ 2π λ 2 2 X mn ½ðhþmnÞ2þðh−mnÞ2½Ji2ðπDkþmnÞ þJi2ðπDk−mnÞþ8hþmnh−mnJiðπDkþmnÞJiðπDk−mnÞ þ X m0n0 mn≠m0n0 ½hþmnhþ m0n0 þh−mnh− m0n0½JiðπDkþmnÞJiðπD EQ-TARGET;temp:intralink-;e040;326;236 hIΦð~rÞi ¼ 2π λ 2 hjhmnj2i½PSFð~r −~kmnλÞ þ PSFð~r þ ~kmnλÞ: (40) (40) þJiðπDk−mnÞJiðπDk− m0n0Þ þ½hþmnh− m0n0 þh−mnhþ m0n0½JiðπDkþmnÞJiðπDk− m0n0Þ þJiðπDk−mnÞJiðπDkþ m0n0Þ ; ( This is an assertion that the the complete evaluation of Eq. (37) in the long-exposure limit is equivalent to the modal variance calculation on the aperture with Eq. (28). Acknowledging that we have not actually proven this, we demonstrate its validity with numerical experiments in the following section. (37) The amplitude component of the postcoronagraph long- exposure intensity, hIAð~rÞi, is evaluated in nearly identical fashion. which is the instantaneous intensity at a position ~r and time t in the postcoronagraph image plane. Now, we seek the long expo- sure, or expected, value of this expression. Though this appears quite intractable, we see that this will require evaluating We define the “raw PSF contrast” as the ratio of the intensity at ~r to the peak intensity of a noncoronagraphic long-exposure Journal of Astronomical Telescopes, Instruments, and Systems 019001-6 Jan–Mar 2018 • Vol. 4(1) of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Fig. 4 Illustration of the relationship between a phase aberration defined by a Fourier mode with spatial frequency ~kmn and coefficient hmn, to the long-exposure contrast of the resultant speckles in the post- coronagraph image plane. This relationship does not fully define the contrast in the presence of many speckles. Fig. 4 Illustration of the relationship between a phase aberration defined by a Fourier mode with spatial frequency ~kmn and coefficient hmn, to the long-exposure contrast of the resultant speckles in the post- coronagraph image plane. This relationship does not fully define the contrast in the presence of many speckles. Fig. 4 Illustration of the relationship between a phase aberration defined by a Fourier mode with spatial frequency ~kmn and coefficient hmn, to the long-exposure contrast of the resultant speckles in the post- coronagraph image plane. This relationship does not fully define the contrast in the presence of many speckles. image of the star, hINCð0Þi. This is related to the PSF by the long-exposure Strehl ratio hSi, defined by the relationship arbitrary parameters. This method uses the residual PSD, as in Eq. Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . (27), and so requires a convolution. Hence, this tests the convolution solution for the discrepancy between the true variance and the simple PSD estimate highlighted above. EQ-TARGET;temp:intralink-;e041;63;414hINCð0Þi ¼ hSiPSFð0Þ: (41) EQ-TARGET;temp:intralink-;e041;63;414hINCð0Þi ¼ hSiPSFð0Þ: (41) The fourth model consisted of directly measuring the vari- ance of each modal coefficient. The coefficient of each mode was measured by inner product with the mean-subtracted phase and amplitude on the aperture for each randomly gener- ated screen. In this case, the aperture is already taken into account so Eq. (40) describes the results. The contrast is then The contrast is then EQ-TARGET;temp:intralink-;e042;63;370hCð~rÞi ¼ hIΦð~rÞi þ hIAð~rÞi hSiPSFð0Þ : (42) (42) The final comparison coronagraph was an apodized pupil Lyot complex mask coronagraph (APLCMC35). We included this as an example of a coronagraph that can be implemented in the real world. In this case, the focal plane mask was 1.29 λ∕D in diameter at 800 nm, with a complex transmission of −0.458. The optimum pupil apodization gave a net intensity throughput of 0.587. Journal of Astronomical Telescopes, Instruments, and Systems 019001-7 Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms nals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/ter 4.2 Numerical Verification 4(1) Journal of Astronomical Telescopes, Instruments, and Systems 019001-7 nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Sy Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Fig. 5 Numerical verification of coronagraph models. Here, we compare PSF contrasts for the perfect coronagraph,32 the coronagraph defined by Eq. (34), the APLCMC,35 the contrasts C1 þ C2 from Guyon,15 and the variance of the Fourier modes as in Eq. (40). The left graph is for residual atmospheric phase aberrations only, and the right includes amplitude errors. The bottom graph shows the fractional errors of the model coronagraph w.r.t. the APLCMC. In general, the analytic coronagraph models match the physically realizable APLCMC well. This shows that such models are valid for analyzing the potential of ground-based AO-fed coronagraphs. Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Fig. 5 Numerical verification of coronagraph models. Here, we compare PSF contrasts for the perfect coronagraph,32 the coronagraph defined by Eq. (34), the APLCMC,35 the contrasts C1 þ C2 from Guyon,15 and the variance of the Fourier modes as in Eq. (40). The left graph is for residual atmospheric phase aberrations only, and the right includes amplitude errors. The bottom graph shows the fractional errors of the model coronagraph w.r.t. the APLCMC. In general, the analytic coronagraph models match the physically realizable APLCMC well. This shows that such models are valid for analyzing the potential of ground-based AO-fed coronagraphs. 5.1 Temporal PSD of Fourier Modes in Wind-Driven Turbulence applied the five comparison coronagraph models. The results for these coronagraphs are shown in Fig. 5, where we see that, in general, these various coronagraph models match the APLCMC. We note that the “perfect coronagraph” and direct propagation using Eq. (34) are nearly identical to each other. The residual-PSD+convolution analysis also matches well, as does the direct calculation of modal variance. Restating Eq. (8), the spatial PSD of a Fourier mode on an unobstructed circular aperture is given as (as noted above, we are suppressing p) EQ-TARGET;temp:intralink-;e043;326;400Pmnð~kÞ ¼ Poð~kÞjQmnð~kÞj2: (43) (43) 4.3 Long-Exposure Contrast and the PSD We next invoke the Taylor or frozen-flow hypothesis, which for our purposes states that we can treat the time-domain behav- ior of turbulence as if fixed turbulent phase screens are blowing across the telescope in discrete layers. With this assumption we can calculate the temporal PSD of a turbulent mode with21,22 We can summarize the results of this section with the following points: • The postcoronagraph contrast is not simply the residual PSD, due to the effect of the aperture. This is easily addressed by an unnormalized convolution with the PSF. EQ-TARGET;temp:intralink-;e044;326;307T mnðf; VÞ ¼ 2 V Z ∞ −∞ Pmn f V ; kv dkv; (44) (44) • If the variance is calculated with the aperture included, then the convolution is not needed. where f is temporal frequency, V is the wind-speed, and T mn denotes the temporal PSD of the mode specified by mn. With no loss of generality, we have chosen coordinates such that the wind vector ~V ¼ V ^u. We have included a factor of 2 since we will consider only f > 0. • Ideal coronagraph models are valid in the regime of ground-based, well-corrected, AO-fed coronagraphy, and are useful proxies for real-world coronagraphs. Our result that the postcoronagraph long-exposure image is a convolution of the PSF with the residual PSD is nearly identical to that developed by Herscovici-Schiller et al.27 It is fully equiv- alent if one uses a position-dependent PSF and includes internal aberrations, both details we neglect since we are analyzing an ideal system. The difference between this case, and the case when the aperture is included, is important when the modal variances are individually calculated as we do in the following sections. Now to account for an arbitrary wind direction, let Θ be the direction of the wind with respect to the ^u axis. We then derotate the basis functions by this angle to align with the axes of the integral in Eq. (44) by defining EQ-TARGET;temp:intralink-;e045;326;163 m 0 ¼ m cosðΘÞ −n sinðΘÞ n 0 ¼ m sinðΘÞ þ n cosðΘÞ: (45) (45) The temporal PSD for arbitrary wind direction is then simply The temporal PSD for arbitrary wind direction is then simply 4.2 Numerical Verification We conducted a set of numerical experiments to verify our long- exposure coronagraph model, compare it to other models, and to assess its relevance to real-world coronagraphy. Five distinct model coronagraphs were considered, in each case both for phase aberrations only and with the amplitude errors included. We generated a series of phase screens from the von Kármán PSD using Fourier-space convolution of the PSD with Gaussian white noise fields. Scintillation was included by multiplying the PSD by the X and Y functions, Eq. (12), resulting in phase and amplitude screens. AO correction of the phase was approxi- mated as in Eq. (30). We used a Fried parameter of r0 ¼ 0.2 m, an outer scale L0 ¼ 25 m, and windspeed of V ¼ 10 m∕s. The total time delay was set to 2.5 frames at 2000 Hz (1.25 ms). We set telescope diameter to D ¼ 6.5 m and actuator spacing to d ¼ 13.5 cm (48 across) and assumed a wavelength of λ ¼ 800 nm. The phase screens were 2048 pixels across, and we used a circular unobstructed aperture 128 pixels across. We assumed an arbitrarily bright star so WFS measurement noise was ignored. The Strehl ratio of a noncoronagraphic image with these parameters was ∼94%. The first comparison coronagraph model was the so-called “perfect coronagraph.”32 In this model, the complex plane wave that minimizes the energy in the pupil is subtracted, and the result is propagated to the image plane. It has been shown that analysis with the perfect coronagraph produces results that match the real-world four quadrant phase mask coronagraph.33,34 The second coronagraph model consisted of propagating the complex wavefronts using Eq. (34) directly, employing the FFT. The phase and amplitude components were treated separately, then combined after taking the modulus squared. The third coronagraph model corresponds to the measure- ment error and time delay limited contrast C2 of Guyon,15 com- bined with the contrast from uncorrected amplitude C1 due to atmospheric scintillation. In Appendix B, we present a deriva- tion of C2 utilizing our present notation and generalizing it for 10,000 realizations of the AO-corrected phase and uncor- rected amplitude were generated. For each realization, we Journal of Astronomical Telescopes, Instruments, and Systems 019001-7 Jan–Mar 2018 • Vol. 5 Temporal Power Spectra EQ-TARGET;temp:intralink-;e046;326;103T mnðf; V; ΘÞ ¼ 4 V Z ∞ −∞ Pm 0n 0 f V ; kv dkv: (46) (46) Next, we describe a process for calculating the temporal PSDs of the Fourier basis in wind-driven von Kármán turbulence. s, Instruments, and Systems 019001-8 Jan–Mar 2018 • Vol. 4(1) Journal of Astronomical Telescopes, Instruments, and Systems 019001-8 Jan–Mar 2018 • Vol. 4(1) Jan–Mar 2018 • Vol. 4(1) Journal of Astronomical Telescopes, Instruments, and Systems 019001-8 d From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Table 2 LCO turbulence profile for median conditions assumed in this work. Based on the GMT site survey.37,38 Table 2 LCO turbulence profile for median conditions assumed in this work. Based on the GMT site survey.37,38 Layer no. Layer heighta z (m) Layer strengthb C2n Wind Dirc Θ (deg) Wind speed V (m∕s) 1 250 0.42 60 10 2 500 0.03 60 10 3 1000 0.06 75 20 4 2000 0.16 75 20 5 4000 0.11 100 25 6 8000 0.10 110 30 7 16,000 0.12 100 25 aHeight above the observatory. bNormalized. cAngle east of north. Finally, the temporal PSD of a Fourier mode for a turbulence profile is the C2n weighted sum of the single layer profiles EQ-TARGET;temp:intralink-;e047;63;730T mnðfÞ ¼ X i C2nðziÞT mnðf; Vi; ΘiÞ: (47) EQ-TARGET;temp:intralink-;e047;63;730T mnðfÞ ¼ X i C2nðziÞT mnðf; Vi; ΘiÞ: (47) (47) 5.3 Temporal PSD of Measurement Noise We also need the PSD of the WFS measurement noise. The vari- ance at a spatial frequency due to photon noise in the WFS is15 EQ-TARGET;temp:intralink-;e053;326;341 Z 1∕2τwfs 0 T ph;mnðfÞdf ¼ σ2 ph;mn; (53) (53) EQ-TARGET;temp:intralink-;e048;63;307σ2 ph;mn ¼ β2pð~kmnÞ Nph ðrad2 rmsÞ; (48) (48) assuming white noise (a flat PSD) we have EQ-TARGET;temp:intralink-;e054;326;287T ph;mnðfÞ ¼ σ2 ph;mnτwfs: (54) (54) where the factor βpð~kmnÞ quantifies the sensitivity of the WFS to photon noise. Nph is the total number of photons available for wavefront sensing where the factor βpð~kmnÞ quantifies the sensitivity of the WFS to photon noise. Nph is the total number of photons available for wavefront sensing Note that in the absence of detector noise, T ph;mnðfÞ is independent of τwfs. EQ-TARGET;temp:intralink-;e049;63;220Nph ¼ Fγτwfs; (49) (49) EQ-TARGET;temp:intralink-;e049;63;220Nph ¼ Fγτwfs; 5.2 Numerical Calculation The improper integral in Eq. (46) does not in general have a simple closed-form solution, so we must calculate it numeri- cally. In this work, we used the GSL gsl_integration_ qagi36 routine. We found that a relative tolerance of 10−4 and an absolute tolerance of 10−10 in double precision gave good results. Though these choices noticeably extend cal- culation time, less demanding tolerances can produce numeri- cally unsatisfying results. For example, modes with similar spatial frequencies should have similar total variance (integral of the PSD), but setting numerical tolerance too low can intro- duce occasional 50% jumps. We also find that temporal frequency sampling is important, settling on Δf ¼ 0.1 Hz as the largest sampling that should be used in general. Larger samplings tend to also produce jumps, caused by moving on and off sharp peaks associated with indi- vidual wind-layers. If such a peak falls between two frequencies and the sampling is too coarse, the power in the peak will be missed. cAngle east of north. EQ-TARGET;temp:intralink-;e051;326;511S∕N ¼ Fγτwfs ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Fγτwfs þ npxFbgτwfs þ npxσ2ron q ; (51) (51) We illustrate calculations of multilayer turbulence using a seven layer turbulence model based on the GMT site survey at Las Campanas Observatory (LCO).37,38 The parameters of this profile are given in Table 2. The calculated temporal PSDs for several Fourier modes are shown in Fig. 6. Due to the identical wind velocities of several layers, we expect only four distinct peaks. The number of peaks seen at a given spatial frequency depends on fpk;i ¼ ~Vi · ~km;n. If the frequency of two peaks is not well separated, they appear blended at the resolution of the calculations and plots. where npx is the number of pixels involved in measuring Nph, each pixel having readout noise with variance σ2ron, and Fbg is the photon rate per pixel from the background. Now the WFE due to photon noise at the WFS wavelength can be written as EQ-TARGET;temp:intralink-;e052;326;422σ2 ph;mn ¼ β2pð~kmnÞ ðS∕NÞ2 ðrad2 rmsÞ: (52) (52) This variance is spread over the sampling bandwidth of the WFS, 1∕2τwfs. We can determine the temporal PSD of measure- ment noise from 6.1 Transfer Functions (57) EQ-TARGET;temp:intralink-;e057;326;415HτðsÞ ¼ e−sτ: First, we need the error and noise transfer functions (NTF) of the AO control system. Here, we closely follow the development of Poyneer et al.,39 and see also the treatment by Madec.40 Our goal is to calculate the error transfer function (ETF) of the control system, which is defined as The system forms an estimate of the coefficient of a mode, ˜h. In closed-loop, the WFS measures the change in the coefficient of a given mode, Δh. This is combined with the current estimate of the total coefficient to form a new estimate of ˜h, which is then applied to the DM. At time-step ti, the coefficient is specified in terms of the current and previous Δh and ˜h, by a linear combination EQ-TARGET;temp:intralink-;e055;63;342jETFðfÞj2 ¼ T mn;outðfÞ T mn;inðfÞ : (55) (55) EQ-TARGET;temp:intralink-;e058;326;312˜hðtiÞ ¼ X J j¼1 aj ˜hðti−jÞ þ g X L l¼0 blΔhðti−lÞ; (58) That is, the ETF describes the AO system as a temporal filter which acts on the input PSD. The NTF is similarly defined, except that it acts on the measurement noise PSD. The ETF and NTF are constructed using the frequency responses, or transfer functions, of the various components of the system, which we will denote as H. (58) where g is the loop gain. Below we consider in depth how to choose the parameters of this control filter. The frequency response of this general linear filter is41 The WFS measurement is an average of the turbulence over the integration time. Likewise, the deformable mirror (DM) is assumed to move in discrete steps, holding its position between updates. The frequency response of such components is called a sample and hold, which has the functional form EQ-TARGET;temp:intralink-;e059;326;238Hconðz; gÞ ¼ g PL l¼0 blz−l 1 þ PJ j¼1 alz−j (59) (59) and we can map from the z-domain to the s-domain by the sub- stitution z →esT. EQ-TARGET;temp:intralink-;e056;63;176HwfsðsÞ ¼ HdmðsÞ ¼ 1 −e−sT sT ; (56) With the above component frequency responses, we can write the open-loop ETF as (56) EQ-TARGET;temp:intralink-;e060;326;152ETFolðs; gÞ ¼ HwfsðsÞHconðs; gÞHτðsÞHdmðsÞ: (60) (60) where T ¼ 1∕fs, fs being the loop sampling frequency, and s ¼ i2πf. There will be a delay τ due to sensor readout, data transfer, calculations, etc. Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Fig. 6 Calculated temporal PSDs in a seven-layer turbulence model at four different modes, correspond- ing to discrete spatial frequencies. Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Fig. 6 Calculated temporal PSDs in a seven-layer turbulence model at four different modes, correspond- ing to discrete spatial frequencies. EQ-TARGET;temp:intralink-;e057;326;415HτðsÞ ¼ e−sτ: (57) 6 Closed-Loop Control where Fγ is the photon rate (photons s−1) sensed by the WFS, and τwfs is the integration time of the WFS. Fγ depends on star brightness through where Fγ is the photon rate (photons s−1) sensed by the WFS, and τwfs is the integration time of the WFS. Fγ depends on star brightness through Now that we have established the temporal PSDs of the Fourier modes and of WFS measurement noise, we can use them to pre- dict the residual wavefront variance in a closed-loop AO system. Here, we follow the standard model used throughout the AO literature and so only briefly introduce the concepts and our notation. Note that though here we apply the following to the frozen-flow turbulence PSDs we just derived, the techniques are general and could be applied to PSDs in any basis set and to PSDs including additional error sources such as telescope vibrations and nonfrozen-flow. EQ-TARGET;temp:intralink-;e050;63;153Fγ ¼ Fγ;010−0.4×mag; (50) (50) where mag is the Vega magnitude of the star in the WFS band- pass, and Fγ;0 is the photon rate of a 0 mag star in the WFS bandpass including total system throughput. Here, we expand this to include background and readnoise. The signal-to-noise ratio is Jan–Mar 2018 • Vol. 4(1) Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems o nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . 6.2.1 Simple integrator At time step ti, the WFS measures the residual wavefront, Δhi. We combine this with the last estimate, ˜hi−1, which is the result of the previous cycle. The simplest estimate at the current step is the SI The solution vector ~c ¼ ½c0; c1; · · · ; cN−1 contains the opti- mal LP coefficients, which minimize the least-squares error of the output of Eq. (66). The matrix R is a symmetric Toeplitz matrix, and so the Normal equations can be solved very effi- ciently using Levinson’s recursion44. A nice result of using Levinson’s recursion to solve the Yule–Walker equations is that the resultant filter is guaranteed to be stable.42 EQ-TARGET;temp:intralink-;e065;63;354˜hi ¼ ˜hi−1 þ gΔhi: (65) EQ-TARGET;temp:intralink-;e065;63;354˜hi ¼ ˜hi−1 þ gΔhi: (65) g To apply the LP in closed-loop, we use~c as the coefficients in Eq. (58). That is, L ¼ N −1, bl ¼ cl and J ¼ N, aj ¼ cj−1. We can then to determine the optimal loop gain g. This corresponds to a choice of J ¼ 1; a1 ¼ 1 and L ¼ 0; b0 ¼ 1 in Eq. (58). It is also common to choose a1 < 1 (slightly), a so-called leak term, making this the “leaky integra- tor” control law. Here we will confine our analysis to the SI control law. 6.1 Transfer Functions It is common to discuss the “total delay” including 1/2 the WFS sample and hold (for the midpoint of the integration) and 1/2 the DM sample and hold. That is the total delay is T þ τ.40 The transfer function of the pure delay is From this it follows that the closed-loop ETF is From this it follows that the closed-loop ETF is EQ-TARGET;temp:intralink-;e061;326;113ETFclðs; gÞ ¼ 1 1 þ ETFolðs; gÞ : (61) (61) opes, Instruments, and Systems 019001-10 Jan–Mar 2018 • Vol. 4(1) Journal of Astronomical Telescopes, Instruments, and Systems 019001-10 From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . amplitude, RmnðtÞ, where t is the lag, by solving the Normal or Yule–Walker equations41,42 amplitude, RmnðtÞ, where t is the lag, by solving the Normal or Yule–Walker equations41,42 The closed-loop NTF is EQ-TARGET;temp:intralink-;e062;63;741NTFclðs; gÞ ¼ HdmðsÞHτðsÞHconðs; gÞETFclðs; gÞ: (62) EQ-TARGET;temp:intralink-;e062;63;741NTFclðs; gÞ ¼ HdmðsÞHτðsÞHconðs; gÞETFclðs; gÞ: (62) (62) EQ-TARGET;temp:intralink-;e067;326;730R~c ¼ −~r; (67) where (67) The residual PSD in closed-loop control is then the product of the modulus-squared ETF and the modal PSD, and the modu- lus-squared NTF and WFS noise PSD where where EQ-TARGET;temp:intralink-;e068;326;688R ¼ 2 666664 Rmnð0Þ Rmnð1Þ · · · RmnðN −1Þ Rmnð1Þ Rmnð0Þ .. . RmnðN −2Þ .. . · · · .. . .. . RmnðN −1Þ RmnðN −2Þ · · · Rmnð0Þ 3 777775 (68) EQ-TARGET;temp:intralink-;e063;63;677 T cl;mnðf; gÞ ¼ T mnðfÞjETFclðs; gÞj2 þ T ph;mnðfÞjNTFclðs; gÞj2: (63) (63) The residual variance in this mode is just the integral of the residual PSD (68) EQ-TARGET;temp:intralink-;e064;63;601jσmnðgÞj2 ¼ Z fs 0 T cl;mnðf; gÞdf: (64) and (64) and EQ-TARGET;temp:intralink-;e069;326;583~r ¼ 2 66664 Rmnð1Þ Rmnð2Þ ... RmnðNÞ 3 77775 : (69) Note that, since we started with Eq. (43), this estimate of the variance explicitly includes the aperture and so directly describes the postcoronagraph intensity. (69) To employ these calculations, we next need to determine the parameters of Eq. (58) and then find the optimum gain. We can find the autocorrelation of the time-series of a mode from its PSD using the Wiener–Khinchin theorem,43 which states that the autocorrelation is the Fourier transform of the PSD 6.2 Control Laws Our next step in analyzing a closed-loop AO system is to choose the control law used to feed WFS measurements back to the DM. Here we analyze the simple integrator (SI) and the linear predictor (LP). EQ-TARGET;temp:intralink-;e070;326;462RmnðtÞ ¼ FfT mnðfÞg: (70) (70) In Appendix C, we give a recipe for calculating the autocor- relation from a numerical PSD. In Appendix C, we give a recipe for calculating the autocor- relation from a numerical PSD. Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use nals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/ter Journal of Astronomical Telescopes, Instruments, and Systems 019001-11 8. Calculate the contrast according to Eq. (42). 8. Calculate the contrast according to Eq. (42). The first four steps are illustrated in Fig. 8. The PSD of a Fourier mode is shown in Fig. 8(a). The gain-optimized ETFs and NTFs, for both SI and LP controllers, are shown in Fig. 8(b). Figure 8(a) also shows the residual PSDs after applying the ETFs and NTFs to the input turbulence PSD. Fig. 7 Nyquist plot demonstrating the stability of our two control laws. Fig. 7 Nyquist plot demonstrating the stability of our two control laws. The open-loop ETFs are plotted in the imaginary plane for both the SI and LP control laws. Note that frequency increases as the lines move out from the center. The controllers are stable so long as they do not encircle ð0; −1Þ. The value of gain g which causes them to intersect ð0; −1Þ, and hence become unstable, is here called the maximum stable gain gmax. The black circle has a radius of 1. We show the open-loop ETFs for each controller, with optimum gains for both a zeroth and an eighth magnitude star. We illustrate the complete framework in Fig. 9. The fre- quency response of each component is applied to the input PSD, and to the noise PSD, according to the ETF and NTF. The output residual PSD is then input to the coronagraph. e output residual PSD is then input to the coronagrap We applied our method to the cases of a 6.5-m circular aper- ture, here motivated by the in-development MagAO-X system.48 We also consider a 25.4-m telescope, for which we used the col- lecting area of the GMT (seven 8.4 m segments), but ignored details of the aperture in PSD and contrast calculations. The parameters of our calculations for these telescopes are listed in Table 3. EQ-TARGET;temp:intralink-;e071;63;456gopt ¼ arg min 0≤g<gmax jσmnðgÞj2: (71) (71) This is a straightforward optimization problem, which we solve using Brent’s method as implemented in the boost c++ library. Figure 10 shows the Strehl ratio versus guide star magnitude predicted for the 6.5-m AO system at 800 nm. Only residual turbulence is accounted for, including the uncorrected power (a.k.a. fitting error). Integrator control with optimal gains is implemented in the SPHERE SAXO system, which uses a Karhunen–Loéve basis with optimal modal gains.47 The GPI instrument likewise uses optimal gains but with Fourier modes.39 In Fig. 8. Calculate the contrast according to Eq. (42). 11, we show the contrast maps for the 6.5-m system observing 5th, 8th, and 10th magnitude stars, for both control- lers at the WFS wavelength. To estimate the PSF contrast at other wavelengths, all of these results can be scaled by 1∕λ2. The figure shows, as expected, deeper contrasts on brighter stars. The SI controller (left-hand column) exhibits the well known “wind butterfly,” and we see that the gain optimi- zation process turns off modes on fainter stars, changing the shape of the dark hole. The LP controller does not show the “wind butterfly,” rather predictive control essentially eliminates it. It also results in deeper contrasts. Figure 12 shows the PSF contrast profiles both along and across the wind direction. 6.2.2 Linear predictor All that remains is to describe how to choose the optimum value of the gain. The first step is to identify the maximum stable gain, gmax, which is the value of g at which the system becomes unsta- ble (this is sometimes called the “ultimate gain.”) Following Dessenne et al.,45 we evaluate stability of the control law using a Nyquist plot of the open-loop ETF, which plots the real and imaginary components of Eq. (60). In this plane, as long as the ETF does not encircle the point ð0; −1Þ, then the system is stable—a result known as Nyquist’s stability criterion. We then can find the value of gmax by finding the value of g which causes the curve to intersect ð0; −1Þ. This is illustrated in Fig. 7. 25 46 In general, the estimate formed by the SI controller will be slightly incorrect when actually applied due to the finite loop delay. Here, we consider a conceptually straightforward exten- sion of the integrator to include a prediction. We note that more sophisticated methods exist, which we discuss after presenting our results. One way to improve the accuracy of the estimate formed by the controller is with a filter of the form EQ-TARGET;temp:intralink-;e066;63;145˜hi ¼ X N−1 j¼0 cjð˜hi−j−1 þ Δhi−jÞ: (66) (66) From here we follow the standard recipe25,46 for using the input (or “open-loop”) PSD to optimize the closed-loop gain. The per-mode variance-minimizing optimum gain is given by solving We can determine a set of cj which is optimal in the least- squares sense from the temporal autocorrelation of the modal Jan–Mar 2018 • Vol. 4(1) Jan–Mar 2018 • Vol. 4(1) Journal of Astronomical Telescopes, Instruments, and Systems 019001-11 Jan–Mar 2018 • Vol. 4(1) nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . 6. Calculate the intensity: 6. Calculate the intensity: Journal of Astronomical Telescopes, Instruments, and Systems 019001-12 Journal of Astronomical Telescopes, Instruments, and Systems 019001-12 Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Fig. 7 Nyquist plot demonstrating the stability of our two control laws. The open-loop ETFs are plotted in the imaginary plane for both the SI and LP control laws. Note that frequency increases as the lines move out from the center. The controllers are stable so long as they do not encircle ð0; −1Þ. The value of gain g which causes them to intersect ð0; −1Þ, and hence become unstable, is here called the maximum stable gain gmax. The black circle has a radius of 1. We show the open-loop ETFs for each controller, with optimum gains for both a zeroth and an eighth magnitude star. Males and Guyon: Ground based ada a. For the corrected modes, the variance is properly sampled and directly describes the intensity according to Eq. (40). b. For the uncorrected modes, we must use Eq. (38) and then Eq. (39). Note that we include the cor- rected-mode variances in the convolution. 7. Calculate the long-exposure Strehl ratio as the sum of variance over all spatial frequencies to find σ2tot and use the extended Marechal approximation 7. Calculate the long-exposure Strehl ratio as the sum of variance over all spatial frequencies to find σ2tot and use the extended Marechal approximation EQ-TARGET;temp:intralink-;e073;326;639hSi ¼ e−σ2 tot: (73) (73) Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use 7 Closed-Loop Contrast Calculations Given the temporal PSD, the ETF and NTF, and the optimal gain, Eq. (64) describes the residual variance at a given spatial frequency in a closed-loop AO system. We showed how this is related directly to the PSF contrast behind a coronagraph in Sec. 4. So, calculating the closed-loop long-exposure postcor- onagraph contrast consists of the following steps: 1. Calculate the temporal PSDs at each ðm; nÞ according to Secs. 5.1 and 5.2. Due to the Hermitian symmetry of the Fourier basis, this needs to be done in only half the plane. The same calculations were made for the 25.4 GMT-like telescope, shown in Figs. 13 and 14. The results are qualitatively similar, but show a nearly ×10 improvement due to the larger aperture. This can be understood as the larger diameter causing the spatial-frequency sampling interval, 1∕D2, to impose less power per λ∕D, resulting in less residual power per mode. 2. Calculate the temporal PSD of measurement noise according to Sec. 5.3. 3. Determine the optimum controller coefficients per Sec. 6.2, and the associated optimum gains per Sec. 6.3. 3. Determine the optimum controller coefficients per Sec. 6.2, and the associated optimum gains per Sec. 6.3. 4. Determine the ETF and NTF per Sec. 6.1 For both the 6.5- and the 25.4-m telescopes, the gain in con- trast at small separations provided by the LP controller is remarkable. On the 6.5 m observing a 0 mag star, the LP contrast is over 200 times better at 1 λ∕D and is nearly 10 times better for an eighth mag star. On the 25.4 m, the LP contrast is over 1400 times better at 1 λ∕D on a 0-mag star and is over 30 times better for an eighth mag star. 5. Populate the residual PSD as follows: EQ-TARGET;temp:intralink-;e072;63;116σ2mn ¼ jσmnðgoptÞj2; if gopt > 0: Pmnð~kÞ∕D2; if gopt ¼ 0: (72) EQ-TARGET;temp:intralink-;e072;63;116σ2mn ¼ jσmnðgoptÞj2; if gopt > 0: Pmnð~kÞ∕D2; if gopt ¼ 0: (72) (72) Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . ion Dessenne et al 45,50 presented a method usin Fig. 8 (a) The input turbulent PSD for Fourier mode ðm ¼ 12; n ¼ 12Þ (black), along with the residual PSDs in closed-loop control for the SI (blue) and LP (red) controllers. The WFS noise PSD (dashed) corresponds to an eighth mag star on a 6.5-m telescope. (b) The ETF (solid) and NTF (dashed) for both SI (blue) and LP (red) controllers, optimized for the input PSDs shown in (a). Fig. 9 Illustration of our framework, using one input PSD as an example. The input wavefront, described by its temporal PSD, is corrected by the DM. The WFS measures the residual, and noise is added as described by a temporal PSD. A controller, represented as a linear filter, determines an estimate of the wavefront at the next time-step. After a delay, this estimate determines the new shape of the DM. The control loop acts on the input PSD according to the ETF and on the noise PSD according to the NTF. The resultant residual temporal PSD determines the residual variance of this mode, which is then input to the coronagraph. Blue curves are for the SI and red curves are for the LP. Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Fig. 8 (a) The input turbulent PSD for Fourier mode ðm ¼ 12; n ¼ 12Þ (black), along with the residual PSDs in closed-loop control for the SI (blue) and LP (red) controllers. The WFS noise PSD (dashed) corresponds to an eighth mag star on a 6.5-m telescope. (b) The ETF (solid) and NTF (dashed) for both SI (blue) and LP (red) controllers, optimized for the input PSDs shown in (a). Fig. 8 (a) The input turbulent PSD for Fourier mode ðm ¼ 12; n ¼ 12Þ (black), along with the residual PSDs in closed-loop control for the SI (blue) and LP (red) controllers. The WFS noise PSD (dashed) corresponds to an eighth mag star on a 6.5-m telescope. (b) The ETF (solid) and NTF (dashed) for both SI (blue) and LP (red) controllers, optimized for the input PSDs shown in (a). Fig. 9 Illustration of our framework, using one input PSD as an example. The input wavefront, described by its temporal PSD, is corrected by the DM. Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . The WFS measures the residual, and noise is added as described by a temporal PSD. A controller, represented as a linear filter, determines an estimate of the wavefront at the next time-step. After a delay, this estimate determines the new shape of the DM. The control loop acts on the input PSD according to the ETF and on the noise PSD according to the NTF. The resultant residual temporal PSD determines the residual variance of this mode, which is then input to the coronagraph. Blue curves are for the SI and red curves are for the LP. Fig. 9 Illustration of our framework, using one input PSD as an example. The input wavefront, described by its temporal PSD, is corrected by the DM. The WFS measures the residual, and noise is added as described by a temporal PSD. A controller, represented as a linear filter, determines an estimate of the wavefront at the next time-step. After a delay, this estimate determines the new shape of the DM. The control loop acts on the input PSD according to the ETF and on the noise PSD according to the NTF. The resultant residual temporal PSD determines the residual variance of this mode, which is then input to the coronagraph. Blue curves are for the SI and red curves are for the LP. 8 Discussion Dessenne et al.45,50 presented a method using a linear filter, essentially identical to the technique we used. Their method for optimizing the parameters was based on an explicit least squares minimization using past data, rather than the PSD, and allowed the coefficients a and b to differ. This technique was demon- strated on sky. Journal of Astronomical Telescopes, Instruments, and Systems 019001-12 Jan–Mar 2018 • Vol. 4(1) Jan–Mar 2018 • Vol. 4(1) Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems o nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us 8.1 Predictive Control Predictive control has been discussed in many other studies. Here, we used a relatively simple form of it, rather than more sophisticated options. The LP controller provides a con- ceptually simple extension of the integrator controller and pro- vides an efficient and easy to understand method for calculating its coefficients since we know the open-loop PSD. However, it is not the most sophisticated method proposed to-date in the liter- ature and our intent here is not to propose it as the best one. We recently presented a related predictive control strategy, based on empirical orthogonal functions (EOFs).51 EOFs are essentially a time-and-space Karhunen–Loève basis, and this method allows for including non-WFS measurements, such as accelerometers. Filter optimization is done on past time-domain Journal of Astronomical Telescopes, Instruments, and Systems 019001-13 Journal of Astronomical Telescopes, Instruments, and Systems 019001-13 Jan–Mar 2018 • Vol. 4(1) nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us m: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of U Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems o Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Table 3 Parameters. Symbol Parameter Value Units Notes f s Max sampling frequency 2000 Hz τ Loop delay 1.5 Frames d Actuator spacing 0.135 m 48 across 6.5 m βp WFS sensitivity ﬃﬃﬃ 2 p For unmodulated pyramid λ WFS wavelength 800 nm Based on the MagAO WFS49 WFS filter width 357 nm Based on the MagAO WFS49 WFS throughput 0.1 ratio τwfs WFS exposure time 0.0005 s 2 kHz σron WFS readout noise 0.1 Electrons 0 mag flux density @ 800 nm 5.0 × 107 Photons∕sec ∕m2∕nm Vega based Sky background @ 800 nm 20 mags∕arcsec2 r 0 Fried param. 0.16 m Median 0.62 in. at 0.5 μm38 ¯V Weighted mean wind speed 18.7 m/s 5∕3 moment19 Magellan clay D Telescope diameter 6.5 m F γ; 0 0 mag photon flux 5.9 × 1010 Photons∕s Unobstructed aperture npx WFS pixels 7280 Pixels Four quadrants with D∕d across F bg Background flux 0.22 Photons∕pix∕s Assumes 2 in. FOV for WFS GMT D Telescope diameter 25.4 m Edge to edge F γ; 0 0 mag photon flux 6.6 × 1011 Photons∕s Vega, using GMT aperture npx WFS pixels 111,208 Pixels Four quadrants with D∕d across F bg Background flux 0.01 Photons∕pix∕s Assumes 2 in. FOV for WFS Table 3 Parameters. data. We found essentially the same level of improvement in simulations. Fig. 10 Strehl ratio versus guide star magnitude with optimized gains for the two controllers at the WFS wavelength (800 nm). Only residual modal variance and fitting error are included in this plot. The LP con- troller improves Strehl at all magnitudes, with the benefit being great- est between 8th and 10th magnitude. Johnson et al.52 developed a control strategy based on predicting the wind-driven translation of turbulent layers. With simulations incorporating on-sky data from the ViLLaGEs AO system,53 they showed significant increase in Strehl on fainter stars and predicted large gains in Strehl for larger telescopes. Much recent work has been in the area of linear quadratic Gaussian (LQG) controllers. Journal of Astronomical Telescopes, Instruments, and Systems 019001-13 Poyneer et al.24 proposed a Fourier-mode by Fourier-mode optimization of a Kalman filter controller, and using end-to-end simulations showed large gains in contrast similar to what we have shown. They presented their method as a control filter much as we did here. They pointed out that any filter could be cast in the direct form as in Eq. (59), though it is then subject to errors due to numerical precision if actually used to implement the filter. Fig. 10 Strehl ratio versus guide star magnitude with optimized gains for the two controllers at the WFS wavelength (800 nm). Only residual modal variance and fitting error are included in this plot. The LP con- troller improves Strehl at all magnitudes, with the benefit being great- est between 8th and 10th magnitude. More broadly, the determination of optimum control filters is of great interest in the AO-related literature. In the context of AO, this has been studied extensively for LQG controllers.54,55 rom: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use nals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/ter Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Fig. 11 Postcoronagraph long-exposure contrast maps for a 6.5-m telescope at 800 nm. Here, we com- pare results for the SI and LP controllers, for 5th, 8th, and 10th magnitude stars. As we do not address chromaticity here, the WFS and observation wavelengths are both 800 nm. Fig. 11 Postcoronagraph long-exposure contrast maps for a 6.5-m telescope at 800 nm. Here, we com- pare results for the SI and LP controllers, for 5th, 8th, and 10th magnitude stars. As we do not address chromaticity here, the WFS and observation wavelengths are both 800 nm. Fig. 12 Contrast profiles for a 6.5-m telescope observing at 800 nm, using the parameters shown in Tables 2 and 3. As we do not address chromaticity here, the WFS and observation wavelengths are both 800 nm. (a) is along the main wind direction (up-and-down in Fig. 11), and (b) is perpendicular. The dashed lines are for the SI controller, standard in current AO systems. The solid lines are for the LP controller, the form of predictive control we analyze in this work. Since we are conducting a mode-by-mode analysis in discrete spatial frequencies, the lowest spatial frequency we analyze is 1∕D, so the smallest separation we show is 1 λ∕D. Fig. 12 Contrast profiles for a 6.5-m telescope observing at 800 nm, using the parameters shown in Tables 2 and 3. As we do not address chromaticity here, the WFS and observation wavelengths are both 800 nm. (a) is along the main wind direction (up-and-down in Fig. 11), and (b) is perpendicular. The dashed lines are for the SI controller, standard in current AO systems. The solid lines are for the LP controller, the form of predictive control we analyze in this work. Since we are conducting a mode-by-mode analysis in discrete spatial frequencies, the lowest spatial frequency we analyze is 1∕D, so the smallest separation we show is 1 λ∕D. Fig. 12 Contrast profiles for a 6.5-m telescope observing at 800 nm, using the parameters shown in Tables 2 and 3. As we do not address chromaticity here, the WFS and observation wavelengths are both 800 nm. (a) is along the main wind direction (up-and-down in Fig. 11), and (b) is perpendicular. The dashed lines are for the SI controller, standard in current AO systems. The solid lines are for the LP controller, the form of predictive control we analyze in this work. Journal of Astronomical Telescopes, Instruments, and Systems 019001-14 Journal of Astronomical Telescopes, Instruments, and Systems 019001-14 Jan–Mar 2018 • Vol. 4(1) Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Since we are conducting a mode-by-mode analysis in discrete spatial frequencies, the lowest spatial frequency we analyze is 1∕D, so the smallest separation we show is 1 λ∕D. Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Fig. 13 Postcoronagraph long-exposure contrast maps for a 25.4-m telescope at 800 nm, with collecting area defined by the GMT aperture. Here, we compare results for the SI and LP controllers, for 5th, 8th, and 10th magnitude stars. Note that the color scale is ×10 lower than in Fig. 11. As we do not address chromaticity here, the WFS and observation wavelengths are both 800 nm. Fig. 13 Postcoronagraph long-exposure contrast maps for a 25.4-m telescope at 800 nm, with collecting area defined by the GMT aperture. Here, we compare results for the SI and LP controllers, for 5th, 8th, and 10th magnitude stars. Note that the color scale is ×10 lower than in Fig. 11. As we do not address chromaticity here, the WFS and observation wavelengths are both 800 nm. Fig. 14 Contrast profiles for a 25.4-m telescope with the collecting area of the GMT aperture, observing at 800 nm, using the parameters shown in Tables 2 and 3. As we do not address chromaticity here, the WFS and observation wavelengths are both 800 nm. (a) is along the main wind direction (up-and-down in Fig. 13), and (b) is perpendicular. The dashed lines are for the SI controller, standard in current AO systems. The solid lines are for the LP controller, the form of predictive control we analyze in this work. Since we are conducting a mode-by-mode analysis in discrete spatial frequencies, the lowest spa- tial frequency we analyze is 1∕D, so the smallest separation we show is 1 λ∕D. Fig. 14 Contrast profiles for a 25.4-m telescope with the collecting area of the GMT aperture, observing at 800 nm, using the parameters shown in Tables 2 and 3. As we do not address chromaticity here, the WFS and observation wavelengths are both 800 nm. (a) is along the main wind direction (up-and-down in Fig. 13), and (b) is perpendicular. The dashed lines are for the SI controller, standard in current AO systems. The solid lines are for the LP controller, the form of predictive control we analyze in this work. Since we are conducting a mode-by-mode analysis in discrete spatial frequencies, the lowest spa- tial frequency we analyze is 1∕D, so the smallest separation we show is 1 λ∕D. Fig. Journal of Astronomical Telescopes, Instruments, and Systems 019001-15 Jan–Mar 2018 • Vol. 4(1) Journal of Astronomical Telescopes, Instruments, and Systems 019001-15 From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . 9 Conclusion We have developed a semianalytic framework for predicting the raw PSF contrast in an AO-fed coronagraphic image under closed-loop control. Instead of the usual spatial-filter analysis, we explicitly calculated temporal PSDs and analyzed AO per- formance in the temporal-frequency domain. Our technique takes into account closed-loop dynamics as well as a multilayer frozen-flow turbulence model. We also analyzed performance of ideal coronagraphs, showing how to account for the subtle dif- ference between the residual PSD and the residual modal vari- ance on an aperture. Using our model, we showed that predictive control will improve raw contrast by up to a factor of 1400 on bright stars, and by a factor of 30 on eighth magnitude stars, compared to the SI control law. Assuming a photon-noise lim- ited observation, the exposure time required to characterize an exoplanet will decrease by the same large factors. This has sig- nificant implications for the potential of the coming ground- based GSMTs to search for life outside our solar system and makes predictive control one of the key techniques that must be perfected for exoplanet characterization. 8.3 Impact on Exoplanet Characterization Our motivation for this study was to analyze the limits of ground-based coronagraphic exoplanet characterization, and to assess the impact that predictive control will have on it. Consider the coupling of an ExAO-fed coronagraph to a high-dispersion spectrograph,57 a technique recently dubbed “high-dispersion coronagraphy” (HDC).58,59 We focus on this technique because it is, in principle, photon-noise limited, meaning we do not have to consider speckle noise. Neglecting detector and background noise, the signal-to-noise ratio (S∕N) in the HDC technique is given as57 Appendix A: Mathematics of the Fourier Basis We derive the normalizations and Fourier transforms of the Fourier basis on a circular aperture. We derive the normalizations and Fourier transforms of the Fourier basis on a circular aperture. 8.2 Sensitivity to Frozen Flow Our model of input turbulence was based on the frozen flow model. However, our semianalytic method does not require fro- zen flow. That is, any source of the temporal PSDs could be used as input to the analysis. We could incorporate boiling or disturb- ances derived from telescope models (e.g., vibrations due to wind shake). This could also be extended to assess the impact of time-variable turbulence profiles. It should be expected that inclusion of additional sources of error will reduce projected performance. In the case of, e.g., tele- scope vibrations this is simply because there is more variance in the system. In the case of time-variable turbulence, i.e., chang- ing wind and seeing, the reduced performance will result from suboptimality of the filter coefficients. Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . 14 Contrast profiles for a 25.4-m telescope with the collecting area of the GMT aperture, observing at 800 nm, using the parameters shown in Tables 2 and 3. As we do not address chromaticity here, the WFS and observation wavelengths are both 800 nm. (a) is along the main wind direction (up-and-down in Fig. 13), and (b) is perpendicular. The dashed lines are for the SI controller, standard in current AO systems. The solid lines are for the LP controller, the form of predictive control we analyze in this work. Since we are conducting a mode-by-mode analysis in discrete spatial frequencies, the lowest spa- tial frequency we analyze is 1∕D, so the smallest separation we show is 1 λ∕D. Journal of Astronomical Telescopes, Instruments, and Systems 019001-16 Jan–Mar 2018 • Vol. 4(1) Journal of Astronomical Telescopes, Instruments, and Systems 019001-16 wnloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . 8.4 Code Availability See also the review of various system identification strategies presented by Kulcsár et al.56 The code we developed for this analysis is freely available in a Github repository: https://github.com/jaredmales/aoSystem. It is written in c++, is highly parallelized, and compiles to a command line application which accepts configuration files to set up the analysis. It depends on the library available in a Github repository: https://github.com/jaredmales/mxlib, as well as several easily available open-source libraries. No licenses are needed. While this paper was under review, Correia et al. presented an analysis of predictive control using the distributed Kalman filter method.14 Their analysis employed the spatial filtering method (as opposed to our temporal frequency method) and showed postcoronagraph contrast gains for the Keck AO system at least as large as we show here. Any of these control methods can be represented by their temporal transfer functions, and so could be included in further studies using our technique. In summary, the LP is not the only, nor the most advanced, technique we have available, but it is apparent that predictive control offers significant gains for high-contrast imaging of exoplanets. (75) (75) Using the exponential form of the cosine Using the exponential form of the cosine So, in terms of required integration times, the potential ben- efits of predictive control are quite profound. Using just the relatively simple form of it considered in this study, we find that predictive control of atmospheric turbulence could make a GSMT up to 30 times more efficient at exoplanet characteri- zation around a star as bright as Proxima Centauri, and up to 1400 times more efficient on brighter stars. EQ-TARGET;temp:intralink-;e078;326;176 j cosð2π~kmn · ~qÞj2 ¼ Z D∕2 0 Z 2π 0 1 2 þ 1 4 e4πm Dq cos θ þ e−4πm Dq cos θ dθq dq: (78) (78) s, Instruments, and Systems 019001-17 Jan–Mar 2018 • Vol. 4(1) urnal of Astronomical Telescopes, Instruments, and Systems 019001-17 Jan–Mar 2018 • Vol. 4(1) Jan–Mar 2018 • Vol. 4(1) EQ-TARGET;temp:intralink-;e075;63;173Δt ∝hCi: Using the exponential form of the cosine A.1 Normalization The L2 norm of the cosine mode on the aperture is EQ-TARGET;temp:intralink-;e074;63;286S∕N ¼ FpΔt ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ FΔthCi p ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Nlines p ; (74) EQ-TARGET;temp:intralink-;e076;326;310j cosð2π~kmn · ~qÞj2 ¼ Z Að~qÞcos2ð2π~kmn · ~qÞd~q: (76) (74) (76) where Fp is the photon rate from the planet and F is the photon rate from the star (both in one spectral resolution element), and Nlines is the number of resolved lines in the planet spectrum used for crosscorrelation template matching. hCi is the raw PSF con- trast as we have derived here. Δt is the integration time needed to reach a desired S∕N. This leads directly to where Fp is the photon rate from the planet and F is the photon rate from the star (both in one spectral resolution element), and Nlines is the number of resolved lines in the planet spectrum used for crosscorrelation template matching. hCi is the raw PSF con- trast as we have derived here. Δt is the integration time needed to reach a desired S∕N. This leads directly to For a symmetric aperture, we can restrict ourselves to n ¼ 0 with no loss of generality, which simplifies the integral to EQ-TARGET;temp:intralink-;e077;326;245jcosð2π~kmn·~qÞj2¼ 4 πD2 Z D∕2 0 Z 2π 0 cos2 2πm Dqcosθ dθqdq: (77) (77) (77) EQ-TARGET;temp:intralink-;e075;63;173Δt ∝hCi: (75) A.2 Fourier Transforms on an Aperture (93) We need the Fourier transform of the Fourier modal basis over the aperture of the telescope, that is Similarly for the sine modes EQ-TARGET;temp:intralink-;e085;63;398Qcmnð~kÞ ¼ Z ∞ −∞ Z ∞ −∞ Aðu;vÞcosð2π~kmn · ~qÞei2πðkuuþkvvÞdudv: (85) EQ-TARGET;temp:intralink-;e094;326;376Qsmnð~kÞ ¼ i J1ðπDkþmnÞ πDkþmn −J1ðπDk−mnÞ πDk−mn : (94) (94) (85) The unobstructed circular aperture is described by Eq. (2). The unobstructed circular aperture is described by Eq. (2). Now switching to polar coordinates and collecting terms we have The transform can now be written EQ-TARGET;temp:intralink-;e080;63;687j cosð2π~kmn · ~qÞj2 ¼ 1 2 þ 4 D2 Z D∕2 0 J0 4π m D q qdq: (80) The transform can now be written EQ-TARGET;temp:intralink-;e089;326;677 Qcmnð~kÞ ¼ 2 πD2 Z D∕2 0 Z 2π 0 ½ei2πqkþ mn cosðθ−αÞ EQ-TARGET;temp:intralink-;e080;63;687j cosð2π~kmn · ~qÞj2 ¼ 1 2 þ 4 D2 Z D∕2 0 J0 4π m D q qdq: (80) The transform can now be EQ-TARGET;temp:intralink-;e089;326;677 Qcmnð~kÞ ¼ 2 πD2 Z D∕2 0 Z 2π 0 ½ei EQ-TARGET;temp:intralink-;e080;63;687j cosð2π~kmn · ~qÞj2 ¼ 1 2 þ 4 D2 Z D∕2 0 J0 4π m D q qdq: (80) Next using the identity The transform can now be written EQ-TARGET;temp:intralink-;e089;326;677 Qcmnð~kÞ ¼ 2 πD2 Z D∕2 0 Z 2π 0 ½ei2πqkþ mn cosðθ−αÞ þ e−i2πqk−mn cosðθ−βÞdθq dq: EQ-TARGET;temp:intralink-;e089;326;677 Qcmnð~kÞ ¼ 2 πD2 Z D∕2 0 Z 2π 0 ½ei2πqkþ mn cosðθ−αÞ þ e−i2πqk−mn cosðθ−βÞdθq dq: (89) Next using the identity (89) EQ-TARGET;temp:intralink-;e081;63;631zJ1ðzÞ ¼ Z zJ0ðzÞdz; (81) With the following identity: With the following identity: (81) EQ-TARGET;temp:intralink-;e090;326;6052πJ0ðzÞ ¼ Z 2π 0 eiz cosðϕÞdϕ; (90) (90) (cf. Ref. 60 5.52 #1) and a change of variables we have EQ-TARGET;temp:intralink-;e082;63;577j cosð2π~kmn · ~qÞj2 ¼ 1 2 þ Jið2πkmnDÞ: (82) (cf. Ref. 60 8.411 #7) the transform is equivalent to (cf. Ref. 60 8.411 #7) the transform is equivalent to (82) EQ-TARGET;temp:intralink-;e091;326;551Qcmnð~kÞ ¼ 4 D2 Z D∕2 0 ½J0ð2πkþmnqÞ þ J0ð2πk−mnqÞqdq; (91) (91) Similarly, the L2 norm of the sine mode on the aperture is Similarly, the L2 norm of the sine mode on the aperture is EQ-TARGET;temp:intralink-;e083;63;525j sinð2π~kmn · ~qÞj2 ¼ 1 2 −Jið2πkmnDÞ (83) where JnðxÞ are the cylindrical Bessel functions of the first kind. With the identity (83) and the norm of the product is EQ-TARGET;temp:intralink-;e084;63;473j sinð2π~kmn · ~qÞ cosð2π~kmn · ~qÞj ¼ 0: (84) and the norm of the product is EQ-TARGET;temp:intralink-;e092;326;486zJ1ðzÞ ¼ Z zJ0ðzÞdz; (92) (92) (84) (cf. Ref. 60 5.52 #1) we finally have (cf. Ref. 60 5.52 #1) we finally have (cf. Ref. 60 5.52 #1) we finally have EQ-TARGET;temp:intralink-;e093;326;431Qcmnð~kÞ ¼ J1ðπDkþmnÞ πDkþmn þ J1ðπDk−mnÞ πDk−mn : (93) Journal of Astronomical Telescopes, Instruments, and Systems 019001-17 From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use /www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . With the following identity: EQ-TARGET;temp:intralink-;e079;63;7412πJ0ðzÞ ¼ Z 2π 0 eiz cosðϕÞdϕ: (79) EQ-TARGET;temp:intralink-;e088;326;752 cos α ¼ ku þ m D ∕kþmn sin α ¼ kv þ n D ∕kþmn cos β ¼ ku −m D ∕k−mn sin β ¼ kv −n D ∕k−mn: (88) With the following identity: EQ-TARGET;temp:intralink-;e079;63;7412πJ0ðzÞ ¼ Z 2π 0 eiz cosðϕÞdϕ: (79) cos α ¼ ku þ D ∕kþmn sin α ¼ kv þ D ∕kþmn cos β ¼ ku −m D ∕k−mn sin β ¼ kv −n D ∕k−mn: (88) (88) (cf. Ref. 60 8.411 #7), the integral evaluates as (cf. Ref. 60 8.411 #7), the integral evaluates as (cf. Ref. 60 8.411 #7), the integral evaluates as The transform can now be written The transform can now be written Appendix B: Contrast C2 of Guyon Here we present a derivation of the contrast due to time delay and measurement error from Ref. 15, there called C2. We extend the analysis of Ref. 15 to include finite loop delays, detector, and background noise, and make it consistent with the results of Sec. 4. A spatial frequency component of the wavefront phase at time t can be written as EQ-TARGET;temp:intralink-;e086;63;308 Qcmnð~kÞ ¼ 4 2πD2 Z D∕2 0 Z 2π 0 8 < :e i2πq h ðkuþm DÞ cos θþðkvþn DÞ sin θ i þ e −i2πq h ðku−m DÞ cos θþðkv−n DÞ sin θ i9 = ;dθq dq; (86) (86) EQ-TARGET;temp:intralink-;e095;326;238Φmnð~q; tÞ ¼ 2π λ h† mnðtÞ cos½~kmn · ~q þ ϕ† mnðtÞ; (95) (95) where we have made use of the complex exponential form of the cosine. We next introduce the notational simplification where the coefficient h† mnðtÞ is the combined amplitude of the Fourier mode, and ϕ† mnðtÞ is its phase. The † superscript is to differentiate this unnormalized basis from the normalized Fourier modes presented in Sec. 3.3.1. Now at a time τtl later the wavefront will be EQ-TARGET;temp:intralink-;e087;63;189 kþmn ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ku þ m D 2 þ kv þ n D 2 s k−mn ¼ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ ku −m D 2 þ kv −n D 2 s (87) EQ-TARGET;temp:intralink-;e096;326;141Φmnð~q; t þ τtlÞ ≈Φmnð~q; tÞ þ ∂Φmnð~q; tÞ ∂t τtl: (96) (96) (87) The residual variance after applying the wavefront correction will be and the associated angles defined by Th will b and the associated angles defined by Journal of Astronomical Telescopes, Instruments, and Systems 019001-18 Jan–Mar 2018 • Vol. 4(1) s, Instruments, and Systems 019001-18 Jan–Mar 2018 • Vol. 4(1) Journal of Astronomical Telescopes, Instruments, and Systems 019001-18 Journal of Astronomical Telescopes, Instruments, and Systems 019001-18 From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Appendix C: Calculating the Autocorrelation EQ-TARGET;temp:intralink-;e099;63;601 dh† mnðtÞ dt ¼ 0 dϕ† mnðtÞ dt ¼ 2π~kmn · ~V; (99) The autocorrelation R can be calculated from a numerical PSD using the following recipe: (99) which leads to the residual variance due to time lag which leads to the residual variance due to time lag 1. Start with temporal PSD T mnðfÞ at N discrete frequencies fj, where f1 ¼ Δf and fN ¼ 1 2 fs. 1. Start with temporal PSD T mnðfÞ at N discrete frequencies fj, where f1 ¼ Δf and fN ¼ 1 2 fs. 1. Start with temporal PSD T mnðfÞ at N discrete frequencies fj, where f1 ¼ Δf and fN ¼ 1 2 fs. EQ-TARGET;temp:intralink-;e100;63;546σ2† tl;mn ≈ 2π λ 2 hh†2 mnðtÞið2π~kmn · ~VÞ2τ2 tl: (100) (100) 2. Form the two-sided temporal PSD of length 2N by augmenting with 0 at the beginning, and augmenting with the reverse at the end 2. Form the two-sided temporal PSD of length 2N by augmenting with 0 at the beginning, and augmenting with the reverse at the end Now the variance of each mode will be given by the spatial PSD according to Now the variance of each mode will be given by the spatial PSD according to EQ-TARGET;temp:intralink-;e107;326;485 T 0mnðfj0Þ ¼ 8 < : 0; j0 ¼ 1 T mnðfjÞ; j0 ¼ 2:::N þ 1;j ¼ 1:::N T mnðfjÞ; j0 ¼ N þ 2:::2N;j ¼ N −1:::1 : (107) EQ-TARGET;temp:intralink-;e107;326;485 T 0mnðfj0Þ EQ-TARGET;temp:intralink-;e101;63;484hh 2 mnðtÞi ¼ Pð~kmnÞ D2 λ0 2π 2 ðm2 rmsÞ: (101) ¼ 8 < : 0; j0 ¼ 1 T mnðfjÞ; j0 ¼ 2:::N þ 1;j ¼ 1:::N T mnðfjÞ; j0 ¼ N þ 2:::2N;j ¼ N −1:::1 : (107) (101) Employing the wavefront variance from measurement noise given by Eq. (52), the total residual variance at the science wavelength will be Employing the wavefront variance from measurement noise given by Eq. (52), the total residual variance at the science wavelength will be (107) 3. Calculate the DFT of the augmented PSD using the FFT algorithm, giving the autocorrelation at lag τ 3. Appendix B: Contrast C2 of Guyon EQ-TARGET;temp:intralink-;e097;63;752σ2 tl;mn ≈ ∂Φmnð~q; tÞ ∂t 2 τ2 tl ; (97) EQ-TARGET;temp:intralink-;e105;326;752C2ð~rÞ ¼ hI† Φð~rÞi hSiPSFð0Þ : (105) 2 l ; (97) EQ-TARGET;temp:intralink-;e105;326;752C2ð~rÞ ¼ hI† Φð~rÞi hSiPSFð0Þ : (105) (105) (97) which becomes which becomes The Strehl ratio can be calculated by summing the total WFE after the convolution and then employing the Marechal approximation EQ-TARGET;temp:intralink-;e098;63;705 σ2 tl;mn ≈ 2π λ 2 dh† mnðtÞ dt 2 þ h† mnðtÞ dϕ† mnðtÞ dt 2 τ2 tl: (98) EQ-TARGET;temp:intralink-;e106;326;682hSi ¼ e−σ2: (106) (106) (98) It is straightforward to derive the other contrasts, C0;1;3;4;5;6, of Ref. 15 using similar arguments. We next employ the frozen flow hypothesis, assuming that the amplitude does not change and that the phase changes only due to wind-driven flow. This gives Appendix C: Calculating the Autocorrelation Calculate the DFT of the augmented PSD using the FFT algorithm, giving the autocorrelation at lag τ EQ-TARGET;temp:intralink-;e102;63;403 σ†2 mn ¼ λ0 λ 2 Pð~kÞ D2 ð2π~kmn · ~VÞ2τ2 tl þ β2pð~kmnÞ S∕N2 λwfs λ 2 ðrad2 rmsÞ: (102) EQ-TARGET;temp:intralink-;e108;326;361RmnðtÞ ¼ FFTfT 0mnðfj 0Þg: (108) (108) (102) Note that this is will be a circular autocorrelation wrapping around after N points. N should be chosen to be larger (by at least a factor of 2) than the needed number of points of R. In this open-loop framework, the equivalent time lag is τtl ¼ τwfs þ τ where τ is the closed-loop delay defined in Sec. 6.1, and τwfs accounts for the sample-and-hold of the WFS and DM.40 We calculate the optimum frame rate that minimizes the variance by differentiating Eq. (102) with respect to τwfs, which yields a quartic equation 4. 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Early steps in this work were performed under con- tract with the California Institute of Technology (Caltech)/Jet Propulsion Laboratory (JPL) funded by NASA through the Sagan Fellowship Program executed by the NASA Exoplanet Science Institute. The authors acknowledge support from NSF awards 1506818 and 1625441. Each mode will have its own optimum τwfs, which is somewhat analogous to modal gains in closed-loop control. Since hhp2 mni ¼ hhc†2 mn i þ hhs†2 mni ¼ hh†2 mni, we can use the results of Sec. 4 to write EQ-TARGET;temp:intralink-;e104;63;133hI† Φ;mnð~rÞi ¼ σ†2 mn½PSFð~r −~kmnλÞ þ PSFð~r þ ~kmnλÞ: (104) (104) The summation in Eq. (39) defines hI† ΦðrÞi, which leads to the expression for the contrast C2 Journal of Astronomical Telescopes, Instruments, and Systems 019001-19 Journal of Astronomical Telescopes, Instruments, and Systems 019001-19 Jan–Mar 2018 • Vol. 4(1) nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use Jared R. Males is an astronomer at the University of Arizona’s Steward Observatory. He received his PhD from the University of References C. Kulcsár et al., “Vibration mitigation in adaptive optics control,” Proc. SPIE 8447, 84470Z (2012). Journal of Astronomical Telescopes, Instruments, and Systems 019001-20 Journal of Astronomical Telescopes, Instruments, and Systems 019001-20 Jan–Mar 2018 • Vol. 4(1) From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Males and Guyon: Ground-based adaptive optics coronagraphic performance. . . Journal of Astronomical Telescopes, Instruments, and Systems 019001-21 Males and Guyon: Ground-based adaptive optics coronagraphic performance. . Arizona, and he was a NASA Sagan Fellow there from 2013 to 2016. His research is focused on exoplanet characterization and high contrast imaging instrumentation. He is the principal investigator of the NSF-funded MagAO-X extreme adaptive optics system. 57. I. Snellen et al., “Combining high-dispersion spectroscopy with high contrast imaging: probing rocky planets around our nearest neighbors,” Astron. Astrophys. 576, A59 (2015). 58. J. Wang et al., “Observing exoplanets with high dispersion coronagra- phy. I. The scientific potential of current and next-generation large ground and space telescopes,” Astron. J. 153, 183 (2017). Olivier Guyon is an astronomer at the Subaru Telescope, the University of Arizona, and the Japanese Astrobiology Center. He spe- cializes in development of optical techniques to detect and study exo- planets. He develops high contrast imaging techniques combining coronagraphs and extreme adaptive optics to image nearby exopla- nets with space and ground telescopes. His research interests also include advanced adaptive optics control techniques and calibration for high contrast imaging. 59. D. Mawet et al., “Observing exoplanets with high-dispersion coronag- raphy. II. Demonstration of an active single-mode fiber injection unit,” Astrophys. J. 838, 92 (2017). 60. I. S. Gradshteyn et al., Table of Integrals, Series, and Products, Academic Press, Oxford (2007). Jared R. Males is an astronomer at the University of Arizona’s Steward Observatory. He received his PhD from the University of Journal of Astronomical Telescopes, Instruments, and Systems 019001-21 Jan–Mar 2018 • Vol. 4(1) Downloaded From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-use From: https://www.spiedigitallibrary.org/journals/Journal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms nal-of-Astronomical-Telescopes,-Instruments,-and-Systems on 5/8/2018 Terms of Use: https://www.spiedigitallibrary.org/terms-of-us
https://openalex.org/W2088318211	https://figshare.com/articles/journal_contribution/Supplementary_Figure_7_from_Interactions_of_Abiraterone_Eplerenone_and_Prednisolone_with_Wild-type_and_Mutant_Androgen_Receptor_A_Rationale_for_Increasing_Abiraterone_Exposure_or_Combining_with_MDV3100/22394031/1/files/39839640.pdf	unk	null	Interactions of Abiraterone, Eplerenone, and Prednisolone with Wild-type and Mutant Androgen Receptor: A Rationale for Increasing Abiraterone Exposure or Combining with MDV3100	Cancer research	2,012	cc-by	2	7 7
https://openalex.org/W2547461568	https://cuadernoslinguistica.colmex.mx/index.php/cl/article/download/31/29	es		Reseña de <em>Variación y Diversidad Lingüística. Hacia una teoría convergente</em>, editado por Pedro Martín Butragueño y Esther Hernández	Cuadernos de lingüística de el colegio de México	2,016	cc-by	3,083	Reseña s 213 Martín Butragueño, Pedro & Hernández, Esther (eds.), 2015. Variación y Diversidad Lingüística. Hacia una teoría convergente. México: El Colegio de México, 464 pp. Variación y Diversidad Lingüística. Hacia una teoría convergente es el resultado de un intercambio académico entre investigadores de dos instituciones: El Colegio de México y el Consejo Superior de Investigaciones Científicas, de España. En él se reúnen ocho trabajos en torno al tema de la variación y la diversidad lingüísticas, entendiendo por lo primero la existencia de estructuras y patrones alternativos para un mismo contexto al interior de una lengua y por lo segundo la existencia de estructuras y patrones alternativos en lenguas distintas. Esta distinción, por cierto, será sometida a discusión, pero por lo pronto se presenta como un contraste posible que le da título al volumen. La variación lingüística es ubicua, evidente e innegable. Toca todos los niveles de la lengua y se explica por factores tan diversos, que, si uno se trata de concentrar en ella, corre el riesgo de perderse en el vértigo de su inevitabilidad, al grado de llegar a preguntarse si hay acaso algo que en la lengua no varíe, si existe algún núcleo estable –aunque sea idealizado– a partir del cual se puedan establecer comparaciones, en fin: si es posible ponerle límites –como si eso fuera necesario. En este libro, ocho 214 Reseña s CLECM 3(1)-2016 autores toman ese toro por los cuernos y muestran que no solo es posible y deseable enfocar fenómenos lingüísticos desde el punto de vista de la variación, sino que en hacerlo reside precisamente el genio de una teoría general del lenguaje de base realista. En este volumen, el tema de la variación, en ambas dimensiones (inter- e intra- lingüística) se aborda desde perspectivas muy diversas. Así, por ejemplo, encontramos, desde luego, variación entre las estructuras de lenguas distintas y variación entre las estructuras, rasgos y patrones manifiestos en hablantes de la misma lengua –que posiblemente pertenecen a distintos estratos sociales, áreas geográficas o puntos temporales. Pero además se incluyen también explicaciones muy atractivas de la existencia de estructuras distintas empleadas posiblemente por un mismo hablante, simplemente porque la lengua misma permite las alternancias. Es el caso de la concordancia en las construcciones partitivas y pseudo-partitivas: casi puedo apostar que, como consignan Demonte y Pérez-Jiménez, cualquier lector aceptaría como gramaticales estructuras del tipo: El resto de los corredores no llegó a la meta / El resto de los corredores no llegaron a la meta, donde la concordancia entre el sujeto y el verbo puede ser tanto en singular como en plural. El lector hablante de maya Yucateco, podría reconocer que algunas cláusulas de propósito en esta lengua se introducen sin subordinante, pero en cuanto aparece una negación es necesario introducir un complementante explícito, como describe Gutiérrez Bravo en su contribución al volumen. Estos ejemplos me sirven para mostrar que el libro aborda la variación desde arenas que han sido clásicas en su estudio (variación diacrónica, diatópica) pero también desde ámbitos mucho menos comunes (teoría Reseña s 215 fonológica, interfaz semántica/sintaxis o la relación entre prosodia y pragmática). Lo característico de los trabajos que conforman el volumen es que los autores invariablemente buscan en los datos una respuesta a interrogantes teóricas generales. No dan, pues, solo cuenta de la variación por consignar un dato curioso aislado, sino que persiguen los hechos hasta dar con la consecuencia teórica de interés en su disciplina. Esto es quizás lo que más me llama la atención de este libro, y la excusa sobre la que basaré mi breves reseña de los contenidos de cada artículo. El artículo de Violeta Demonte e Isabel Pérez Jiménez, “Construcciones partitivas y pseudo partitivas en español, concordancia híbrida y variación en la interficie sintaxis-semántica”, nos presenta ante un caso de variación regido por factores intrínsecos al sistema de lengua. Se trata del patrón de concordancia híbrida de número que disparan las construcciones partitivas (como La mayoría de los asistentes era afroamericana / La mayoría de los asistentes eran afroamericanos) y pseudo-partitivas (como Un centenar de artistas se ofrecieron a actuar gratis / Un centenar de artistas se ofreció a actuar gratis). Las autoras analizan varios posibles factores determinantes de estos patrones de concordancia en un corpus extenso (por ejemplo, la posición del sujeto respecto al verbo, la presencia de adjetivos, etc.), de los cuales solo los siguientes parecen ser significativos: (i) la categoría de la cabeza: si la cabeza de la construcción partitiva es cuantificacional o sustantiva, y (ii) si la frase nominal hace referencia a grupos o a sus átomos o particiones. Cuando la cabeza es cuantificacional, la concordancia es siempre singular (Ninguno de los corredores reportó / *reportaron problemas), mientras que si es un sustantivo (la mayoría, la mitad, una tercera parte), la concordancia tiende a ser plural. En las construcciones pseudo-partitivas, 216 Reseña s CLECM 3(1)-2016 los sustantivos numerales colectivos (centenar) y multiplicativos (el doble) tienden a concordar en plural, mientras que los que denotan contenedores (caja de galletas) y medidas fijas (kilo) concuerdan en singular. Ahora bien, los casos que permiten la concordancia tanto plural como singular no lo hacen aleatoriamente, sino que ahí la alternancia se explica por un factor semántico: la referencia a un grupo como entidad impone la concordancia singular, mientras que la referencia a los sub-átomos (o particiones) de la pluralidad se correlaciona con la concordancia plural. El lector encontrará una explicación sintáctica de estos hechos en términos de la distinción entre distintos tipos de rasgos de concordancia: unos semánticos (rasgos index) y otros morfológicos (rasgos concord). Otro caso de variación sintáctica es el que presenta Gutiérrez-Bravo, en “Los límites del análisis cartográfico. El caso de las cláusulas de propósito en el Maya Yucateco”. A diferencia de las cláusulas completivas y relativas del maya, las cláusulas de propósito (CPR) en esta lengua no pueden introducirse por un complementante fonéticamente nulo. Esto se aprecia en el hecho de que, aunque en su forma afirmativa las CPR pueden introducirse sin subordinador explícito, cuando aparecen negadas requieren de un complementante. El autor presenta este hecho como evidencia en contra de la hipótesis fuerte de la teoría cartográfica en sintaxis (Rizzi 1997; Cinque 1999), según la cual todas las cláusulas de todas las lenguas constan de las mismas proyecciones funcionales, y solo varían en el hecho de que en algunos núcleos se expresan de manera encubierta o fonéticamente nula. El análisis de las cláusulas de propósito del maya yucateco muestra que para este tipo de cláusulas subordinadas (a diferencia de las relativas y las de complemento), no hay c omplementantes Reseña s 217 nulos disponibles y, por lo tanto, su estructura no corresponde a la de una Frase Complementante, sino a una proyección menor. Así, pues, estos datos del maya yucateco y el contraste entre diferentes tipos de cláusulas subordinadas abren la posibilidad, a decir del autor, de que “incluso dentro de una misma lengua no todas las cláusulas tienen ni el mismo patrón oracional ni el mismo conjunto de frases funcionales” (p.125). Continuando en la línea de los artículos que tratan la variación de estructuras, esta vez a partir de la comparación entre lenguas distintas, está el trabajo de Esther Herrera, “De la simplicidad a la complejidad en los inicios silábicos: el caso de tres lenguas mexicanas”. El artículo se basa en datos provenientes del amuzgo, el mazahua y el tsotsil para poner a prueba dos teorías sobre la estructura de la sílaba: por un lado, la teoría que predice el acomodo de los segmentos con base en la escala de sonoridad y, por otro, la teoría que propone una organización jerárquica en la que el tamaño de la sílaba está determinado por un principio de binariedad. En amuzgo se permiten inicios silábicos de más de dos segmentos sin que su ordenamiento obedezca a la escala de sonoridad, pero los puntos de articulación involucrados en el inicio silábico es máximo de dos. Se argumenta, así, que la teoría de la escala de sonoridad no puede explicar el acomodo de los segmentos del inicio de la sílaba en amuzgo, y este argumento se verá reforzado con datos del tsotsil. La comparación entre las posibles estructuras de inicio de estas tres lenguas aporta evidencia de que la binariedad opera en el nivel melódico de las representaciones segmentales, impidiendo de que estén involucrados más de dos puntos de articulación en este ámbito. En conclusión, la comparación de las estructuras de los inicios silábicos en lenguas distintas permite probar los 218 Reseña s CLECM 3(1)-2016 alcances explicativos de dos teorías. Los datos del amuzgo, el mazahua y el tsotsil llevan a descartar a una de ellas como explicación y, en cambio, permiten reformular los dominios posibles del principio de binariedad. Pedro Martín Butragueño, en “Acercamiento a la prosodia de los actos de habla expresivos. Datos del español de México”, nos ofrece un estudio en un ámbito poco explorado, como lo es el de la prosodia asociada a actos de habla particulares. Se analiza la relación entre prosodia y actos expresivos bajo el aparato de la Teoría de la Optimidad en términos de jerarquías de restricciones de alineamiento prosódico, de asociación entre patrones tonales y unidades portadoras de tono con restricciones de fidelidad, de buena formación y de fraseo. El autor avanza algunas hipótesis sobre las posibilidades prosódicas de los actos expresivos, como el hecho de que la expresividad en general está asociada a determinados parámetros prosódicos, mientras que los tipos expresivos específicos se asocian a ciertos parámetros pragmáticos. La riqueza de datos y detalles del artículo me hacen imposible resumir en unas líneas sus contribuciones centrales, así que me limito a destacar el interés que reviste el estudio de la pragmática en relación con la prosodia. Es un hecho que los hablantes reconocen la emotividad, las actitudes y las intenciones del interlocutor con base en patrones entonacionales específicos, y cualquier estudio que arroje luz sobre los universales que rigen la asociación entre pragmática y prosodia, así como la posible variación entre lenguas o entre dialectos con respecto a estos patrones, es un aporte invaluable a nuestra comprensión general del lenguaje. Los otros trabajos en este libro describen fenómenos de variación en ejes más clásicos, relacionados uno con el eje diatópico, otros con el diacrónico. Pilar García Mouton en su artículo “Reflexiones metodológicas Reseña s 219 sobre lengua y espacio” explica la metodología de la geografía lingüística, cuyo objetivo es la publicación de atlas que permitan visualizar y comparar un fenómeno lingüístico en una determinada área. Nos presenta un recorrido histórico en la producción de atlas que revela cómo los métodos para el diseño de mapas y la recopilación de datos para los atlas lingüísticos ha cambiado notablemente con los años. Por ejemplo, si bien anteriormente se prefería aplicar encuestas a hablantes rurales de variantes conservadoras, en los últimos años la preferencia se ha volcado hacia el hablante común, lo cual implica considerar condiciones reales y corrientes como los movimientos de población y las situaciones de contacto. Además, los nuevos atlas no le conceden importancia primordial o única al eje diatópico, sino que lo combinan con otros ejes. El Atlas Lingüístico de la Península Ibérica contiene información sobre fonética, morfología, sintaxis y léxico. El reto de su edición consiste en respetar los principios metodológicos originales y a la vez combinarlos con las posibilidades que brindan las tecnologías digitales nuevas, que permiten acceder remotamente a los datos, realizar búsquedas cruzadas, disponer tanto de las transcripciones originales como de una transcripción actual unificada, y tener acceso a las anotaciones y la información etnográfica (dibujos, planos y fotografías) recabados durante las encuestas. La conclusión de estas reflexiones plantea a la geolingüística como una metodología que se adapta a nuevas investigaciones, pero sigue vinculándose con los atlas anteriores, que no pierden su validez como registros de variación lingüística en un momento determinado del tiempo. Dejé para el final los dos estudios diacrónicos del libro. En el capítulo “La variación de luego y su evolución histórica”, Esther Hernández 220 Reseña s CLECM 3(1)-2016 describe la variación que ha sufrido este adverbio, tanto en variedades peninsulares como americanas, en sus dimensiones fonológica (lugo, logo, luego), morfológica (aluego, luego), sintáctica (luego de, luego que, luego de que), y la variación léxico-sintáctica, que ha dado lugar a locuciones adverbiales (como desde luego), conjuntivas (luego como), interjectivas (hasta luego) y nuevos lexemas derivados (lueguito). Respecto a la variación semántica, en la que se concentra más a detalle, se consigna que en América y algunas partes del occidente de España, luego mantiene el sentido de ‘inmediatamente’, además del de ‘después’, que es el que tiene en español peninsular. Para explicar esta variación, la autora propone que el significado de los adverbios de tiempo no es estable, sino que depende del concepto de tiempo del hablante o la comunidad que lo maneja. Por esta razón, se incluye a luego dentro de los adverbios deícticos. La descripción histórica de Hernández da testimonio de un cambio que involucra variación en múltiples niveles, típica de procesos de gramaticalización: variación fonética, morfológica, léxica y semántica –del significado locativo del latín ‘en lugar oportuno’ hacia un significado temporal, ‘sin dilación’ (ya presente en latín, pero generalizado más adelante). También se ha documentado como adverbio para “ordenar” el discurso, según la gramática de Nebrija, lo que explica su transferencia del espacio-tiempo hacia el espacio discursivo, como indicador de sucesión. En resumen, la diversidad de construcciones en las que aparece luego se explica por su evolución semántica de expresión espacial a temporal, y de ahí a marcador de sucesión discursiva, en un proceso histórico típico de la gramaticalización y pragmaticalización al que también se añade la variación entre la norma peninsular y las otras normas hispánicas. Reseña s 221 En “Cambio semántico y reestructuración del sistema de los determinantes definidos”, Julia Pozas Loyo apoya, con base en datos diacrónicos, un análisis según el cual el artículo indefinido un contrasta con el determinante indefinido algún en que este último implica no-especificidad epistémica, mientras que el primero puede emplearse tanto con sentido específico como no específico. El contraste, sin embargo, no se extiende a sus contrapartes plurales, pues los datos de Pozas Loyo nos muestran que, desde el siglo XVII, los determinantes plurales unos y algunos no se oponen en términos de especificidad. Más bien, su oposición reside en que unos se ha gramaticalizado en un artículo y, por lo tanto, ha perdido su fuerza cuantificacional, mientras que algunos, que se mantiene como un cuantificador, puede aparecer en construcciones partitivas. Además del interés de las afirmaciones centrales, este capítulo es de un gran valor metodológico, pues la autora hace explícitas las decisiones que debe tomar para considerar si la referencia de una frase nominal es específica o no lo es, con las limitaciones que implica el obtener los datos de un corpus (a diferencia, por ejemplo, de los juicios que se pueden obtener de un hablante). El trabajo presenta un buen modelo de cómo los datos diacrónicos pueden confirmar los análisis semánticos sincrónicos (como, en este caso, la propuesta de Menéndez-Benito y Alonso-Ovalle sobre la no-especificidad de algún), así como orientar análisis sobre contrastes que en sincronía no se tenían hasta la fecha muy claros (como el contraste actual entre unos y algunos). En suma, no solo los temas tratados son valiosos como estudios de caso particulares, sino que emplean la descripción de fenómenos puntuales como base para evaluar teorías y metodologías sobre el estudio del 222 Reseña s CLECM 3(1)-2016 lenguaje en general. La variación es la fibra que las hilvana, pero esta se enfoca desde múltiples áreas y con distintos fines. Esta tendencia culmina con lo que considero la parte más disfrutable del libro, que es una discusión tipo panel en la que participan Esther Hernández, Pedro Martín Butragueño, Rebeca Barriga, Violeta Demonte, Pilar García Mouton, Rodrigo Gutiérrez Bravo, Esther Herrera, Isabel Pérez-Jiménez y Julia Pozas Loyo. En ella, cada uno de los participantes, desde su disciplina y áreas de interés específicas, contesta a preguntas como ¿Qué es la variación lingüística? ¿Cuáles son los conceptos clave de la variación (para cada ámbito de estudio)? ¿Qué es diversidad? ¿Hay universales lingüísticos? ¿Se puede comparar la variación entre lenguas con la variación al interior de las lenguas? ¿Cómo se manifiesta la variación en los distintos niveles lingüísticos? ¿Cuáles son los métodos para su estudio? ¿Cuál es el papel de los corpus en el estudio de la variación? ¿Se pueden tender puentes entre la variación y la teoría formal? Considero a esta parte del libro “interactiva” no solo porque es el producto de un ingenioso intercambio entre los panelistas, sino porque también es la que anima al lector a plantearse esas mismas preguntas y considerar posibles respuestas. En las respuestas de los autores atestiguamos una diversidad de opiniones y enfoques. Hay quienes, por ejemplo, conciben la distinción entre variación inter-lingüística y variación intra-lingüística como una diferencia prácticamente de grado –donde la variación entre lenguas es simplemente más notoria porque involucra más parámetros, pero no por que sea de una naturaleza distinta a la variación dialectal–, y que en el fondo ambas están regidas por los mismos principios. Algunos Reseña s 223 conciben la variación como indisociablemente ligada al cambio; otros afirman que se puede tener una sin tener el otro –es decir, que no toda variación implica cambio, aunque todo cambio implica variación. Las preguntas sobre variación van inevitablemente ligadas a la pregunta sobre la existencia de universales. Martín Butragueño propone replantear esta pregunta, de manera que no se dirija a la presencia de hechos invariables en las lenguas (o entre las lenguas), los cuales serán difíciles de encontrar en términos absolutos, sino enfocarla en la universalidad de los procesos y las propiedades del cambio. Me llama particularmente la atención –porque me tranquiliza, pensando en el vértigo del que hablaba al inicio de esta reseña– la siguiente afirmación de Violeta Demonte: “estudiar la variación desde distintos ángulos permite comprobar que las lenguas son más similares unas de otras de lo que parece” (p. 406). En fin, el lector encontrará en esta discusión la confirmación de que la preocupación central de los autores es teórica en el mejor sentido del término: que apuntan a la formulación crítica de una teoría general del lenguaje, desde un punto de vista realista y novedoso, sin ataduras a principios preconvenidos, sino desde la perspectiva creativa y fresca que aporta el estudiar a la lengua desde su naturaleza más primordial y evidente: su permanente y omnipresente variación. Violeta Vázquez Rojas Maldonado El Colegio de México vazquezrojas@colmex.mx
https://openalex.org/W4231651402	https://figshare.com/articles/journal_contribution/Subjective_wellbeing_in_parents_during_the_COVID-19_pandemic_in_Australia/20671461/1/files/36881316.pdf	English	null	Subjective wellbeing in parents during the COVID-19 Pandemic in Australia	null	2,020	cc-by	10,475	AUTHOR(S) Elizabeth Westrupp, Mark Stokes, Matthew Fuller-Tyszkiewicz, Tomer Berkowitz, Tanja Capic, Sarah Khor, Christopher Greenwood, Antonina Mikocka-Walus, Emma Sciberras, George Youssef, Craig Olsson, Delyse Hutchinson Subjective wellbeing in parents during the COVID-19 pandemic in Australia A B S T R A C T Keywords: Australia COVID-19 pandemic Families Parent Subjective wellbeing Keywords: Australia COVID-19 pandemic Families Parent Subjective wellbeing Objectives: To examine (1) the subjective wellbeing of Australian parents raising children and adolescents (0–18 years) during April 2020 ‘stage three’ COVID-19 restrictions, in comparison with parents assessed over 18-years prior to the pandemic; and (2) socio-demographic and COVID-19 predictors of subjective wellbeing during the pandemic. p Methods: Cross-sectional data were from the COVID-19 Pandemic Adjustment Survey (CPAS, N = 2365 parents of a child 0–18 years, 8-28th April 2020); and a pre-pandemic national database containing 18 years of annual surveys collected in 2002–2019 (N = 17,529 parents). y Results: Levels of subjective wellbeing during the pandemic were considerably lower than ratings prior to the pandemic (Personal Wellbeing Index, mean[SD] = 65.3 [17.0]; compared to [SD] = 75.8 [11.9], p < 0.001). During the pandemic, lower subjective wellbeing was associated with low education (adjusted regression coef ficient, 95% confidence interval [95% CI] = −5.19, −0.93), language other-than-English (95% CI = -7.22, −1.30), government benefit (95% CI = -6.99, −0.96), single parents (95% CI = -8.84, −4.59), child neuro developmental condition (95% CI = -3.44, −0.76), parent physical/mental health problems (95% CI = -3.23, −0.67), COVID-environmental stressors (95% CI = -3.48, −2.44), and fear/worry about COVID-19 (95% CI = -8.13, −5.96). Unexpectedly, parent engagement with news media about the pandemic was associated with higher subjective wellbeing (95% CI = 0.35, 1.61). g j g Conclusion: Subjective wellbeing in parents raising children aged 0–18 years appears to be disproportionately impacted by the COVID-19 pandemic and restrictions in Australia. Specific at-risk groups, for which government intervention may be warranted, include parents in socially disadvantaged contexts, parents with pre-existing mental health difficulties, and parents facing significant COVID-19-related work changes. The global spread of SARS-Cov-2 (i.e., the virus that causes COVID- 19) has led to the rapid implementation of population-wide lockdown measures which have raised widespread public health and clinical concern about impacts on health, and mental health, in particular. Ev idence describing the impact of the pandemic has indicated increased rates of mental health problems in Australia [20,37] and internationally [36,41]. A review of 68 studies (N = 288,830 participants, 19 countries) found the prevalence of adult anxiety and depression during the pandemic to be 30–33%, with women, younger adults, and low socio- economic status most vulnerable to experiencing higher levels of psy chological distress [49]. Available online 1 April 2021 0022-3999/© 2021 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jpsychores.2021.110482 Received 1 December 2020; Received in revised form 2 March 2021; Accepted 29 March 2021 Subjective wellbeing in parents during the COVID-19 pandemic in Australia Elizabeth M. Westrupp a,b,c,, Mark A. Stokes a, Matthew Fuller-Tyszkiewicz a, Tomer S. Berkowitz a, Tanja Capic a, Sarah Khor a, Christopher J. Greenwood a, Antonina Mikocka-Walus a, Emma Sciberras a,b,c,d, George J. Youssef a, Craig A. Olsson a,b,d, Delyse Hutchinson a,b,d,e a Deakin University, Centre for Social and Early Emotional Development, School of Psychology, Victoria, Australia b University of Melbourne, Department of Paediatrics, Victoria, Australia c La Trobe University, Judith Lumley Centre, Victoria, Australia d Murdoch Children’s Research Institute, Melbourne Royal Children’s Hospital, Victoria, Australia e The University of New South Wales, The National Drug and Alcohol Research Centre, Australia a Deakin University, Centre for Social and Early Emotional Development, School of Psychology, Victoria, Australia b University of Melbourne, Department of Paediatrics, Victoria, Australia c La Trobe University, Judith Lumley Centre, Victoria, Australia d Murdoch Children’s Research Institute, Melbourne Royal Children’s Hospital, Victoria, Australia e The University of New South Wales, The National Drug and Alcohol Research Centre, Australia Contents lists available at ScienceDirect Contents lists available at ScienceDirect Corresponding author at: School of Psychology, Deakin University, 221 Burwood Highway, Burwood, VIC 3125, Australia. E-mail address: elizabeth.westrupp@deakin.edu.au (E.M. Westrupp). 10536/DRO/DU:30149942 Downloaded from Deakin University’s Figshare repository Deakin University CRICOS Provider Code: 00113B Journal of Psychosomatic Research 145 (2021) 110482 * Corresponding author at: School of Psychology, Deakin University, 221 Burwood Highway, Burwood, VIC 3125, Australia. E-mail address: elizabeth.westrupp@deakin.edu.au (E.M. Westrupp). https://doi.org/10.1016/j.jpsychores.2021.110482 Received 1 December 2020; Received in revised form 2 March 2021; Accepted 29 March 2021 A B S T R A C T Less is known about broader impacts on overall subjective wellbeing; yet widespread subclinical mental health diffi culties carry significant implications for the subjective wellbeing of the population particularly in the absence of sufficient resources [12,21]. The population level of subjective wellbeing is relatively stable and positive, yet vulnerable population groups such as carers or single par ents typically report lower than normal levels [11,27]. One notable group missing from COVID research undertaken so far is that of the Journal of Psychosomatic Research 145 (2021) 110482 E.M. Westrupp et al. family unit. This is despite families representing one of the largest de mographics of any given population, one of the most resource-intensive periods of the life course, and a key point of exchange between gener ations that can have intergenerational consequences (both positive and negative). The current study thus compares parent subjective wellbeing during lockdown to pre-pandemic normative data from a series of cross- sectional surveys of Australian adults collected annually over 18 years [27].Australia had early success in slowing the infection rate of SARS- Cov-2 via social distancing measures implemented from March through May 2020, with a ‘stage three’ lockdown requiring that Aus tralians avoid leaving their house except for four reasons: (1) shopping for food and supplies, (2) care and caregiving, (3) exercise, and (4) study or work – if unable to do so from home [15]. In the first week of lock down (and of our data collection), on the 5th April 2020, Australia had experienced 5687 cases, including 34 deaths; by the end of our data collection on 28th April 2020, Australia had a total of 6731 cases, including 84 deaths. Although the stay-at-home orders were applied nationally, individual states lowered lockdown restrictions at different rates progressively throughout early May to mid-June 2020 [14]. The period of lockdown and restrictions was accompanied by a rapid in crease in job losses and unemployment, with two-thirds of Australians having their employment affected (Roy [35]). [16], and has tended to focus on child outcomes. Data from the first lockdown in Spain demonstrates links between higher levels of parenting distress, less structured parenting, and child mental health problems [42]. A study in the USA showed that parent anxiety and depression were associated with parent stress and child abuse potential [7]. A B S T R A C T However, these studies examined associations at the time of the pandemic, and did not estimate whether there were changes in family functioning prior to, compared to during, the pandemic. Just one pre print has examined this to-date; findings from the Born in Bradford study (N = 1860) show increases in parent depression and anxiety assessed before and during the first COVID-19 lockdown in the United Kingdom, and also that parent loneliness, financial insecurity, lack of physical activity poor partner relationship were predictors of poorer mental health functioning [16]. Here, we address a need to estimate the impact of the SARS-Cov-2 containment measures on parent subjective wellbeing, by comparing parent-report of subjective wellbeing using the Personal Wellbeing Index, in: (1) cross-sectional pandemic data from 2365 parents of a child 0–18 years in the COVID-19 Pandemic Adjustment Survey (CPAS), collected in April 2020 during ‘level three’ restrictions; and, (2) national pre-pandemic data from 17,529 parents living with children of all ages in the Australian Unity Wellbeing Index (AUWI), collected in 36 annual cross-sectional surveys over 2002–2019 [13,28]. Specifically, our aims were threefold: Based on data from Life in Australia (~N = 3000), a representative longitudinal sample of Australian residents aged 18 years and over [24], there is emerging evidence of lower rates of adult subjective wellbeing during the pandemic [4,5]. Specifically, pre-pandemic, Global Life Satisfaction, i.e., measured on a 0–10 scale where 0 is ‘not at all satisfied’ and 10 is ‘completely satisfied’, changed from 7.05 in October 2019, to 6.90 in January 2020. This period in January saw the most widespread and destructive bushfire season ever to occur in Australia. Global Life Satisfaction then dropped again to 6.51 in April 2020 (coinciding with restrictive COVID-19 social distancing measures), rising to 6.86 in May 2020, at a period when social distancing measures were on the cusp of being relaxed. i 1. To compare subjective wellbeing assessed in CPAS parents during the pandemic with subjective wellbeing assessed in AUWI parents prior to the pandemic. 2. Within CPAS parents, to investigate whether subjective wellbeing in parents reporting ‘high mental health risk’ is lower compared to other parents, with ‘high mental health risk’ defined according to pre-existing or current parent physical or mental health problem, and/or having a child with a neurodevelopmental or mental health condition; all of which have been associated with lower parent subjective wellbeing in previous research [10,30]. 1. Method Families, in particular, have been hard hit by SARS-Cov-2 contain ment lockdowns and restrictions. Parent mental health, parenting practices, and the quality of the couple relationship are all fundamental to parent subjective wellbeing and healthy child development [40,54]. However, their protection depends on access to high quality supports, which have been substantially reduced in the pandemic. During lock down, playgrounds and campus-based schooling were closed, requiring that parents supervise children and/or manage children’s distance ed ucation from home, often while juggling their own paid work [15]. Emerging findings from Australian and USA research suggest that many parents, and particularly mothers, were forced to reduce their paid work hours during lockdown [9,31,43]. Further, pandemic data from 1500 parents of children aged 4–16 years in the United Kingdom’s Co-SPACE Study showed that two thirds of parents reported they were not meeting the needs of both work and their child(ren) during lockdown in late March [48]. Data from Singapore also shows associations between work- family conflict, parenting stress and couple conflict in parents juggling work while supervising children during lockdown [8]. i A B S T R A C T This finding was not replicated in neighbouring New Zealand over a similar time period, and under a more restrictive ‘stage four’ lockdown. Specifically, Sibley et al. [44] assessed subjective wellbeing from March to April 2020, in a nationally representative sample of 1003 New Zea landers, and found no change over this period on items from both the Personal Wellbeing Index [23] or the Satisfaction With Life Scale [17]. The reason for differential impacts between Australia and New Zealand remains unclear, but may be related to overall lower infection rates in New Zealand. It is also likely that impacts on subjective wellbeing are being felt in both countries, but are concentrated in more particular populations characterised by higher demands (or higher needs) prior to the pandemic, and not modelled in either study. 3. Within CPAS parents, to examine the extent to which pre-pandemic factors (demographic, socio-economic, parent and child diagnosis) and COVID-related stressors (i.e., environmental risks such as financial or housing insecurity; working from home with children; food shortages; media use, as well as feelings and attitudes about COVID-19) are associated with compromised parent subjective wellbeing during the pandemic. 1.1. Participants and study design 1.1.1. The COVID-19 pandemic adjustment survey (CPAS) 1.3.2. Population weighting i We derived post-stratification weights in the CPAS dataset to compensate for differences between the final sample and the national population of parents. We generated post-stratification weights through a raking approach [29], using six demographic factors: (1) geographic location (major city, inner and outer regional areas, and remote areas); (2) child age groups (0–4, 5–9, 10–12, 13–14, and 15–18 years); (3) parent gender (male, female), (4) family structure (single parent, couple family), (5) parent education (Did not complete high school, high school completion; and (6) parent employment status (employed, unem ployed). The CPAS datasets was weighted to be equivalent to a sub population of Australian adults; i.e., parents of a child 0–18 years, with an estimated total population size of 8.4 million parents. Australian Bureau of Statistics population level statistics for parents of dependent children were included for comparison to the AUWI and CPAS de mographic characteristics [2]. Education level (i.e., non-high school completion versus completion), household income (low income defined as AU $52,000 per year or less), receipt of government benefits. Participants were also asked about money shortages in the 12 months prior to the pandemic (e.g., unable to pay bills, mortgage or rent, unable to heat home, went without meals, pawned or sold something, asked for financial help). These 7 items were summed to form a financial deprivation index [51]. Parents also reported whether they have a pre-existing physical or mental health condition, and whether their child has a neurodevelopmental or mental health condition (Attention Deficit Hyperactivity Disorder; Autism, Asperger’s, or other Autism Spectrum; Oppositional Defiant or Conduct Disorder; Speech or Language Disorder; Reading or Learning Disorder; Head Injury; Epilepsy/Head Injury/Other Neurological Diagnosis; Disability). High pre-existing mental health risk was defined as parents with any of the following risk factors: in the severe range for current anxiety or depression symptoms, reporting a previous physical or mental health diagnosis, or having a child with a pre-existing neurodevelopmental or mental health condition. Four items were adapted from the CoRonavIruS Health Impact Survey (CRISIS) V1.0 [34] to measure: (1) COVID-19 participant or family member diagnosis, hospitalisation, self-quarantine, family member passed away; (2) financial problems; (3) housing; and (4) food insecurity related to COVID-19. In addition, participants were asked whether they had experienced (5) job loss; (6) reduced employment; or, (7) redeployment to new roles and responsibilities in their work. COVID-19 environmental risk index Parent and child diagnosis (CPAS only) Social disadvantage (CPAS only) Demographic factors (CPAS only) 1.3.1. Overview The aims of the present study emphasize comparison of several datasets (Aims 1 and 2) and further modelling of wellbeing outcomes for the CPAS dataset (Aim 3). Accordingly, datasets were not pooled, and preparatory steps for intended analyses were conducted for each dataset separately. Participants were instructed to complete the following items as they pertained to their situation prior to the pandemic: their own and their child’s age and gender, parent country of birth and Aboriginal and Torres Strait Islander status, whether a language other than English was spoken at home, geographical location (i. e., postcode), and number of children in the household, single parent status (i.e., have no partner or not living with partner). Table 1 Table 1 Study measures in the COVID-19 pandemic adjustment survey (CPAS). The Personal Wellbeing Index (PWI) [23] (7 items) comprises seven domains measuring satisfaction with Standard of Living, Health, Achieving in Life, Relationships, Safety, Community-Connectedness, and Future Security. Example item: “How satisfied are you with… your standard of living?”. The PWI correlates strongly with the Satisfaction with Life Scale [17] (r = 0.78; α = 0.70–0.85) [23]. The items are intended to be rated on an end-defined, unipolar, 11-choice scale from zero ‘no satisfaction at all’ to 10 ‘completely satisfied’. The CPAS applied a 10-choice scale, ranging from 1 ‘no satisfaction at all’ to 10 ‘completely satisfied’. For consistency with the AUWI datasets, the CPAS version of the PWI scale was transformed to a 11- point scale. Domain scores are converted to a percentage of scale maximum and summing them together to produce a PWI score on a scale from 0 to 100 percentage points. Participant data with a score 0 or 100 percentage points on the PWI were removed [23]. High mental health risk (CPAS only) 1.3.2. Population weighting i Each of the 7 COVID-19 risk factors was converted to a binary variable (0 = no risk; 1 = risk), and then summed to form a COVID-19 environmental risk index score. Participants were also asked about the frequency of their use of media news sources (newspapers, television, social media, radio, rated on 6-point scale from ‘not at all’ to ‘multiple times per day’); two items assessed participants’ appraisals of COVID-19 as a serious health risk, whether they were likely to catch COVID-19 (both items rated on a 7- point scale from ‘strongly disagree’ to ‘strongly 1.3.3. Missing data The AUWI dataset had minimal missing data (0 to 0.8% on PWI variables), thus no missing data treatment was applied. In the CPAS dataset, item level missing data ranged from 0 to 8% on individual variables. In the CPAS dataset, multivariate multiple imputation using chained equations was performed to account for missing data. All var iables from the final analytic models and weights were included in the multiple imputation model to create 100 imputed datasets. All CPAS reported results are from the multiply imputed datasets. 1.1.1. The COVID-19 pandemic adjustment survey (CPAS) Demographic factors (CPAS only) Participants were instructed to complete the following items as they pertained to their situation prior to the pandemic: their own and their child’s age and gender parent country of birth and Aboriginal and Torres Strait Islander status, whether a language other than English was spoken at home, geographical location (i e., postcode), and number of children in the household, single parent status (i.e., have no partner or not living with partner). Social disadvantage (CPAS only) Education level (i.e., non-high school completion versus completion), household income (low income defined as AU $52,000 per year or less), receipt of government benefits. Participants were also asked about money shortages in the 12 months prior to the pandemic (e.g., unable to pay bills, mortgage or rent, unable to heat home, went without meals, pawned or sold something, asked for financial help). These 7 items were summed to form a financial deprivation index [51]. Parent and child diagnosis (CPAS only) Parents also reported whether they have a pre-existing physical or mental health condition, and whether their child has a neurodevelopmental or mental health condition (Attention Deficit Hyperactivity Disorder; Autism, Asperger’s, or other Autism Spectrum; Oppositional Defiant or Conduct Disorder; Speech or Language Disorder; Reading or Learning Disorder; Head Injury; Epilepsy/Head Injury/Other Neurological Diagnosis; Disability). High mental health risk (CPAS only) High pre-existing mental health risk was defined as parents with any of the following risk factors: in the severe range for current anxiety or depression symptoms, reporting a previous physical or mental health diagnosis, or having a child with a pre-existing neurodevelopmental or mental health condition. COVID-19 environmental risk index Four items were adapted from the CoRonavIruS Health Impact Survey (CRISIS) V1.0 [34] to measure: (1) COVID-19 participant or family member diagnosis, hospitalisation, self-quarantine, family member passed surveys was carried out via telephone interview. The sample for each survey was stratified to match the demographic distribution of the population by gender and geographic location. Participants were aged 18 years and older and fluent in English. The AUWI was approved by the Deakin University Human Ethics Advisory Group (Project number: HEAG-H 45). 1.2. Measures Please see Table 1 for a summary of parent, socio-demographic and COVID-19 risk measures used in this study. 1.1.1. The COVID-19 pandemic adjustment survey (CPAS) We used baseline data from a longitudinal cohort study of Australian parents, the COVID-19 Pandemic Adjustment Survey (CPAS, N = 2365). Data were collected over 3 weeks from the 8th to the 28th of April 2020 (see study protocol, [50]). Parents were eligible to participate if they were a parent of a child aged 0–18 years, an Australian resident, and 18 years or over. Parents were recruited via paid and unpaid social media advertisements, which contained a web link directing participants to a Qualtrics survey. The study was approved by the Deakin University Human Ethics Advisory Group (Project number: HEAG-H 52_2020). 1.1.2. Normed data from the Australian Unity Wellbeing Index (AUWI) We used normative data on subjective wellbeing from 36 cross- sectional surveys collected annually over the period 2002–2019 as part of the Australian Unity Wellbeing Index (for access to cross- sectional data, see: www.acqol.com.au). We analysed data from a sub- sample of parents with one or more child living in the same household (total sub-sample, N = 17,529) [26]. Data collection for the AUWI Despite being at high risk, research specifically examining parents’ wellbeing and functioning throughout the pandemic has been limited 2 Journal of Psychosomatic Research 145 (2021) 110482 E.M. Westrupp et al. Table 1 Study measures in the COVID-19 pandemic adjustment survey (CPAS). Construct Measure Parent subjective wellbeing The Personal Wellbeing Index (PWI) [23] (7 items) comprises seven domains measuring satisfaction with Standard of Living, Health, Achieving in Life, Relationships, Safety, Community-Connectedness, and Future Security. Example item: “How satisfied are you with… your standard of living?”. The PWI correlates strongly with the Satisfaction with Life Scale [17] (r = 0.78; α = 0.70–0.85) [23]. The items are intended to be rated on an end-defined, unipolar, 11-choice scale from zero ‘no satisfaction at all’ to 10 ‘completely satisfied’. The CPAS applied a 10-choice scale, ranging from 1 ‘no satisfaction at all’ to 10 ‘completely satisfied’. For consistency with the AUWI datasets, the CPAS version of the PWI scale was transformed to a 11 point scale. Domain scores are converted to a percentage of scale maximum and summing them together to produce a PWI score on a scale from 0 to 100 percentage points. Participant data with a score 0 or 100 percentage points on the PWI were removed [23]. Table 2 l Table 2 Sample characteristics for the Australian Unity Wellbeing Index (AUWI) and COVID-19 Pandemic Adjustment Survey (CPAS) samples. each indicator of parent subjective wellbeing, while simultaneously accounting for the contribution of all the other pre-pandemic and COVID-related stressor variables in the model. Variables were included in adjusted models where there was evidence for an unadjusted associ ation with the outcome (p < 0.1). In line with Perneger [39], we describe all results without adjusting for multiple comparisons. 2.1. Sample characteristics Characteristics of the CPAS and AUWI samples are shown in Table 2. Parents in the CPAS sample were on average 38 years with a primary school aged child. The majority of the CPAS sample were cisgender women; and just over half of their children were cisgender boys. Parents in the AUWI norm sample were on average 46 years of age, with an even distribution of cisgender men and women. The CPAS sample was broadly representative of the Australian parent population in terms of geographic location, number of children, parents born overseas, and single parent households, but was somewhat under-representative of families with a low income and low education [2]. 1.3.4. Data analysis Analyses were conducted in Stata version 16 [45]. To address Aims 1 and 2, means and standard deviations were calculated for the CPAS and AUWI samples, overall, and for CPAS parents with and without high mental health risk. CPAS weighted data are presented to ensure reported outcomes are as close to population representation as possible, but we primarily focus on unweighted results in our interpretation. Data from the two studies were not pooled but rather analysed separately. We conducted a series of one sample t-tests (CPAS compared to AUWI norms), and independent samples t-tests (CPAS high risk versus low risk groups) and calculated Cohen’s d effect sizes to assess unweighted dif ferences between samples/groups on each domain. To address Aim 3, a series of unadjusted linear regression analyses were conducted (unweighted) with the total PWI score and PWI domains entered as dependent outcome variables, and pre-pandemic and COVID- related stressors separately entered as independent variables into each univariate model. Next, adjusted models were conducted to assess the unique contribution of each of these independent variables in relation to (continued on next page) 3 3 Journal of Psychosomatic Research 145 (2021) 1104 Table 1 (continued) Construct Measure agree’). Participants were also asked about their feelings about COVID-19 (worry, fear, confidence, hope) rated on a 4-point scale from ‘not at all’ to ‘a great deal’. The two positive feelings were reverse coded and the 4 items were summed to a total score with higher scores reflecting more negative feelings. Participants reported on whether they were working from home, and whether children who were usually in a childcare or a formal education setting were also at home with them. Table 2 Sample characteristics for the Australian Unity Wellbeing Index (AUWI) an COVID-19 Pandemic Adjustment Survey (CPAS) samples. Australianpopulation$ AUWI Norms% CPAS§ N (%) % Parent age, m(sd) n/a 45.9 (11.8) 38.3 (7.1) Child age, m(sd) n/a n/a 8.7 (5.1) Parent gender Cisgender men 46% 8255 19% E.M. Westrupp et al. Table 1 (continued) Construct Measure agree’). Participants were also asked about their feelings about COVID-19 (worry, fear, confidence, hope) rated on a 4-point scale from ‘not at all’ to ‘a great deal’. The two positive feelings were reverse coded and the 4 items were summed to a total score with higher scores reflecting more negative feelings. 2.2. Measurement sensitivity analysis As explained in the attached Supplementary Analysis, we conducted two sensitivity analyses related to the measurement of parent subjective wellbeing to explore whether the estimates of subjective wellbeing were influenced by slight methodological changes in the presentation of items (see Supplementary Analysis for details). First, we assessed the place ment of the PWI items after emotive measures within the CPAS survey, which may have influenced PWI scores. We found a small placement effect of 0.75 PWI units (t = 36.15, df = 2107.60, p < 0.001). Second, we assessed the restricted range of the CPAS PWI response scale and found that the variance had not been reduced by the reduced score range. Given the difference in the gender balance between the CPAS and AUWI samples, we conducted a third sensitivity analysis to investigate socio- demographic differences between cisgender women and men within the CPAS sample. Compared to men, we found that women were more likely to report COVID-19 diagnosis, hospitalisation, employment changes, and financial impacts, to be a single parent, and to be receiving a government benefit, and less likely to report that they had not completed high school, and were supervising a child at home while they worked from home. There were no differences in reported rates of pre- pandemic financial deprivation. Notes: m(sd) = Mean (standard deviation); AUWI = Australian Unity Wellbeing Index; CPAS = COVID-19 Pandemic Adjustment Survey. $ Data from the Australian Bureau of Statistics summarising characteristics of Australian parents living with a dependent child (usually defined as 0–14 years). i % Norms from 2002 to 2019, N = 17,529 parents living with children (in cludes children of all ages living in the same household as their parent/s). § Data collected 8th–28th April 2020, N = 2365 parents of a child 0–18 years, data are multiply imputed and thus can only be presented as percentages. deviation lower than the norm data, t(2159) = −29.92, p < 0.001, Cohen’s d = -62. There was evidence for differences across all domains, with small differences for Standard of Living and Personal Safety (Cohen’s d −0.21; −0.32 respectively, both p < 0.001), moderate differences for Achieving in Life, Personal Relationships, Community Connectedness, and Future Security (Cohen’s d −0.50 to −0.55, all p < 0.001), and a large difference for Health (Cohen’s d = −0.60, p < 0.001). 1.3.4. Data analysis Participants reported on whether they were working from home, and whether children who were usually in a childcare or a formal education setting were also at home with them. E.M. Westrupp et al. Journal of Psychosomatic Research 145 (2021) 110482 E.M. Westrupp et al. Table 2 Sample characteristics for the Australian Unity Wellbeing Index (AUWI) and COVID-19 Pandemic Adjustment Survey (CPAS) samples. Australianpopulation$ AUWI Norms% CPAS§ N (%) % Parent age, m(sd) n/a 45.9 (11.8) 38.3 (7.1) Child age, m(sd) n/a n/a 8.7 (5.1) Parent gender Cisgender men 46% 8255 (47%) 19% Cisgender women 54% 9274 (53%) 81% Transgender or nonbinary n/a n/a <1% Child gender Cisgender boy n/a n/a 51% Cisgender girl n/a n/a 49% Transgender or nonbinary n/a <1% Geographic location Major Cities of Australia 74% 70% Inner Regional Australia 17% 23% Outer Regional Australia 7% 6% Remote Australia 2% 1% Number of children 1 child 42% n/a 28% 2 children 39% n/a 46% 3 children 14% n/a 18% 4 or more children 5% n/a 7% Single parent household 11% 11% Parent born overseas 21% n/a 18% Aboriginal or Torres Strait Islander 4% n/a 2% Did not complete high school 40% n/a 9% Receiving government benefit n/a n/a 6% Low household income $52,000 or less per year 21% 14% $60,000 or less per year 13% Parent mental health condition n/a n/a 37% Parent chronic health condition n/a n/a 30% Child neurodevelopmental or mental health condition n/a n/a 31% COVID-19 related factors Deprivation index, m(sd) n/a n/a 0.4 (1.0) Child home while working n/a n/a 50% COVID-19 environmental risk index, m(sd) n/a n/a 1.4 (1.2) 2.2. Measurement sensitivity analysis CPAS weighted results showed consistently lower subjective wellbeing than the unweighted results, suggestive of even greater differences between pre-pandemic and pandemic figures. -62. There was evidence for differences across all domains, with small differences for Standard of Living and Personal Safety (Cohen’s d −0.21; −0.32 respectively, both p < 0.001), moderate differences for Achieving in Life, Personal Relationships, Community Connectedness, and Future Security (Cohen’s d −0.50 to −0.55, all p < 0.001), and a large difference for Health (Cohen’s d = −0.60, p < 0.001). CPAS weighted results showed consistently lower subjective wellbeing than the unweighted results, suggestive of even greater differences between pre-pandemic and pandemic figures. 2.2.1. Aim 1: comparing parent subjective wellbeing before and during the pandemic Fig. 1 and Supplementary Table 1 compares parent PWI total and individual domain scores for the AUWI norms (pre-pandemic) and CPAS results (during the pandemic). According to CPAS unweighted results, subjective wellbeing levels in parents during the pandemic were notably lower than pre-pandemic AUWI norms. The largest difference was evident for the PWI, where CPAS results were close to one standard 2.2.2. Aim 2: associations with parent and offspring physical and mental health Table 3 presents comparisons for CPAS parents with heightened risk Table 3 presents comparisons for CPAS parents with heightened risk 4 Journal of Psychosomatic Research 145 (2021) 110482 Fig. 1. Means and standard deviations (i.e., error bars) for the Australian Unity Wellbeing Index (AUWI) norm and unweighted and weighted COVID-19 Pandemic Adjustment Survey (CPAS) samples on the Personal Wellbeing Index (PWI) and PWI individual domains. E.M. Westrupp et al. E.M. Westrupp et al. E.M. Westrupp et al. wellbeing across the PWI, t(609) = −9.05, p < 0.001, Cohen’s d = −0.36) and six of the seven domains (Cohen’s d, −0.09 to −0.39), whereas no differences were evidence for Standard of Living, t(609) = −1.50, p = 0.135. wellbeing across the PWI, t(609) = −9.05, p < 0.001, Cohen’s d = −0.36) and six of the seven domains (Cohen’s d, −0.09 to −0.39), whereas no differences were evidence for Standard of Living, t(609) = −1.50, p = 0.135. 2.2.3. Aim 3: associations with socioeconomic and COVID-19 stressors Tables 4–6 present results from unadjusted and adjusted regression analyses testing the associations between parent subjective wellbeing on the PWI and subdomains, and a range of demographic, socio-economic, parent and child diagnosis, and COVID-19 related stressors. 3. Discussion We found that subjective wellbeing in CPAS parents during the April 2020 ‘level three’ lockdown in Australia was lower (0.5–1 SD) than pre- pandemic levels, as assessed annually over the previous two decades in AUWI parents. Pre-pandemic to pandemic differences equated to more than a 10-point reduction in scores on the total PWI in CPAS parents compared with AUWI parents. Within the CPAS parent sample, the most profound differences in subjective wellbeing were in parents with pre- existing challenges that predated the pandemic; including physical or mental health problems, having a child with a neurodevelopmental or mental health condition, living in a socially disadvantaged context, and being a young parent, as well as parents reporting current challenges; including anxiety and depression, and direct impacts of COVID-19, such as job loss. We also found that parents from Aboriginal and Torres Strait Islander backgrounds reported lower subjective wellbeing in the CPAS sample. Notes: Data are multiply imputed. sd = Standard deviation; CPAS = COVID-19 Pandemic Adjustment Survey. High risk defined as parents with any of the following risk factors, including severe range for current anxiety or depression, reporting a previous physical or mental health diagnosis, or having a child with a pre-existing neurodevelopmental or mental health condition. We consistently found that indicators of pre-pandemic socio-eco nomic disadvantage, such as financial deprivation, government benefit, single parent status, speaking a language other than English, and low education, were associated with lower subjective wellbeing. Results also indicated that younger parent age was associated with lower ratings of subjective wellbeing, personal relationships, and community connect edness. These results suggest that already vulnerable families struggling with socio-economic disadvantage prior to the pandemic, or parents in disadvantaged demographic groups, are likely to have suffered the most during the April 2020 ‘level three’ restrictions in Australia. Further, parents directly impacted by the COVID-19 pandemic reported consis tently lower subjective wellbeing, over and above the influence of pre- pandemic socio-economic disadvantage. Direct COVID-19 impacts included the experience of COVID-19-related illness, job loss or for compromised subjective wellbeing, i.e., current parent anxiety or depression symptoms, a pre-existing mental and/or physical health diagnosis, or child neurodevelopmental or mental health conditions (72%), compared to other parents (28%). Parents at risk consistently reported lower subjective wellbeing across all domains. Table 3 Differences for COVID-19 Pandemic Adjustment Survey (CPAS) participants with and without high pre-existing mental health risk: Unweighted and weighted comparisons for the Personal Wellbeing Index (PWI). Differences for COVID-19 Pandemic Adjustment Survey (CPAS) participants with and without high pre-existing mental health risk: Unweighted and weighted comparisons for the Personal Wellbeing Index (PWI). CPAS: High risk (72%) CPAS: Not high risk (28%) Weighted Unweighted Weighted Unweighted Mean (sd) Mean (sd) Mean (sd) Mean (sd) Personal Wellbeing Index 59.88 (18.61) 63.42 (17.50) 68.65 (14.59) 70.40 (14.80) Standard of Living 67.76 (22.02) 71.76 (21.08) 72.98 (19.12) 75.92 (18.77) Health 56.22 (22.39) 60.10 (21.87) 68.15 (19.52) 69.91 (18.99) Achieving in Life 57.06 (23.33) 60.81 (22.33) 64.95 (19.91) 67.37 (20.20) Personal Relationships 65.23 (24.83) 66.78 (23.60) 73.35 (20.09) 74.23 (20.11) Personal Safety 69.79 (24.11) 71.76 (22.68) 78.22 (18.55) 78.80 (18.82) Community Connectedness 50.87 (27.25) 56.12 (25.73) 60.35 (23.37) 62.45 (23.54) Future Security 52.24 (26.63) 56.63 (25.14) 62.54 (21.48) 64.13 (22.19) Notes: Data are multiply imputed. sd = Standard deviation; CPAS = COVID-19 Pandemic Adjustment Survey. High risk defined as parents with any of the following risk factors, including severe range for current anxiety or depression, reporting a previous physical or mental health diagnosis, or having a child with a pre-existing neurodevelopmental or mental health condition. 2.2. Measurement sensitivity analysis Parent Aboriginal and Torres Strait Islander status, speaking a language other than English at home, receiving a government benefit, low education, single parents, younger child age, and higher financial deprivation prior to the pandemic were all associated with lower subjective wellbeing. Parents living in regional areas,raising a child with a neuro developmental or mental health condition, and having a physical or mental health diagnosis themselves, were also associated with lower subjective wellbeing. The one protective association was with higher frequency of a variety of news media use. The COVID-19 pandemic- related factors were all associated with lower parent subjective well being, including supervising children while working from home, higher levels of COVID-19 risk factors; i.e., diagnosis, hospitalisation, employment changes, and financial impacts; beliefs about being likely to catch COVID-19, more negative feelings about COVID-19; i.e., higher fear and worry, and low confidence and hope; and the belief that COVID- 19 is a serious health risk. Fig. 1. Means and standard deviations (i.e., error bars) for the Australian Unity Wellbeing Index (AUWI) norm and unweighted and weighted COVID-19 Pandemic Adjustment Survey (CPAS) samples on the Personal Wellbeing Index (PWI) and PWI individual domains. These results were mostly consistent across the PWI domains, with some exceptions. Cisgender women were more likely than cisgender men to report higher subjective wellbeing on the Standard of Living, Achieving in Life, and Community Connectedness domains. The most consistent findings were evident for Aboriginal and Torres Strait Islander status, single parents, government benefits, pre-pandemic financial deprivation, the COVID-19 environmental risk index, and negative feelings about COVID-19. 3. Discussion The largest differences were evident for Personal Health, t(2176) = −9.74, p < 0.001, Cohen’s d = −0.47, and the PWI, t(2176) = −8.71, p < 0.001, Cohen’s d = −0.42, but group differences were evident across all do mains (p < 0.001, Cohen’s d −0.20 to −0.33). We compared levels of subjective wellbeing in CPAS parents not reporting high mental health risk with AUWI norms, and found evidence for lower levels of subjective 5 5 Journal of Psychosomatic Research 145 (2021) 110482 E.M. Westrupp et al. E.M. Westrupp et al. Table 4 Unadjusted and adjusted associations between COVID-19 Pandemic Adjustment Survey (CPAS) demographic characteristics prior to the pandemic and parent sub jective wellbeing domains: Personal Wellbeing Index, Standard of Living and Health. 3. Discussion Personal Wellbeing Index Standard of Living Health Unadjusted Adjusted Unadjusted Adjusted Unadjusted Adjusted B B LL UL B B LL UL B B LL UL Demographic factors Parent age −0.01 0.06 −0.04 Child age −0.23** −0.17* −0.32 −0.03 −0.26 −0.12 −0.30 0.05 −0.30 −0.17 −0.36 0.03 Parent gender Cisgender women$ 0.23 1.98 3.83 1.83 5.83 0.25 Transgender or nonbinary$ 18.29 24.53 26.91 −7.83 61.64 7.16 Aboriginal or Torres Strait Islander −13.96 −7.92 −12.12 −3.71 −11.82 −6.25* −11.64 −0.87 −13.10** −6.73* −12.45 −1.00 Language other than English −3.48* −4.26 −7.22 −1.30 −6.92 −7.48 −11.25 −3.70 −1.24 Socio-economic risk factors Receiving government benefit −17.34 −3.98* −6.99 −0.96 −20.42 −7.47 −11.34 −3.60 −20.42 −3.55 −7.63 0.53 Did not complete high school −9.73 −3.06 −5.19 −0.93 −9.64 −2.45 −5.19 0.28 −10.74 −4.35 −7.24 −1.46 Single parent household −13.53 −6.72 −8.84 −4.59 −12.34 −4.49 −7.27 −1.70 −9.91 −2.60 −5.51 0.31 Geographic location Inner Regional Australia% −3.15 −1.03 −2.49 0.43 −2.90 −0.65 −2.55 1.26 −4.95 −3.25** −5.21 −1.28 Outer Regional Australia% −2.99* −0.17 −2.67 2.34 −2.32 0.61 −2.62 3.84 −4.44* −1.58 −4.99 1.83 Remote Australia% 2.71 2.97 −3.26 9.19 4.43 3.12 −4.76 11.01 2.14 3.03 −5.36 11.42 Number of children −0.58 −1.17* −0.16 −1.10 0.77 −0.60 Financial deprivation index −5.83 −3.09 −3.78 −2.41 −7.38 −4.94 −5.82 −4.06 −5.72 −3.28 −4.22 −2.35 Parent and child diagnosis Child neurodevelopmental diagnosis −5.42 −2.10 −3.44 −0.76 −4.58 −1.57 −3.29 0.15 −6.04 −1.83* −3.65 −0.01 Parent physical/mental health diagnosis −6.35 −1.95 −3.23 −0.67 −3.21 0.94 −0.72 2.59 −10.27 −5.85** −7.59 −4.12 COVID-19 related stressors Child home while working −1.70* 1.34 −0.12 2.80 −1.30 −2.69 0.71 −1.28 2.71 COVID-19 environmental risk index −5.00 −2.96 −3.48 −2.44 −5.09 −3.35 −4.02 −2.67 −3.91 −1.50 −2.22 −0.78 Believe likely to catch COVID-19 −0.89 −0.04 −0.43 0.34 −0.25 −1.28 −0.12 −0.65 0.40 Fear and worry about COVID- 19 −10.08 −7.04 −8.13 −5.96 −6.57 −4.09 −5.50 −2.69 −9.13 −4.92 −6.41 −3.43 Believe COVID-19 serious health risk −1.96 −0.45* −0.82 −0.07 −1.34 −0.20 −0.67 0.27 −3.18 −1.86** −2.37 −1.35 Higher frequency of news media use 0.93* 0.98 0.35 1.61 1.27 1.25** 0.45 2.06 0.78 Notes: Data are multiply imputed. Variables were included in adjusted models where there was evidence for an unadjusted association with the outcome (p < 0.1). 3. Discussion B = unweighted regression coefficient; LL = lower limit of a 95% confidence interval; UL = upper limit of a 95% confidence interval; * p < 0.05; ** p < 0.01. $ Compared to cisgender men. % Compared to Major Cities of Australia. Table 4 Unadjusted and adjusted associations between COVID-19 Pandemic Adjustment Survey (CPAS) demographic characteristics prior to the pandemic and parent sub jective wellbeing domains: Personal Wellbeing Index, Standard of Living and Health. Table 4 Unadjusted and adjusted associations between COVID-19 Pandemic Adjustment Survey (CPAS) demographic characteristics prior to the pandemic and parent sub jective wellbeing domains: Personal Wellbeing Index, Standard of Living and Health. Notes: Data are multiply imputed. Variables were included in adjusted models where there was evidence for an unadjusted association with the outcome (p < 0.1). B = unweighted regression coefficient; LL = lower limit of a 95% confidence interval; UL = upper limit of a 95% confidence interval; * p < 0.05; ** p < 0.01. $ Compared to cisgender men Notes: Data are multiply imputed. Variables were included in adjusted models where there was evidence for an unadjusted association with the outcome (p < 0.1). B = unweighted regression coefficient; LL = lower limit of a 95% confidence interval; UL = upper limit of a 95% confidence interval; * p < 0.05; p < 0.01. $ Compared to cisgender men. % Compared to Major Cities of Australia. employment changes, financial hardship, negative feelings and attri butions about COVID-19, and juggling child supervision/remote learning whilst working from home. All of these factors are likely to have placed parents under additional strain as suggested by their low scores on indicators of subjective wellbeing. Together, these findings highlight a need for targeted supports and services for families experiencing socio- demographic risk, both in relation to pre-existing disadvantage, and in relation to risk that has occurred as a result of the pandemic and the related social distancing measures put in place to contain the virus in Australia. health risks did not account for the overall differences in subjective wellbeing evident in parents prior to and during the pandemic; that is, when we excluded parents with high mental health risk, differences remained evident for CPAS and AUWI data on almost all domains of subjective wellbeing. 3. Discussion These findings are suggestive of additional strains related to the pandemic that are not fully explained by pre-existing or concurrent mental health risk in families. Unexpectedly, we found that parents engaging more with news media about the pandemic were more likely to report higher subjective wellbeing. This is counter-intuitive and requires further investigation, but it may be that being informed leads to a higher sense of control, and thus improved subjective wellbeing, in context of a crisis such as the COVID-19 pandemic. This association may also be related to the quality and type of media that parents engage with, factors that were not assessed in this study. We found consistent associations between lower subjective well being and parent report of high mental health risk, demonstrating additional strain associated with parents having pre-existing or con current mental or physical health problems or having a child with a neurodevelopmental or mental health condition. These findings are in line with previous pre-pandemic research showing lower subjective wellbeing in these groups [10,30]. It is also notable that rates of child neurodevelopmental or mental health conditions were higher than other Australian population estimates [1,19,32,33], suggesting that CPAS represents a higher-risk sample of parents from the Australian commu nity. When we compared rates of subjective wellbeing within CPAS parents, we found consistently lower wellbeing in parents with mental health risks compared to other parents. Yet the elevated rates of mental Table 5 Table 5 Unadjusted and adjusted associations between COVID-19 Pandemic Adjustment Survey (CPAS) demographic characteristics prior to the pandemic and parent sub jective wellbeing domains: Achieving in Life, Personal Relationships, and Personal Safety. Unadjusted and adjusted associations between COVID-19 Pandemic Adjustment Survey (CPAS) demographic characteristics prior to the pandemic and parent sub jective wellbeing domains: Achieving in Life, Personal Relationships, and Personal Safety. Table 5 Demographic variables Achieving in Life Personal Relationships Personal Safety Unadjusted Adjusted Unadjusted Adjusted Unadjusted Adjusted B B LL UL B B LL UL B B LL UL Demographic factors Parent age −0.05 −0.25 −0.33 −0.51 −0.15 −0.13 −0.11 −0.27 0.05 Child age −0.30 −0.16 −0.38 0.06 −0.36 0.19 −0.09 0.48 −0.37 −0.17 −0.38 0.04 Parent gender Cisgender women$ 1.94 4.37** 2.15 6.58 −0.61 −2.37* 1.69 −0.44 3.82 Transgender or nonbinary$ 18.72 23.67 −16.02 63.36 22.89 16.39 23.52 −13.38 60.41 Aboriginal or Torres Strait Islander −11.52 −5.42 −11.38 0.54 −8.96 −2.37 −8.69 3.96 −17.37 −10.86 −16.49 −5.23 Language other than English −5.53* −6.45** −10.68 −2.22 −0.20 −5.50* −5.42 −9.41 −1.43 Socio-economic risk factors Receiving government benefit −17.81 −7.62 −11.96 −3.28 −16.52 −1.24 −5.78 3.30 −13.24 −0.67 −4.70 3.36 Did not complete high school −8.85 −2.13 −5.14 0.87 −5.87 −0.12 −3.32 3.08 −7.78 −1.62 −4.49 1.24 Single parent household −10.24 −2.99 −6.07 0.10 −20.41 −16.24 −19.44 −13.04 −13.55 −8.53 −11.41 −5.65 Geographic location Inner Regional Australia% −3.18 −1.26 −3.34 0.82 −2.76* −1.05 −3.23 1.13 −1.53 Outer Regional Australia% −6.03 −3.36 −6.89 0.17 −3.67 −1.88 −5.63 1.86 −0.87 Remote Australia% 0.82 −0.06 −8.66 8.53 −1.03 −1.53 −10.83 7.77 0.50 Number of children −0.87 0.25 −0.81 1.31 0.25 −0.87 −2.00 0.26 −0.16 Financial deprivation index −5.92 −3.33 −4.30 −2.36 −3.33 −2.30 −3.34 −1.26 −4.61 −2.11 −3.04 −1.19 Parent and child diagnosis Child neurodevelopmental diagnosis −5.57** −2.29* −4.19 −0.40 −6.72 −3.46 −5.46 −1.45 −5.21 −1.59 −3.38 0.20 Parent physical/mental health diagnosis −5.73 −1.89* −3.67 −0.10 −6.83 −2.97 −4.88 −1.06 −6.27 −1.69 −3.40 0.02 COVID-19 related stressors Child home while working −3.15 −0.38 −2.48 1.72 −0.38 0.11 −2.14 2.36 −1.24 COVID-19 environmental risk index −4.91 −3.03 −3.76 −2.29 −3.03 −1.81 −2.60 −1.02 −4.87 −2.45 −3.16 −1.75 Believe likely to catch COVID-19 −0.49 0.09 −0.45 0.63 0.09 −0.38 −0.96 0.20 −1.87 −0.75 −1.26 −0.23 Fear and worry about COVID-19 −8.57 −6.59 −8.14 −5.04 −6.59 −5.13 −6.76 −3.51 −14.33 −11.83 −13.31 −10.36 Believe COVID-19 serious health risk −1.38 0.16 −0.37 0.69 0.16 0.44 −0.12 1.00 −2.54 −0.28 −0.78 0.23 Higher frequency of news media use 0.60 0.66 0.26 Notes: Data are multiply imputed. Variables were included in adjusted models where there was evidence for an unadjusted association with the outcome (p < 0.1). Table 5 B = unweighted regression coefficient; LL = lower limit of a 95% confidence interval; UL = upper limit of a 95% confidence interval; * p < 0.05; ** p < 0.01. $ Compared to cisgender men. % Compared to Major Cities of Australia Notes: Data are multiply imputed. Variables were included in adjusted models where there was evidence for an unadjusted association with the outcome (p < 0.1). B = unweighted regression coefficient; LL = lower limit of a 95% confidence interval; UL = upper limit of a 95% confidence interval; * p < 0.05; ** p < 0.01. $ Compared to cisgender men Notes: Data are multiply imputed. Variables were included in adjusted models where there was evidence for an unadjusted association with the outcome (p < 0.1). B = unweighted regression coefficient; LL = lower limit of a 95% confidence interval; UL = upper limit of a 95% confidence interval; * p < 0.05; ** p < 0.01. $ Compared to cisgender men. % Compared to Major Cities of Australia. Research and policy is often, and understandably, focussed on mental illnesses such as depression and anxiety, but these can be viewed as end-states of more mild symptoms that may be captured in measures of general wellbeing. Longitudinal studies could be useful to confirm whether lower than usual (or lower than normal) subjective wellbeing scores are an early marker for later mental health problems. Such in formation could help with prevention and early intervention efforts, which have been argued to be more cost-effective than treating once full blown disorder is present. Further, prospective studies are needed to improve understanding of family outcomes longitudinally. Our data are suggestive of negative impacts associated with the COVID-19 pandemic for parents, children, and families. In particular, the state of Victoria in Australia has experienced one of the world’s most stringent and lengthy periods of social distancing restrictions. It is imperative that future research investigate the longer-term impact of the COVID-19 pandemic on families, with a focus on how impacts vary relative to the nature and targeted support. This is consistent with well-established evidence showing that families with pre-existing social disadvantage are much more likely to experience adversity, and have fewer resources to buffer the negative impact of stressful life events, such as challenges related to the COVID-19 pandemic and social distancing measures [38,53]. 3.1. Policy implications and future research Taken together, the results of the current study suggest that parents are an important group of Australians that need extra government re sources for support in the current (and potential future) pandemics. Results specifically suggest that parents already struggling with socio- economic adversity and/or other disadvantage may benefit from 6 Journal of Psychosomatic Research 145 (2021) 110482 E.M. Westrupp et al. E.M. Westrupp et al. Table 5 Our findings also support recent calls for additional government support during and beyond the pandemic [6], such as extending income support for parents experiencing financial stress. Further, given our findings indicating that juggling child supervision with work was associated with parent depression and irritability, and the documented negative impact of poor parent mental health and irritable parenting on children’s future outcomes [25,40,55], steps to alleviate the stress of working parents are likely to be beneficial. This could include leave entitlements for parents juggling work with home-schooling or caring for children [6], alongside workplace interventions including flexible work arrangements or workload assistance during the pandemic. 7 Journal of Psychosomatic Research 145 (2021) 110482 E.M. Westrupp et al. E.M. Westrupp et al. Table 6 Unadjusted and adjusted associations between COVID-19 Pandemic Adjustment Survey (CPAS) demographic characteristics prior to the pandemic and parent sub jective wellbeing domains: Community Connectedness and Future Security. Declaration of Competing Interest All authors have completed the Unified Competing Interest form at http://www.icmje.org/coi_disclosure.pdf and declare that the authors have no competing interests to report. Acknowledgements We would like to thank all the parents who have contributed valu able data to the project. The current study was supported by funding from the Centre for Social and Early Emotional Development, School of Psychology, Victoria, Australia. EW & AM were supported by Deakin Faculty of Health Mid-Career Fellowships. DH was supported by a NHMRC Investigator Grant (1197488). ES was supported by an NHMRC Career Development Fellowship (1110688), a Medical Research Future Fund Investigator Grant (1194297) and a veski Inspiring Women’s Fellowship. Table 5 B = unweighted regression coefficient; LL = lower limit of a 95% confidence interval; UL = upper limit of a 95% confidence interval; * p < 0.05; p < 0.01. $ % Compared to Major Cities of Australia. duration of social distancing measures worldwide. duration of social distancing measures worldwide. parents experiencing socioeconomic disadvantage prior to the pandemic; and, (3) parents affected by COVID-19 related factors, such as illness, financial or housing insecurity, changes to employment, and juggling childcare with work from home, who were also more vulner able to the pandemic. Our findings specifically point to the need for targeted supports and services for parents and their families in these three high risk categories of Australian parents in the community. 3.2. Limitations Our study had a number of limitations. There were systematic dif ferences between the CPAS sample and the AUWI normed sample, where parents in the normed sample were on average eight years older, more socially advantaged, and with a higher proportion of fathers. It was also not possible to limit the AUWI comparison to parents of children 0–18 years, so this sample included parents living with children of all ages. CPAS participants were recruited online, while AUWI participants were recruited via telephone. There is a precedent for greater enrolment of at- risk individuals via online research [3]; but there may also be emerging bias associated with telephone recruitment in an opposite direction, perhaps resulting in a disproportionately low-risk population, particu larly in younger populations. We did not investigate whether these parents were also more likely to experience COVID-19 related risk fac tors, but this should be considered in future research given established associations between pre-existing health risks, socio-economic adver sity, and vulnerability to crisis events [53]. g g p y Supplementary data to this article can be found online at https://doi. org/10.1016/j.jpsychores.2021.110482. Table 5 Demographic variables Community connectedness Future security Unadjusted Adjusted Unadjusted Adjusted B B LL UL B B LL UL Demographic factors Parent age 0.28 0.26 0.11 0.41 0.04 Child age 0.13 −0.17 −0.12 −0.33 0.09 Parent gender Cisgender women$ 2.16 6.58 3.94 9.22 −1.77 Transgender or nonbinary$ 29.78 34.73 −11.73 81.20 8.57 Aboriginal or Torres Strait Islander −16.09 −9.82 −16.79 −2.85 −18.86 −11.96 −18.11 −5.81 Language other than English −0.67 −4.31 −3.86 −8.10 0.38 Socio-economic risk factors Receiving government benefit −15.17 −2.42 −7.37 2.53 −22.44 −6.46 −10.85 −2.06 Did not complete high school −13.40 −6.99 −10.48 −3.50 −11.82 −3.07 −6.13 0.00 Single parent household −11.75 −6.90 −10.43 −3.37 −16.53 −7.79 −10.86 −4.73 Geographic location Inner Regional Australia% −3.88** −2.88* Outer Regional Australia% −2.11 −1.47 Remote Australia% 9.08 3.00 Number of children −0.26 −0.02 Financial deprivation index −5.13 −2.23 −3.36 −1.09 −7.31 −3.38 −4.37 −2.39 Parent and child diagnosis Child neurodevelopmental diagnosis −4.32 −1.91 −4.08 0.27 −5.49 −1.44 −3.39 0.51 Parent physical/mental health diagnosis −5.85 −1.87 −4.00 0.26 −6.27 −0.09 −1.94 1.77 COVID-19 related stressors Child home while working 1.60 −1.99 1.58 −0.57 3.72 COVID-19 environmental risk index −4.35 −2.25 −3.11 −1.38 −8.41 −6.08 −6.84 −5.31 Believe likely to catch COVID-19 −0.59 0.16 −0.47 0.80 −0.95 0.25 −0.30 0.81 Fear and worry about COVID-19 −10.73 −8.91 −10.73 −7.09 −14.48 −9.99 −11.56 −8.42 Believe COVID-19 serious health risk −1.69 −0.33 −0.94 0.29 −2.61** −0.54* −1.08 0.00 Higher frequency of news media use 2.03 1.89 0.86 2.93 0.94** 1.05* 0.15 1.95 Notes: Data are multiply imputed. 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Brown, J.R. Doom, S. Lechuga-Pe˜na, et al., Stress and parenting during the global COVID-19 pandemic, Child Abuse Negl. 104699 (2020). [35] Roy Morgan, Article No. 8383: Over two-thirds of working Australians have had their employment impacted by the ‘Coronavirus Crisis’, 2020. [8] Chung SKG, Chan XW, Lanier P, et al. (2020) Associations between work-family balance, parenting stress, and marital conflicts during COVID-19 pandemic in Singapore. [36] B.W. Nelson, A.K. Pettitt, J. Flannery, et al., Psychological and Epidemiological Predictors of COVID-19 Concern and Health-Related Behaviors, 2020 (PsyArXiv). 4. Summary Results indicated substantially lower rates of subjective wellbeing in a cohort of Australian parents raising children and adolescents (0–18 years) during ‘level-three’ COVID-19 restrictions in April 2020, compared to pre-pandemic levels. We identified three high-risk groups of parents for whom we recommend direct assistance through govern ment, public health, and clinical services. These include: (1) parents reporting their own or their child as having mental health risk; (2) 8 E.M. Westrupp et al. Journal of Psychosomatic Research 145 (2021) 110482 [28] S. Khor, M. Fuller-Tysziewicz, D. Hutchinson, Australian normative data for subjective wellbeing, in: R.A. Cummins (Ed.), Personal Wellbeing Index Manual: 6th Edition. International Wellbeing Group, Australian Centre on Quality of Life, Deakin University, Melbourne, 2020. http://www.acqol.com.au/publications#pub lications. i References Jorm, Parental factors associated with childhood anxiety, depression, and internalizing problems: a systematic review and meta-analysis, J. Affect. Disord. 175 (2015) 424–440. [26] S. Khor, R.A. Cummins, M. Fuller-Tyszkiewicz, et al., Australian Unity Wellbeing Index: - Report 36: Social Connectedness and Wellbeing, Australian Centre on Quality of Life, School of Psychology, Deakin University, Geelong, 2020. [27] S Kh R A C i M F ll T ki i l A li U i W llb i [55] M.B.H. Yap, P.D. Pilkington, S.M. Ryan, et al., Parental factors associated with depression and anxiety in young people: a systematic review and meta-analysis, J. Affect. Disord. 156 (2014) 8–23. [27] S. Khor, R.A. Cummins, M. Fuller-Tyszkiewicz, et al., Australian Unity Wellbeing Index: - Report 36: Social Connectedness and Wellbeing. Australian Centre on Quality of Life, Deakin University, School of Psychology, 2020.
https://openalex.org/W4299914039	https://zenodo.org/records/6907492/files/Manoj2%402016.pdf	English	null	A CRITICAL STUDY OF THE POSITION OF WOMEN IN VEDIC AND POST VEDIC PERIOD	Zenodo (CERN European Organization for Nuclear Research)	2,016	cc-by	3,463	Abstract: Of all the religious practices and beliefs concerned with women in Indian culture, women granted supreme importance. In this tradition female is raised above the male as Durga described as Shakti, the energy of the cosmos. Without her we are told in one of the texts that the world is lifeless and even, the great Shiva is merely a corpse. An analysis of the legends, doctrines and abstract philosophies indicates the first, the Goddess is portrayed as power, and the female Shakti element is identified as the essence of reality, the male element playing a subservient role. Second she is identified with Prakriti, the primeval matter. As such she is identified with existence or that which underlines all existent things. Thirdly, she is described as giving food to all nourish all life and to cause decay. Fourthly, the Goddess incarnates in herself all the brilliance and power that the Gods collectively possess and her pervasive magic gives them sufficient power to be able to battle with all evil. It is an exuberant celebration of the various forms of women and their role in making and constructing society. As a Nation, we progressed when the Indian woman was accorded her rightful place in society. It is my belief that India will progress much faster if a greater number of women are educated, financially independent and realize that equality does not mean that you listen to yourself only. Let us resolve to allow her to blossom, flower and show her inner beauty. Keywords: Culture, Religion, Vedic, Upanishad, Post Vedic. Through a myriad of cultural and political forces, ideal womanhood came to symbolize the nation. The demarcation of ideal womanhood was crucial to the formation of class and national culture.1 As one commentator has summarized that 'the nations identity lay in the culture and more specifically in its womanhood'.2 Thus women were the epicenter of cultural reconstruction and recreation, much of which centered on the need to recapture the grandeur of India's lost high Aryan past. The construction of an idealized past called for and depended upon the construction of a particular kind of womanhood. The way the women were conceptualized changed the way they could navigate the world. AARHAT MULTIDISCIPLINARY INTERNATIONAL EDUCATION RESEARCH JOURNAL (AMIERJ) (BI-MONTHLY) PEER-REVIEWED JOURNAL VOL NO V ISSUES II ISSN 2278-5655 AARHAT MULTIDISCIPLINARY INTERNATIONAL EDUCATION RESEARCH JOURNAL (AMIERJ) (BI-MONTHLY) PEER-REVIEWED JOURNAL VOL NO V ISSUES II ISSN 2278-5655 A CRITICAL STUDY OF THE POSITION OF WOMEN IN VEDIC AND POST VEDIC PERIOD A CRITICAL STUDY OF THE POSITION OF WOMEN IN VEDIC AND POST VEDIC PERIOD Dr. Manoj Kumar Dubey Iswar Saran Degree College University of Allahabad,Allahabad-211002 University of Allahabad,Allahabad-211002 AARHAT MULTIDISCIPLINARY INTERNATIONAL EDUCATION RESEARCH JOURNAL (AMIERJ) (BI-MONTHLY) PEER-REVIEWED JOURNAL VOL NO V ISSUES II ISSN 2278-5655 books such as Dharma-Shastras, however, basic rules in the laws of Manu (200 B.C.) lays out how a women or wife should behave in the household and towards her husband. Nevertheless women's roles have evolved over time and women are going against the social norms of their tradition and even their way of life. Hinduism is a complex religion and unlike many western religions. It is also a way of life. In Hinduism women play an important role in the tradition. Women are revealed in the sacred scriptures as presenting a duality of being benevolent and malevolent, exposing her with great contrasting powers. In times of prosperity she indeed is Lakshmi, who bestows prosperity in the homes of men, and in times of misfortune, she herself becomes the goddess of misfortune and brings about ruins.4 Because of these changing powers that a women possesses, it is rational that man should want to control this mysterious power. The females nature are like the soil where the male plants his seeds to grow into conjoined images.5 And therefore the male controls the female, that nature is controlled by culture.6 Culture or society controls nature as it is motivated to change and evolve just as the man tries to control the women. Many Hindu scriptures says that women are to be honoured, as stated that 'religious deeds are says to be useless, if women are not honoured and cherished'.7 So, in a small village in North India, women instigate and participate in twenty one of the thirty three annual rites and dominate nine of the twenty one annual rites.8 Although women have developed a stronger religious status, but these consequences of perceiving the significance of women only in relation to men is seen most clearly in Hindu attitudes towards women which is rooted in the religious beliefs and practices.9 The major problems faced by women in Hindu tradition arise because of patriarchal views that affirm the value and significance of women only in relation to men, but women have value and significance, not primarily because of their instrumental roles and relationships to us as wives and mothers, but because, like us, they equally embody the divine. Their worth is intrinsic one and does not come indirectly through males. Abstract: They could more within the public sphere, but only so long as their identity as cultural makers was upheld through symbols such as traditional dress.3 But women have fought for their status and role in communities, religions and the nation for years. And women in Hinduism are no different. Women traditionally would live the life of a mother and a wife following the footsteps of their ancestors. Women's roles were laid out in Hindu law Feb – Mar 2016 Page No 8 Impact factor: 2.125 Page No 8 Feb – Mar 2016 www.aarhat.com www.aarhat.com AARHAT MULTIDISCIPLINARY INTERNATIONAL EDUCATION RESEARCH JOURNAL (AMIERJ) (BI-MONTHLY) PEER-REVIEWED JOURNAL VOL NO V ISSUES II ISSN 2278-5655 others by the worth of commodities that they own. A tradition that affirms unequivocally that the significance of the human being is to be found in the fact that human nature embodies the divine cannot condone the commercialization of human existence. It must champion the dignity of all human beings and attitudes of respect and reverence for all human life. others by the worth of commodities that they own. A tradition that affirms unequivocally that the significance of the human being is to be found in the fact that human nature embodies the divine cannot condone the commercialization of human existence. It must champion the dignity of all human beings and attitudes of respect and reverence for all human life. In the age of Rigveda the frequent reference to unmarried girls speaks in favor of a custom of girls marrying long after they had reached puberty. Among Aryans, marriage among brothers and sisters was prohibited. There seems to have been considerable freedom on the part of young persons in the selection of their life partners as they generally married at a mature age. Approval of the parent or the brother was not essential, the boy and the girl made up their minds and then informed the elders though their participation in the marriage ceremony was essential i.e. the blessings of the elders were sought.11 The wife was respected in her new house and wielded authority over her husband’s family. The wife participated in the sacrificial offerings of her husband. Abundance of sons was prayed for so, naturally so in a patriarchal society since the son performed the last rites and continued the line. Remarriage of widows was permitted under certain conditions. Female morality maintained a high standard although but the same degree of fidelity was not expected from the husband. Net yet women enjoyed much freedom. They took an active part in agriculture, manufacture of bows. They moved around freely, publicly attended feasts and dances. In Later Vedic period freedom of marriage continued and remarriage of widows continued to be allowed. The sale of a daughter was known but viewed with extreme disfavor. Dowries continued to be given but not in the sense that we understand today. The marriage ceremony was the same as in the previous period except that the girl had to mount a stone before the boy caught her hand. AARHAT MULTIDISCIPLINARY INTERNATIONAL EDUCATION RESEARCH JOURNAL (AMIERJ) (BI-MONTHLY) PEER-REVIEWED JOURNAL VOL NO V ISSUES II ISSN 2278-5655 To counteract such role of stereotyping and exclusivity, we need to highlight the many women in history of Hinduism who liberated themselves from traditional roles and made their own destinies. Most of them come from the devotional traditions of Hinduism that emphasize the centrality of love in the divine human relationship and that dispense with the role of priestly intermediaries. These movements have also employed vernacular languages of India as their mode of religious expressions. Prominent among these women are like Andal (12th Century B.C.) and Mirabai (14th Century A.D.).10 The doctrine of divine immanence and the sanctity and worth of the human person that follows from it must become the foundation of a Hindu challenge and critique of all attitudes, values and actions that demean and trivialize women and reduce them to sexual object. Hinduism must resist and respond to the challenge of materialistic and consumer oriented culture in which people determine their own value and the values of Feb – Mar 2016 Page No 9 Impact factor: 2.125 Page No 9 Feb – Mar 2016 Page No 9 Feb – Mar 2016 www.aarhat.com www.aarhat.com www.aarhat.com AARHAT MULTIDISCIPLINARY INTERNATIONAL EDUCATION RESEARCH JOURNAL (AMIERJ) (BI-MONTHLY) PEER-REVIEWED JOURNAL VOL NO V ISSUES II ISSN 2278-5655 AARHAT MULTIDISCIPLINARY INTERNATIONAL EDUCATION RESEARCH JOURNAL (AMIERJ) (BI-MONTHLY) PEER-REVIEWED JOURNAL VOL NO V ISSUES II ISSN 2278-5655 2. When the bride’s father gives his formal consent, the bridegroom performs a sacrifice. 3. Early in the morning of the first day of marriage celebrations, the bride is bathed. 4. A sacrifice is offered by the high priests of the bride’s family and a dance of 4/8 women takes place as part of the Indrani karman. 5. The bridegroom goes to the girl’s house and makes the gift of a garment, mirror to the bride who has been bathed earlier. 5. The bridegroom goes to the girl’s house and makes the gift of a garment, mirror to the bride who has been bathed earlier. 6. The Kanya-pradana, formal giving away of the bride takes place now followed by. 7. The clasping of the bride’s right hand by the bridegroom’s own right hand takes place now. 8. The treading on stone. 9. The leading of the bride round the fire by the bridegroom. 9. The leading of the bride round the fire by the bridegroom. 9. The leading of the bride round the fire by the bridegroom. 10. The sacrifice of the fried grains. 11. The Saptapadi i.e. the couple walking seven steps together as a symbol of their livelong concord. 12. Finally, the bride is taken to her new house. 13. After the bride came home, the couple is expected to observe celibacy for three days after which the marriage was consummated. The logic was to emphasize at the outset that self-control was very much part of married life. The women held an honored position in the household. She was allowed to sing, dance and enjoy life. Sati was not generally prevalent. Widow Remarriage was allowed under certain circumstances. On the whole the Dharma-sutras take a more lenient attitude than the Smritis of a later age.13 The Apastamba imposes several penalties on a husband who unjustly forsakes his wife. On the other hand, a wife who forsakes her husband has to only perform penance. In case a grown up girl was not married at a proper time by her father, she could choose her husband after three years of waiting. The most pleasing feature of this period is the presence of women teachers, many of whom possessed highest spiritual knowledge. AARHAT MULTIDISCIPLINARY INTERNATIONAL EDUCATION RESEARCH JOURNAL (AMIERJ) (BI-MONTHLY) PEER-REVIEWED JOURNAL VOL NO V ISSUES II ISSN 2278-5655 As in the previous period the picture of an ideal family life continued. Gradually religious ceremonies increasingly were conducted by the priests resulting in losing her preeminent position in the household. This was the period during which the importance of rituals increased and so did the importance of the Brahmans. Desire for sons continued, but the position of women was not as high as it was in the Rig Vedic period. Female workers were involved in dying, embroidery and basket making. In the age of Upanishad’s, the anuloma system of marriage i.e. between the male of a higher caste and female of a lower caste prevailed during this period. The rules of Panini regarding Abhi-vadana ( salutation as a mark of respect to elderly persons in the house ) show that the presence of wives of the lower caste in a house and their association with ladies of a higher caste brought down the general level of womanly culture and led to a deterioration in their status.12 The Grihya-sutras give detailed rules regarding the proper seasons for marriage, qualifications of bride and bridegroom. The various stages of a marriage ceremony are: Feb – Mar 2016 Page No 10 Feb – Mar 2016 Page No 10 Impact factor: 2.125 Page No 10 www.aarhat.com www.aarhat.com www.aarhat.com become a daughter’s parent became a source of misery. In the post Vedic period, the professions open to woman in higher sections of society were teaching, medical doctors and business. They suffered from no disabilities in doing business and could even pledge their husband’s credit and enter into contracts on their behalf. Purdah system was not prevalent during this period. There is nothing in our tradition or literature to suggest that the father/elder brother in-law could not see the face of the daughter-in-law as is the case in North India today.Man is only one half, says a Vedic passage; he is not complete till he is united with his wife and gives birth to children. The husband is to treat his wife as his dearest friend. The wife is a companion friend of a man, says a Vedic passage. The Mahabharat and Buddhist thinkers concur with this view. Marriage between the same caste was preferred although inter caste marriages were prevalent. Of the eight forms of marriage prescribed by the Dharma-sutras, the Arhsa form of marriage was most popular which was the father gave his daughter after receiving from the bridegroom a cow and a bull or two pairs. The bridegroom was selected by the girl’s father or guardian. According to Nearchus the Indians “marry without giving or taking dowries but the girls, as soon as they are marriageable, are brought forward by their fathers and exposed in public, to be selected by a person who excels in some form of physical exercise”. This indicates a modified form of Svayamvara.14 While girls continued to be married around 16, there was a tendency to marry them before they attained puberty. It was probably due to the anxiety to maintain their body purity. Lowering of the marriage age affected their education and culture adversely. After all, if she got married early then how could she study? Extreme emphasis was now laid on the physical chastity of women which discouraged widow remarriage, divorce and encouragement of sati. We must remember that India faced its first foreign invasion ie Greeks during this period. During the earlier part of this period, there were highly educated women holding an honorable position in society and household. There were lifelong students of sacred texts or those who pursued their study till marriage. Women also read training in arts, music, painting and for some military training also. Female bodyguards are referred to in Kautilya’s Arthasastra. AARHAT MULTIDISCIPLINARY INTERNATIONAL EDUCATION RESEARCH JOURNAL (AMIERJ) (BI-MONTHLY) PEER-REVIEWED JOURNAL VOL NO V ISSUES II ISSN 2278-5655 The famous dialogue between Yajnavalkya and his wife Maitreyi and Gargi Vachaknavi show how enlightened the women of that age were. According to the Sarvanukramanika, there were as many as 20 women among the authors of the Rig Veda. These stories stand in contrast to the later age when the study of Vedic literature was forbidden to women under the most severe penalty. The feeling of dejection on the birth of a daughter did not lead to Female Infanticide in ancient India. This custom crept into India during the medieval period. Once the disappointment on the birth of a daughter was over, the family did not distinguish between their son and daughter. In subsequent periods, growing incidence of Sati meant that parents saw their daughters jumping on to funeral pyres or if she became a widow, live a chaste life since widow remarriage was not permitted. In such an environment to Impact factor: 2.125 Feb – Mar 2016 Page No 11 www.aarhat.com www.aarhat.com Feb – Mar 2016 Feb – Mar 2016 AARHAT MULTIDISCIPLINARY INTERNATIONAL EDUCATION RESEARCH JOURNAL (AMIERJ) (BI-MONTHLY) PEER-REVIEWED JOURNAL VOL NO V ISSUES II ISSN 2278-5655 AARHAT MULTIDISCIPLINARY INTERNATIONAL EDUCATION RESEARCH JOURNAL (AMIERJ) (BI-MONTHLY) PEER-REVIEWED JOURNAL VOL NO V ISSUES II ISSN 2278-5655 invasion like the Greeks. Rituals came to be performed without the Vedic mantras; the Vedic sacrifices were tabooed for the wife. Widow Remarriage and divorce were discouraged. On the other hand, Kautilya, in the matters of divorce, placed man and women on an equal footing. But as people with lax morals came into the social framework on account of the expanding frontiers of Dharma, the marital tie assumed greater sanctity. Their status deteriorated considerably during this period. With time and progress, one would expect the condition of people to improve but in this case it was the opposite. The introduction of slavery revolutionized the position of women in the classical period of Greek history, they lost esteem in society. The same thing happened in India when a semi servile status came to be assigned to the Sudra class whose only duty was service of the higher castes. Over time and due to various factors, inter caste marriages started happening during the period 1000 to 500 BC. The introduction of non-Aryan women into the Aryan household is the starting point to the deterioration in a women’s status. Unfamiliar with religious customs, rituals and Sanskrit the non Aryan wife would have goofed making the priests angry. To avoid this problem it was decided that the whole class of woman were ineligible for Vedic studies and religious duties. Another reason was that Vedic sacrifices became complex making it difficult for the wife to have mastery over them. Although, the view that women must not be allowed to perform sacrifices was opposed by some parts of society. become a daughter’s parent became a source of misery. Buddhist and Jain nuns renounced the world for the sake of spiritual salvation. Jain texts refer to Jayanti who carried on discussions with Mahavira himself and later on became a nun. In spite of the progress, there were growing disabilities. Earlier the girls went through the Upanayana ceremony but now it was only a formality. Manu laid down that marriage was equal to Upanayana while Yajnavalkya took the step of prohibiting Upanayana ceremony for girls.15 The wife who performed Vedic sacrifices was denied the right to do so. Narada is however, more considerate towards women. Greek writers have stated that sati existed, was in vogue in Punjab, possibly confined to the warrior class only. Social customs are a product of the environment we live in. India had never seen, til Social customs are a product of the environment we live in. India had never seen, till 327 BC, an Feb – Mar 2016 Page No 12 Impact factor: 2.125 Page No 12 Feb – Mar 2016 www.aarhat.com www.aarhat.com A. S. Altekar, Position of Women in Hindu Civilization, Orient Book Distributors, 1978, pg. 178-180 113. Ibid., pg. 115 Ibid., pg. 115-16 M.W., Pinkham, Women in the Sacred Scriptures of Hinduism, New York, 1967, pg. 190. Susan, Wadley, op.cit., pg. 123. W., Pinkham, Women in the Sacred Scriptures of Hinduism, New York, 1967, pg. 190. See, Manu Smriti, 9 : 138. See, David Kinsley, Hinduism : A Cultural Perspective, Prentice Hall, 1993, Chapter 7. Sanga Sangari and Vaid, Recasting Women : An Introduction, pg. 15. Ibid., pg. 18. S. Tharu and T. Niranjana, Problems for a Contemporary Theory of Gender, Social Scientist 22, 1994, pg. 93-117. Uma Chakravarti, Whatever Happened to the Vedic Dasi; in Recasting Women in India; Essays in Colonial History, Rutges University Press, 1990, pg. 28. Amrita Basu, Feminism Inverted : The Gender Imagery and Real Women of Hindu Nationalism, New Delhi, 1995, pg. 158-160. Feb – Mar 2016 Page No 1 Impact factor: 2.125 Page No 13 www.aarhat.com Kumkum Sangari and Sudesh Vaid, Recasting Women in India; Essays in Colonial History. Rutgers University Press, 1990, pg. 5. A. S. Altekar, Position of Women in Hindu Civilization, Orient Book Distributors, 1978, pg. 178-180 Feb – Mar 2016 Page No 14 Impact factor: 2.125 Page No 14 Feb – Mar 2016 www.aarhat.com www.aarhat.com
https://openalex.org/W3140927395	https://bmcpediatr.biomedcentral.com/track/pdf/10.1186/s12887-021-02813-x	English	null	Tinea capitis in an immigrant pediatric community; a clinical signs-based treatment approach	BMC pediatrics	2,021	cc-by	5,335	Tinea capitis in an immigrant pediatric community; a clinical signs-based treatment approach Riad Kassem1,2, Yahel Shemesh3, Orna Nitzan3,4, Maya Azrad5 and Avi Peretz3,5* Abstract Orna Nitzan, who passed away on 10 September 2020. 3The Azrieli Faculty of Medicine, Bar-Ilan University, Safed, Israel 5Clinical Microbiology Laboratory, Baruch Padeh Medical Center, Poriya, Israel Full list of author information is available at the end of the article Kassem et al. BMC Pediatrics (2021) 21:363 https://doi.org/10.1186/s12887-021-02813-x Kassem et al. BMC Pediatrics (2021) 21:363 https://doi.org/10.1186/s12887-021-02813-x * Correspondence: aperetz@poria.health.gov.il The authors dedicate this article to the memory of Dr. Orna Nitzan, who passed away on 10 September 2020. 3The Azrieli Faculty of Medicine, Bar-Ilan University, Safed, Israel 5Clinical Microbiology Laboratory, Baruch Padeh Medical Center, Poriya, Israel Full list of author information is available at the end of the article Abstract Background: Tinea capitis is a common cutaneous infection of the scalp and hair follicles, typically diagnosed by direct examination and culture. Treatment with oral antifungals is usually withheld until mycology results are available. In Israel, African refugee children demonstrate higher susceptibility to Tinea capitis and generally fail to undergo follow-up evaluations. Methods: This study aimed to identify the clinical characteristics and treatment responses of refugee children in Israel with Tinea capitis, in order to formulate a treatment plan for primary care physicians. To this end, demographic, clinical and laboratory data were extracted from the electronic medical records of 76 refugee children presenting with Tinea capitis during 2016–2017. All measured variables and derived parameters are presented using descriptive statistics. The correlation between background clinical and demographic data and Tinea capitis diagnosis was assessed using the chi-squared and Wilcoxon tests. Correlations between demographic/ clinical/laboratory characteristics and other types of fungi or other important findings were assessed using a T-test. Results: Scaling was the most common clinical finding. Cultures were positive in 64 (84%) and direct examination in 65 (85%) cases, with a positive correlation between the methods in 75% of cases. The most common fungal strain was T. violaceum. Fluconazole treatment failed in 27% of cases. Griseofulvin 50 mg/kg/day was administered to 74 (97%) children, and induced clinical responses. No side effects were reported. Conclusions: The key aim of this study was to emphasize the importance of diagnosis and treatment of these immigrant children by their primary pediatric doctor since it takes, an average of 4.3 months until they visit a dermatologist. During this critical time period, the scalp can become severely and permanently damaged, and the infection can become systemic or cause an outbreak within the entire community. In conclusion, we recommend to relate to scaly scalp in high-risk populations as Tinea capitis, and to treat with griseofulvin at a dosage of up to 50 mg/kg/day, starting from the first presentation to the pediatrician. Keywords: Tinea capitis, Dermatophyte, Children of refugees, Empiric treatment * Correspondence: aperetz@poria.health.gov.il The authors dedicate this article to the memory of Dr. Orna Nitzan, who passed away on 10 September 2020. 3The Azrieli Faculty of Medicine, Bar-Ilan University, Safed, Israel 5Clinical Microbiology Laboratory, Baruch Padeh Medical Center, Poriya, Israel Full list of author information is available at the end of the article * Correspondence: aperetz@poria.health.gov.il The authors dedicate this article to the memory of Dr. Background Refugee communities are generally of a low socioeco- nomic status and are associated with dense living condi- tions, which facilitate the spread of Tinea infections. In Israel, although refugees are medically insured up to the age of 18, they often receive suboptimal Tinea capitis care, partly due to the low availability of dermatologists in the southern part of Tel-Aviv, where most of them reside. Most cases are treated by pediatricians or family doctors, who generally have minimal expertise with this indication. As a result, the infection is frequently mis- diagnosed, and when properly diagnosed, topical treat- ments are prescribed, which are rarely effective. Tinea capitis is an infection of the scalp and hair folli- cles, caused by dermatophytes [1]. Dermatophytes is a common name of three genera of fungi – Trichophyton, Microsporum and Epidermophyton, with the first two species being most dominant. Dermatophytes are also classified by host preference and natural habitat, with anthropophilic dermatophytes affecting humans, zoo- philic dermatophytes affecting animals, and geophilic dermatophytes mainly affecting the soil. The latter two are relatively rare [2, 3]. All dermatophytes can exploit keratin (hair, skin, and nails) for growth and their clin- ical manifestations are named after the affected area of the body (e.g., Tinea manum, Tinea pedis, Tinea capitis) [4]. Tinea capitis is one of the most common cutaneous infections in pre-pubertal children [1, 5–8], mainly from 6 months to 10–12 years of age [6]. The epidemiology of Tinea capitis varies across geographical regions through- out the world and changes over time [8, 9]. The infec- tion spreads among family members and classmates1. Treatment of Tinea capitis in refugee children poses a challenge, since optimal treatment demands repeated lab exams, yet compliance with such measures is very low because most parents work long hours and cannot afford to miss work days. In addition, a child diagnosed with Tinea capitis is forbidden to return to his daycare facility for several weeks, which requires that a parent miss more workdays. Thus, our aim was to simplify the diag- nosis of Tinea capitis by primary physicians and speed initiation of appropriate treatment, by developing tools to accurately identify and treat the disease based on clin- ical finding and on physical examination. © 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. Page 2 of 8 Page 2 of 8 Kassem et al. BMC Pediatrics (2021) 21:363 What is Known Diagnosis can be confirmed by direct microscopy, with use of standard potassium hydroxide (KOH) prepara- tions. Its disadvantages include the time-consuming process, requirement of an expert and equipment not necessarily available in every clinic, and a false negative rate of up to 40% [12]. In addition, scalp scrapings and hair fragments from the affected area can be cultured, but can involve up to 3 weeks until results are provided [6, 12]. Molecular techniques such as polymerase chain reaction (PCR), provide for faster and more accurate identification of dermatophyte infections [6]. Tinea capitis is a contagious infection, common among refugee children. Treatment with oral antifungals is usually withheld until mycology results are available. y gy This is a critical period, because these children may infect others, and trigger epidemics in their densely populated communities. Methods Tinea capitis is often misdiagnosed, thus delaying ap- propriate treatment and enabling spread of infection [7]. The diagnosis can be made in several ways. First and foremost, anamnesis and physical examination are cru- cial. In recent years, the dermatoscope has become more commonly used for close inspection of the scalp [11]. What is New Systemic antifungal agents are indicated for all cases of Tinea capitis. While topical agents do not pene- trate the hair shaft [7, 13], they are used as adjuncts to reduce the risk of transmission [1]. The common drugs in use are griseofulvin, terbinafine, itraconazole and fluconazole. The type and duration of treatment, as well as the dosage are determined by the dermato- phyte strain [1, 2, 6, 14]. In African refugee children with scalp scales suspected of Tinea capitis, a culture and direct examination and empiric treatment with griseofulvin 50 mg/kg/day are recommended at first presentation to the primary physician. Background To this end, the clinical and demographic characteristics of 76 Tinea capitis-infected pediatric refugees living in the southern district of Tel Aviv, Israel, were analyzed and compared to the current knowledge in the field. The clinical manifestations of Tinea capitis are vari- able, depending on the type of hair invasion, the level of host resistance, the immune system and the degree of inflammatory host response [6, 7], but can grossly be classified as alopecic or inflammatory. Inflamma- tory Tinea capitis includes kerion, which manifests as a tender mass with pustules, purulent discharge, lymphadenopathy, malaise, fever, and favus, with yel- low cup-shaped crusts around the hair, inflammation and scarring [3, 10]. Results The medical records of 76 children fulfilling the eligibil- ity criteria were reviewed (Table 1). Out of 76 cases, 55 (72.4%) were boys and 21 (27.6%) were girls. The average age was 3.1 years (range 0–8 years). In total, 30 children (39.9%; 9 girls (30%) and 21 boys (70%)) had a family The most common cultured dermatophyte species was Trichophyton violaceum, which was found in 25 (39%) samples. Out of the positive cultures, T. violaceum was found in 33% of samples collected from girls versus 41% of the samples collected from boys. The distribution of identified fungal species is shown in Fig. 3a. Distribution by host preference is presented in Fig. 3b. Table 1 Demographic and baseline characteristics of pediatric patients with suspected tinea capitis Table 1 Demographic and baseline characteristics of pediatric patients with suspected tinea capitis Criteria All cases n (%) Culture Direct examination Positive n (%) Negativea n (%) Positive n (%) Negative n (%) Sex 76 64 (84) 12 (16) 65 (85) 11 (15) Male 55 (72.4) 46 (84) 9 (16) 49 (89) 6 (11) Female 21 (27.6) 18 (86) 3 (14) 16 (76) 5 (24) Age (years) 3.3 0–2 13 (17.1) 10 (77) 3 (23) 12 (92) 1 (8) 2.1–4 38 (50) 31 (81) 7 (9) 33 (87) 5 (13) 4.1–6 24 (31.6) 22 (92) 2 (8) 19 (79) 5 (21) 6.1–8 1 (1.3) 1 (100) 0 1 (100) 0 Eosinophilia 23 (30) 21 (91) 2 (9) 21 (91) 2 (9) High IgE levels 8 (10) 8 (100) 0 8 (100) 0 a Contaminated culture was considered negative Tinea capitis cases in children in this community prior to this study, were treated with griseofulvin as first-line therapy at a maximum dose of 25 mg/kg/day for 6–8 weeks, which induced a minimal response, if any. In order to eradicate and prevent spread of the infection, the recommended drug dose was gradually elevated to a dose of 50 mg/kg/day, for 6–8 weeks, which efficiently eradicated the infection. From then on, all children sus- pected to have Tinea capitis were treated with griseoful- vin at this doubled dose. During the study period, due to unavailability of griseofulvin for some time, 21 patients were treated with fluconazole, at a maximum dose of 7.5 mg/kg/day, for 4 weeks. None of these children showed a clinical re- sponse, even when the dose was raised to 10 mg/kg/day for 6 weeks. Study design This was a retrospective study, conducted in the district of Tel-Aviv, Israel. The study analyzed the records of children aged 0–8 years (2 months – 7.7 years) with a clinical manifestation or positive culture of Tinea capitis, Page 3 of 8 Kassem et al. BMC Pediatrics (2021) 21:363 Page 3 of 8 who were referred to a dermatologist in a secondary re- ferral clinic of the Meuhedet sick fund and treated by the same dermatologist between January 2016 and De- cember 2017. Data extracted from patient electronic re- cords included: age, sex, country of birth, origin of parents, duration of lesion until contact with the derma- tologist, infected family member (yes/no), high IgE level (yes/no), eosinophil blood count, history of immunosup- pression, medical history, lesion description (alopecia, scaling, pruritus) and morphology, presence of kerion and lymphadenopathy, culture results, outcome of microscopic examination of skin scrapings and hair frag- ments (KOH exam), topical treatment (yes/no) and type and duration of systemic treatment. member infected with Tinea capitis. All children were born in Israel to parents who emigrated from Eritrea. The average time from the initial appearance of the le- sion until contact with a dermatologist was 4.3 months (3 days - 24 month). Out of 76 cases, 64 had a positive culture, 18 of which were collected from girls (28%) and 46 from boys (72%). The remaining 12 cultures were contaminated. In paral- lel, 65 cases had a positive direct examination, 8 of which showed negative cultures (Table 1). Thus, 57 cases had both a positive culture and direct examination, while 72 patients have at least one positive test (Fig. 1). The most common clinical manifestation among pa- tients with a positive culture or a positive direct examin- ation was scaling, and the least common was kerion with lymphadenopathy (Table 2). Kerion with lymphadenop- athy provided the highest diagnostic specificity, while scaling was associated with the lowest diagnostic specifi- city. No single or combination of symptoms provided both high diagnostic sensitivity and specificity (Tables 2 and 3). Pruritus, as well as kerion with lymphadenopathy had a high diagnostic sensitivity but low specificity. Statistical analysis All measured variables and derived parameters are pre- sented using descriptive statistics. The correlation be- tween background clinical and demographic data and Tinea capitis diagnosis was assessed using the chi- squared and Wilcoxon tests. Correlations between demographic/clinical/laboratory characteristics and other types of fungi or other important findings were assessed using a T-test. Out of the 64 children with a positive culture, 13 and 33% had high levels of IgE and eosinophilia, respectively (Fig. 2a) and 9% had both high IgE levels and a high eo- sinophilic count. Similar percentages of high IgE and/or eosinophilia were seen among the children with a posi- tive direct examination (Fig. 2b). Among children with both a positive culture and a positive direct examination, fewer (17%) had eosinophilia as compared to those with only one positive test (Fig. 2c). All tests were two-tailed, and a p-value of 5% or less was considered statistically significant. The data were analyzed using SAS® version 9.1 (SAS Institute, Cary, North Carolina). a Contaminated culture was considered negative a Contaminated culture was considered negative Results After the return of griseofulvin to the Page 4 of 8 Kassem et al. BMC Pediatrics (2021) 21:363 Table 2 Single symptoms at enrollment: relationship to positive dermatophyte culture (DC) and to positive direct examination (PDA) a Table 2 Single symptoms at enrollment: relationship to positive dermatophyte culture (DC) and to positive direct examination (PDA) a Symptom n/N Sensitivity (%) Specificity (%) PPV (%) NPV (%) DC PDA DC PDA DC PDA DC PDA DC PDA Scaling 58/64 63/65 90.63 96.92 8.33 45.45 84.06 91.3 14.29 71.43 Alopecia 25/64 28/65 39.06 43.08 33.33 54.55 75.76 84 9.30 13 Pruritus 5/64 8/65 7.81 12.31 66.67 90.91 55.56 88.89 11.94 14.93 Kerion with lymphadenopathy 5/64 5/65 4.69 7.69 83.33 100 60 15.49 14.08 21.05 a Contaminated culture was considered negative a Contaminated culture was considered negative the southern Tel Aviv area. This population is generally of a low socioeconomic status, and resides in dense liv- ing conditions (data obtained from Tel Aviv municipal- ity) [15]. The children are generally cared for at daycare centers, which can facilitate transmission, and are less likely to visit a tertiary clinic with expert dermatology services, and are therefore susceptible to Tinea capitis epidemics. Due to a focal outbreak of Tinea capitis among children in this community, and in order to pre- vent epidemics affecting other communities, this work attempted to characterize the clinical manifestations and provide primary physicians with appropriate diagnostic and management guidelines, to minimize the need for postponing the treatment until referral to a dermatology service. market, these children were treated with griseofulvin 50 mg/kg/day; clearance was achieved within 6 weeks in most cases, except for 3 children who required 8 weeks of treatment until the condition fully resolved. Five children presented with kerion. In addition to an- tifungal treatment, they were treated with antibiotics and steroids. None required surgical intervention. Two children with both a negative culture and negative direct examination and suspected of having seborrheic derma- titis, were not treated empirically for Tinea capitis. Two other children with uncertainty regarding the possibility of Tinea capitis or seborrheic dermatitis diagnosis, were given empirical treatment, which was discontinued after receiving negative culture results. Four children who showed no response to griseofulvin had a R. mucilaginosa-positive culture and were treated accordingly. In accordance with the literature regarding children of refugees of African descent in the US, Europe and Israel, T. Results violaceum was the most common cause of Tinea capi- tis [5, 8, 16]. Furthermore, 75% of positive cultures showed that the source of infection was an anthropophi- lic fungus, strengthening the conclusion that patients in- fect each other at daycare facilities and at home, due to the dense living conditions and lack of sufficient hygiene. All children received topical treatment as adjunct ther- apy, as recommended, in order to reduce contagion. No clinical side effects or blood test abnormalities were ob- served in the participants during treatment. All treat- ments and responses are summarized in Table 4. Discussion The probability of seeing an Israeli child with Tinea capitis is very low. Mashiah et al. who treated Tinea capitis in a pediatric population between the years 2011–2014 in a central tertiary reference center, found no cases through the year of 2010 [17]. Through the subsequent years, they started to gradually see more and more cases, which was ascribed to the increasing waves Since 2006, more than 60,000 people illegally emigrated from Africa to Israel through its border with Egypt, ar- riving with no belongings. Most of the refugees came from Eritrea, while others arrived from the Republic of Sudan and other African countries. This phenomenon increased considerably between 2007 and the end of 2012. A significant portion of this population settled in at enrollment: relationship to positive dermatophyte culture (DC) or to positive direct examination Table 3 Multiple symptoms at enrollment: relationship to positive dermatophyte culture (DC) or to positive direct examination (PDA) a Symptom (pruritus, scaling, alopecia and kerion with lymphadenopathy) n/N Sensitivity (%) Specificity (%) PPV (%) NPV (%) DC PDA DC PDA DC PDA DC PDA DC PDA No symptoms 3/64 0/65 4.69 0 100 72.73 100 0 16.44 10.96 Any 1 symptom 34/64 31/65 53.13 47.69 88.33 54.55 94.44 86 25 15 Any 2 symptoms 24/64 29/65 37.50 44.62 41.67 81.82 77.42 20 11.11 50 Any 3 symptoms 3/64 5/65 4.69 7.69 75 90.91 50 83.33 15.79 14.29 Kassem et al. BMC Pediatrics (2021) 21:363 Page 5 of 8 Fig. 1 Percentage of positive tests for Tinea capitis out of all cases: 57 cases (75%) had both a positive culture and direct examination, while 72 cases had at least one positive test of refugees entering Israel. In this 4-year period, they had only 15 Israeli pediatric patients with Tinea capitis compared to 130 refugees [17]. The refugee community is estimated to include 60,000 individuals compared to 450,000 Tel-Aviv residents. However, in contrast to this earlier report, that dealt with urban hospital-based general pediatric practice, in the present study, the majority of children with a posi- tive culture or positive direct exam had one positive sign, which was scales (53 and 48%, respectively). Discussion More- over, the previous work found a high association be- tween lymphadenopathy and positive Tinea capitis cultures, which stands in contrast with our results, where only 4.7% of the children with a positive culture The most common clinical finding in children with ei- ther positive culture or positive direct examination was scaling, observed in 91 and 97% of cases, respectively, aligning with a previously published report [12]. Fig. 2 Percentage of children with high levels of IgE and/or eosinophilia out of all cases with positive A. Culture B. Direct examination C. Culture and direct examination Fig. 2 Percentage of children with high levels of IgE and/or eosinophilia out of all cases with positive A. Culture B. Direct examination C. Culture and direct examination Kassem et al. BMC Pediatrics (2021) 21:363 Kassem et al. BMC Pediatrics Page 6 of 8 Fig. 3 A. Percentage of the different types of dermatophyte fungi in culture among children with suspected tinea capitis. B. Percentage of the different types of dermatophyte fungi by host preference re among children with suspected tinea capitis. B. Percentage of the Fig. 3 A. Percentage of the different types of dermatophyte fungi in culture among children with suspected tinea capitis. B. Percentage of the different types of dermatophyte fungi by host preference infection. However, this lack of correlation may have been the result of the small sample size. Alternatively, it may be due to subtle systemic signs in these children when compared to the other pediatric population, which may be rooted in differential immunologic responses to fungi or parasites, with the exception of kerion cases. and 7.7% of those with positive signs on direct examin- ation, presented with lymphadenopathy. A different im- mune response to the same infection may underlie these conflicting observations, and may require an entirely dif- ferent therapeutic approach. In the current study population, many parents who visited a dermatologist with their infected child discon- tinued follow up at various stages, some after only one visit, likely due to long queues, distant clinics, loss of workdays and more. Some of the parents returned to the dermatologist only because the child was not allowed to return to the daycare center or due to development of infection and fever. Thus, treatment should be given to ensure suitable and efficient management from the ini- tial and possibly only encounter with the patient. Consent for publication Not applicable. Consent for publication Not applicable. Authors’ contributions D ll RK YS Data collection: RK, YS and AP; Data analysis: ON, MA, AP; Drafting of the manuscript: RK. Approval of the final manuscript: AP and RK. The authors read and approved the final manuscript. Author details 1 1Dermatology Department, Sheba Medical Center, Tel Aviv, Israel. 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. 3The Azrieli Faculty of Medicine, Bar-Ilan University, Safed, Israel. 4Infectious Disease Unit, Baruch Padeh Medical Center, Poriya, Israel. 5Clinical Microbiology Laboratory, Baruch Padeh Medical Center, Poriya, Israel. It is critical that the physician attempt to establish a relationship of trust with the parents in order to achieve whole-family care, to minimize reinfection cycles. It is also recommended to sterilize all shared hygiene instru- ments and wash bedding frequently. There might be a need for multidisciplinary collaborations (social worker, medical specialists) to coordinate and follow up on treat- ment plans and appointments. Mass testing in the neigh- borhood/schools may be warranted. Received: 28 March 2021 Accepted: 16 July 2021 Received: 28 March 2021 Accepted: 16 July 2021 References 1. Higgins EM, Fuller LC, Smith CH. Guidelines for the management of tinea capitis. Br J Dermatol. 2000;143(1):53–8. https://doi.org/10.1046/j.1365-2133.2 000.03530.x. 1. Higgins EM, Fuller LC, Smith CH. Guidelines for the management of tinea capitis. Br J Dermatol. 2000;143(1):53–8. https://doi.org/10.1046/j.1365-2133.2 000.03530.x. 1. Higgins EM, Fuller LC, Smith CH. Guidelines for the management of tinea capitis. Br J Dermatol. 2000;143(1):53–8. https://doi.org/10.1046/j.1365-2133.2 000.03530.x. 2. Gupta AK, Summerbell RC. Tinea capitis. Med Mycol. 2000;38(4):255–87. https://doi.org/10.1080/mmy.38.4.255.287. 3. Gupta AK, Hofstader SL, Adam P, Summerbell RC. Tinea capitis: an overview with emphasis on management. Pediatr Dermato. 1999;l16(3):171–89. https://doi.org/10.1046/j.1525-1470.1999.00050.x. Abbreviations l b Acknowledgments Acknowledgments Not applicable. Acknowledgment Not applicable. Due to the relative resistance to traditional dosages of griseofulvin observed in this population and poor follow up, these patients should be treated at a higher dosage than usual. Although treatment decisions rely on the identity of the fungus [14, 18, 19], griseofulvin at a dos- age of up to 50 mg/kg/day is recommended for first-line treatment, since it provides a sufficient clinical response. In addition, years of experience with the drug have dem- onstrated its long-term safety [13]; it has the fewest known drug interactions [17], a favorable adverse-effect profile [20] and rarely induces serious adverse-reactions [2]. Griseofulvin treatment has been associated with a small number of minor adverse effects, mainly gastro- intestinal symptoms (vomiting, abdominal pain, diar- rhea). Furthermore, it is the cheapest antifungal drug [13], a critical criterion for maintaining long-term com- pliance in the target population. When considering these benefits against the potential harm, empiric treatment prior to culture results is recommended [21]. As shown in Table 4, the study population seemed to be resistant to fluconazole. It is therefore not recommended to treat them with this drug, as it may result in lower compli- ance. Topical treatment alone failed to achieve clinical resolution, and therefore should only be administered as adjunct therapy. In case of an inflammatory lesion, such as kerion, additional treatment with steroids and anti- biotic is needed. All the medications should be given to- gether, to enhance compliance. Not applicable. Discussion The four children who had no clinical response to griseofulvin, turned out to have a R. mucilaginosa-posi- tive culture. Although rare, particularly in immunocom- petent patients, scalp infection due to these unicellular pigmented yeasts that mimic Tinea capitis, has been ob- served in a refugee population [15]. Several therapeutic approaches have been described, including amphotericin B, ketoconazole, fluconazole, itraconazole and flucyto- sine, however, there is no consensus regarding the pre- ferred treatment for such infections [15]. In previously reported cases of Tinea capitis, insuffi- cient response in skin of color populations was not solely due to lack of compliance, but rather, to a re- duced clinical response of the fungi to the conven- tional griseofulvin doses [17]. As a result, over the past few decades, dosage elevations have been tested and have effectively achieved clearance. This might be due to suboptimal absorption of the drugs, different host response patterns to the same fungi or evolution of resistance to the drugs [17]. Taken together, for a refugee patient, a full physical examination should be performed. If findings like scal- ing, alopecia or pruritis are identified, the physician must rely on his highest degree of suspicion, and provide em- pirical treatment from the first encounter [18]. Prescrip- tions should be given for the entire treatment period, because it might be the only encounter with the patient. Despite the challenges of follow up in this population, and in accordance with the literature, in any case of sus- pected Tinea capitis, the scalp should be cultured prior to treatment [2], at least for epidemiological No specific association was observed between eosino- philia or IgE levels and susceptibility to Tinea capitis Table 4 Anti-mycotic treatment dosage and duration Participants Line Drug Dose Duration (week) Eradication (n/N) Therapy replacement Therapy stopped 53 1 Griseofulvin 50 mg/kg/day 8 49/53 4 1 18 2a 6 18/18 0 0 3 8 3/3 0 0 21 1 Fluconazole 7.5–10 mg/kg/day 10 0/21 20 1 a Griseofulvin as second line was given after therapeutic failure with fluconazole Kassem et al. BMC Pediatrics (2021) 21:363 Page 7 of 8 Page 7 of 8 Abbreviations IgE: Immunoglobulin E; T. violaceum : Trichophyton violaceum investigation. If opportunity arises, follow-up with a re- peat mycology culture at the end of treatment is recom- mended, as a definitive diagnosis of eradication [1]. Ethics approval and Consent to participate The study protocol was approved by the institutional review board of the Health Maintenance Organization Meuhedet (03–29–10-18). No permissions were required to access the raw data from the electronic medical records. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Availability of data and materials N t li bl Availability of data and materials Not applicable. Conclusions 4. Elewski BE. Tinea capitis: a current perspective. J Am Acad Dermatol. 2000; 42(1):1–20. https://doi.org/10.1016/s0190-9622(00)90001-x. The presented findings emphasized the importance of diagnosis and treatment of these immigrant children by their primary pediatric doctor since it takes, as men- tioned, an average of 4.3 months until they visit a derma- tologist. During this critical time period, the scalp can become severely and permanently damaged, and the in- fection can become systemic or cause an outbreak within the entire community. In conclusion, we recom- mend to relate to scaly scalp in high-risk populations as Tinea capitis, and to treat with griseofulvin at a dosage of up to 50 mg/kg/day, starting from the first presenta- tion to the pediatrician. 5. Grigoryan KV, Tollefson MM, Olson MA, Newman CC. Pediatric tinea capitis caused by Trichophyton violaceum and Trichophyton soudanense in Rochester, Minnesota, United States. Int J Dermatol. 2019;58(8):912–5. https://doi.org/10.1111/ijd.14352. 5. Grigoryan KV, Tollefson MM, Olson MA, Newman CC. Pediatric tinea capitis caused by Trichophyton violaceum and Trichophyton soudanense in Rochester, Minnesota, United States. Int J Dermatol. 2019;58(8):912–5. https://doi.org/10.1111/ijd.14352. 6. Hay RJ. Tinea capitis: current status. Mycopathologia. 2017;182(1–2):87–93. https://doi.org/10.1007/s11046-016-0058-8. 7. John AM, Schwartz RA, Janniger CK. The kerion: an angry tinea capitis. Int J Dermatol. 2018;57(1):3–9. https://doi.org/10.1111/ijd.13423. 8. Mapelli ETM, Cerri A, Bombonato C, Menni S. Tinea capitis in the Paediatric population in Milan, Italy: the emergence of Trichophyton violaceum. Mycopathologia1. 2013;76(3–4):243–6. https://doi.org/10.1007/s11046-013- 9637-0. 6. Hay RJ. Tinea capitis: current status. Mycopathologia. 2017;182(1–2):87–93. https://doi.org/10.1007/s11046-016-0058-8. 7. John AM, Schwartz RA, Janniger CK. The kerion: an angry tinea capitis. Int J Dermatol. 2018;57(1):3–9. https://doi.org/10.1111/ijd.13423. 6. Hay RJ. Tinea capitis: current status. Mycopathologia. 2017;182(1–2):87–93. https://doi.org/10.1007/s11046-016-0058-8. 7. John AM, Schwartz RA, Janniger CK. The kerion: an angry tinea capitis. Int J Dermatol. 2018;57(1):3–9. https://doi.org/10.1111/ijd.13423. p g j 8. Mapelli ETM, Cerri A, Bombonato C, Menni S. Tinea capitis in the Paediatric population in Milan, Italy: the emergence of Trichophyton violaceum. Mycopathologia1. 2013;76(3–4):243–6. https://doi.org/10.1007/s11046-013- 9637-0. 9. Ginter-Hanselmayer G, Weger W, Ilkit M, Smolle J. Epidemiology of tinea capitis in Europe: current state and changing patterns. Mycoses. 2007;50(s2): 6–13. https://doi.org/10.1111/j.1439-0507.2007.01424.x. Page 8 of 8 Kassem et al. BMC Pediatrics (2021) 21:363 Kassem et al. BMC Pediatrics (2021) 21:363 10. Castelo-Soccio L. Diagnosis and Management of Alopecia in children. Pediatr Clin N Am. 2014;61(2):427–42. https://doi.org/10.1016/j.pcl.2013.12. 002. 11. Elghblawi E. Idiosyncratic findings in Trichoscopy of tinea capitis: comma, zigzag hairs, corkscrew, and Morse code-like hair. Int J Trichology. 2016;8(4): 180–3. https://doi.org/10.4103/ijt.ijt_92_15. 12. Conclusions Hubbard TW. The predictive value of symptoms in diagnosing childhood tinea capitis. 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https://openalex.org/W2904921539	https://acp.copernicus.org/preprints/acp-2018-1187/acp-2018-1187.pdf	English	null	Estimates of sub-national methane emissions from inversion modelling	null	2,018	cc-by	19,868	1Centre for Atmospheric Science, University of Cambridge, Cambridge, UK 2Met Office, Exeter, UK 3National Centre for Atmospheric Science (NCAS), School of Environmental Sciences, University of East Anglia Norwich, UK Norwich, UK 4 School of Geographical Sciences, University of Bristol 10 5School of Chemistry, University of Bristol, Bristol, UK 6School of GeoSciences, University of Edinburgh, Edinburgh, UK 7Department of Engineering, University of Cambridge, Cambridge, UK 8Centre for Environmental and Informatics, Cranfield University, Cranfield, UK 4 School of Geographical Sciences, University of Bristol 10 5School of Chemistry, University of Bristol, Bristol, UK 6School of GeoSciences, University of Edinburgh, Edinburgh, UK 7Department of Engineering, University of Cambridge, Cambridge, UK 8Centre for Environmental and Informatics, Cranfield University, Cranfi anow at Université Paris Saclay, Paris, 91120, France 15 bnow at Department of Civil and Environmental Engineering, Princeton University, NJ 08540, USA cnow at CSIRO, Oceans and Atmosphere, Aspendale, Australia Correspondence to: Sarah Connors (sarah.connors@universite-paris-saclay.fr) and Neil Harris (Neil.Harris@cranfield.ac.uk) Correspondence to: Sarah Connors (sarah.connors@universite-paris-saclay.fr) and Neil Harris (Neil.Harris@cranfield.ac.uk) Abstract. Methane is a strong contributor to global climate change, yet our current understanding and quantification of 20 its sources and their variability is incomplete. There is a growing need for comparisons between emission estimates produced using ‘bottom-up’ inventory approaches and ‘top-down’ inversion techniques based on atmospheric measurements, especially at higher spatial resolutions. To meet this need, this study presents using an inversion approach based on the Inversion Technique for Emissions Modelling (InTEM) framework and measurements from four sites in Abstract. Methane is a strong contributor to global climate change, yet our current understanding and quantification of 20 its sources and their variability is incomplete. There is a growing need for comparisons between emission estimates produced using ‘bottom-up’ inventory approaches and ‘top-down’ inversion techniques based on atmospheric measurements, especially at higher spatial resolutions. To meet this need, this study presents using an inversion approach based on the Inversion Technique for Emissions Modelling (InTEM) framework and measurements from four sites in East Anglia, United Kingdom. Atmospheric methane concentrations were recorded at 1-2 minute time-steps at each 25 location within the region of interest. These observations, coupled with the UK Met Office's Lagrangian particle dispersion model, NAME (Numerical Atmospheric dispersion Modelling Environment), were used within InTEM2014 to produce methane emission estimates for a 1-year period (June 2013 - May 2014) in this eastern region of the UK (~100 x 150 km) at high spatial resolution (up to 4 x 4 km). Estimates of sub-national methane emissions from inversion modelling Sarah Connors1a, Alistair J. Manning2, Andrew D. Robinson1, Stuart N. Riddick1b, Grant L. Forster3, Anita Ganesan4, Aoife Grant5, Stephen Humphrey3, Simon O’Doherty5, Dave E. Oram3, Paul I. Palmer6, Robert L. Skelton7, Kieran Stanley5, Ann Stavert5c, Dickon Young5, Neil R. P. Harris8 5 Palmer6, Robert L. Skelton7, Kieran Stanley5, Ann Stavert5c, Dickon Young5, Neil R. P. Harris8 5 1Centre for Atmospheric Science, University of Cambridge, Cambridge, UK 2Met Office, Exeter, UK 3National Centre for Atmospheric Science (NCAS), School of Environmental Sciences, University of East Anglia, Norwich, UK Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. 1. Introduction There is thus great interest in reducing its emissions and atmospheric concentrations in the near future. An essential part of this is, first, knowing what and where the emissions are (source, location, and magnitude) and, second, knowing that these emissions are reducing. Atmospheric observations are an ideal way of providing evidence for both as, through their addition into inversion methods, they can identify and quantify emission sources, which then can be monitored over time. 15 National emission inventories are produced as part of the UNFCCC process, which require nations to submit annual estimates of their GHG emissions. These contain detailed information, often available at sub-national scales, and is produced by so-called ‘bottom-up’ methods, which provide national inventories for multiple emission source sectors. The calculations involve using defined emission factors based on recommended values or field measurements together with National emission inventories are produced as part of the UNFCCC process, which require nations to submit annual estimates of their GHG emissions. These contain detailed information, often available at sub-national scales, and is produced by so-called ‘bottom-up’ methods, which provide national inventories for multiple emission source sectors. The calculations involve using defined emission factors based on recommended values or field measurements together with activity data. The UK’s National Atmospheric Emissions Inventory (NAEI - Brown et al. 2018) for methane contains 20 annually averaged estimates on a 1x1 km or 5x5 km grid resolution. Emissions are categorised into different SNAP (Selected Nomenclature for sources of Air Pollution) sectors which include ‘agric’ (SNAP 10 - agriculture, forestry and land-use change), ‘waste’ (SNAP 09 - waste treatment and disposal), and ‘offshore’ (SNAP 05 - extraction and distribution of fossil fuels). While the total uncertainty for UK methane emissions in the NAEI is estimated at 40%., the activity data. The UK’s National Atmospheric Emissions Inventory (NAEI - Brown et al. 2018) for methane contains 20 annually averaged estimates on a 1x1 km or 5x5 km grid resolution. Emissions are categorised into different SNAP (Selected Nomenclature for sources of Air Pollution) sectors which include ‘agric’ (SNAP 10 - agriculture, forestry and land-use change), ‘waste’ (SNAP 09 - waste treatment and disposal), and ‘offshore’ (SNAP 05 - extraction and distribution of fossil fuels). While the total uncertainty for UK methane emissions in the NAEI is estimated at 40%., the sub-national scale the uncertainty is much larger. The NAEI also does not include seasonal variations or natural emissions. 1. Introduction 25 Inversion, or ‘top-down’, techniques provide an alternative way of estimating GHG emissions. Emission fluxes are estimated using atmospheric measurements and a meteorological dispersion model that can simulate source to receptor dispersion. Methane emissions have been estimated using many inversion methods at global (e.g., (Bousquet et al., 2011; Houweling et al., 2014), European (e.g., Bergamaschi et al., 2005, 2018), and national (e.g., Ganesan et al., 2014; Rigby sub-national scale the uncertainty is much larger. The NAEI also does not include seasonal variations or natural emissions. 25 Inversion, or ‘top-down’, techniques provide an alternative way of estimating GHG emissions. Emission fluxes are estimated using atmospheric measurements and a meteorological dispersion model that can simulate source to receptor dispersion. Methane emissions have been estimated using many inversion methods at global (e.g., (Bousquet et al., 2011; Houweling et al., 2014), European (e.g., Bergamaschi et al., 2005, 2018), and national (e.g., Ganesan et al., 2014; Rigby et al., 2011) scales. These approaches provide an independent way of checking the national inventory totals and can assess 30 emission changes over varying timescales (Brown et al., 2018). This study, performed as part of the Natural Environment Research Council’s Greenhouse gAs UK and Global Emissions project (NERC GAUGE) project (Palmer et al., 2018), explores the possibility of producing top-down methane emission estimates on the sub-national scale to be directly compared with the 2012 NAEI. The approach taken is to make project (NERC GAUGE) project (Palmer et al., 2018), explores the possibility of producing top-down methane emission estimates on the sub-national scale to be directly compared with the 2012 NAEI. The approach taken is to make observations at four sites in Eastern England which are tens of km apart. To achieve this, three additional sites measuring 35 atmospheric methane were installed around an existing site, Tacolneston, which is part of the UK DECC (Deriving Emissions linked to Climate Change) network. Emission estimates were synthesised using a top-down sub-national inversion method developed by the UK Met Office (Manning et al., 2003, 2011). This method is a previous version of the approach known as InTEM (Inversion Technique for Emissions Modelling) as used in Arnold et al. (2018) and will observations at four sites in Eastern England which are tens of km apart. 1Centre for Atmospheric Science, University of Cambridge, Cambridge, UK 2Met Office, Exeter, UK Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. 1. Introduction Methane is a potent greenhouse gas (GHG) whose atmospheric concentration has quadrupled over the past 20,000 years and now lies well outside the variability observed over the past 800,000 years in the ice core record (Brook and Buizert, 2018). This rise became appreciable around the time of the Industrial Revolution and continues up to the present day. Methane is a potent greenhouse gas (GHG) whose atmospheric concentration has quadrupled over the past 20,000 years and now lies well outside the variability observed over the past 800,000 years in the ice core record (Brook and Buizert, 2018). This rise became appreciable around the time of the Industrial Revolution and continues up to the present day. and now lies well outside the variability observed over the past 800,000 years in the ice core record (Brook and Buizer 2018). This rise became appreciable around the time of the Industrial Revolution and continues up to the present day There is considerable dispute about what is driving the recent rise with possible causes including tropical wetland 5 expansion, increased fossil fuel emissions, and a decrease in the atmospheric removal rate (e.g., Nisbet et al., 2016; Rice et al., 2016; Rigby et al., 2017; Turner et al., 2017). Anthropogenic emissions (principally fossil fuels, agriculture and waste, and biomass burning) constitute approximately 60% of the current emissions (Saunois et al., 2016) and so reductions in methane emissions are feasible. Methane has a global warming potential of 28 over a 100 year timescale (Harris et al., 2014) and will play a vital role in 10 any attempt to limit global temperature increase to 1.5°C or even 2.0°C (e.g., Comyn-Platt et al. 2018). There is thus great interest in reducing its emissions and atmospheric concentrations in the near future. An essential part of this is, first, knowing what and where the emissions are (source, location, and magnitude) and, second, knowing that these emissions are reducing. Atmospheric observations are an ideal way of providing evidence for both as, through their addition into inversion methods, they can identify and quantify emission sources, which then can be monitored over time. 15 Methane has a global warming potential of 28 over a 100 year timescale (Harris et al., 2014) and will play a vital role in 10 any attempt to limit global temperature increase to 1.5°C or even 2.0°C (e.g., Comyn-Platt et al. 2018). 1Centre for Atmospheric Science, University of Cambridge, Cambridge, UK 2Met Office, Exeter, UK InTEM2014 was able to produce realistic emissions estimates for East East Anglia, United Kingdom. Atmospheric methane concentrations were recorded at 1-2 minute time-steps at each 25 location within the region of interest. These observations, coupled with the UK Met Office's Lagrangian particle dispersion model, NAME (Numerical Atmospheric dispersion Modelling Environment), were used within InTEM2014 to produce methane emission estimates for a 1-year period (June 2013 - May 2014) in this eastern region of the UK (~100 x 150 km) at high spatial resolution (up to 4 x 4 km). InTEM2014 was able to produce realistic emissions estimates for East Anglia, and highlighted potential areas of difference from the UK National Atmospheric Emissions Inventory (NAEI). 30 As this study was part of the UK Greenhouse gAs Uk and Global Emissions (GAUGE) project, observations were included within a national inversion using all eleven measurement sites across the UK to directly compare emission estimates for the East Anglia Region. Results show similar methane estimates for the East Anglia region. Methane emissions from Norfolk and Suffolk show good agreement with the estimates in NAEI, with differences of ~5%. Larger differences are found for Cambridgeshire where our estimate is 22.5% lower than that of NAEI. The addition of the EA 35 Anglia, and highlighted potential areas of difference from the UK National Atmospheric Emissions Inventory (NAEI). 30 As this study was part of the UK Greenhouse gAs Uk and Global Emissions (GAUGE) project, observations were included within a national inversion using all eleven measurement sites across the UK to directly compare emission estimates for the East Anglia Region. Results show similar methane estimates for the East Anglia region. Methane emissions from Norfolk and Suffolk show good agreement with the estimates in NAEI, with differences of ~5%. Larger differences are found for Cambridgeshire where our estimate is 22.5% lower than that of NAEI. The addition of the EA 35 sites within the national inversion system enabled finer spatial resolution and a decrease in the associated uncertainty for that area. Further development of our approach to include a more robust analysis of the methane concentration in the air entering this region and the uncertainty associated with the resulting emissions would strengthen this inverse method. Nonetheless, our results show there is value in high spatial resolution measurement networks and the resulting inversion emission estimates. 40 1 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. 1. Introduction The initial aim of this project was to establish a 'proof of concept' that InTEM2014 (and by implication other inversion schemes) could be used at the sub-national scale. This paper presents the preliminary findings of the work and discusses ways to improve the current setup. Section 2 describes the methodology underlying the measurements and the inverse modelling used. The results are presented and discussed in Section 3, with particular emphasis on the causes of uncertainty in the adopted approach. In addition, the 5 results of a model calculation performed as part of an inversion incorporating all of the UK measurements collected within GAUGE and DECC, i.e., with the East Anglian measurements nested within the DECC/GAUGE tall tower network, are presented as a possible way forward. 2.1.1 Sites The measurement sites for this trial project were located in East Anglia, United Kingdom. This region was chosen for three reasons: 15 a) The relatively flat topography. Turbulence in the boundary layer and low troposphere is hard to model at the high resolution required for this study. East Anglia is flat and low-lying, with a highest elevation of 146 m. Uncertainties in the small scale meteorological turbulence that is parameterised, not explicitly modelled, in the dispersion model (see Section 2.2.1) are reduced in this simpler topography compared to more heterogeneous terrains. The calculated trajectories are thus in principle more accurate than those calculated for areas of the UK with more complex topography. 20 20 b) The existence of gradients in the NAEI emissions fields across East Anglia, provides a better test of the inversion system than would a region with homogenous emissions. c) Its close proximity to Cambridge, and thus has ease of access to the measurement sites for logistical maintenance and calibration. The sites’ locations were nested within the pre-existing UK DECC tall tower network, which has since been expanded to include two new tall tower sites established under GAUGE (Stavert et al., 2018). The aim was to develop a stand-alone inversion scheme in the first instance and then to integrate these East Anglian measurements into a UK-wide inversion analysis, so the ability to link the calibration of the East Anglian and national networks was vitally important. The four measurement locations are shown in Figure 1 and some characteristics of the sites are given in Table 1. Sites 1 30 (Haddenham) and 4 (Tilney) are churches in villages away from the national gas grid and have inlets ~ 25 m above the ground to reduce the influences of local methane sources. The Weybourne Atmospheric Observatory (Site 3, hereafter Weybourne) is coastal, to the North of East Anglia, and has a 10 m mast for its inlet. Two instruments were run in tandem at the Weybourne site and the data combined to ensure data collection in case of instrument failure (Section 2.1.2). Finally, Site 2 is the tall tower measurement site at Tacolneston which has inlets at three heights, 54 m, 100 m and 185 m above 35 the ground. This study uses an average of the 54 m and 100 m observations as a method to reduce local source influences. 1. Introduction To achieve this, three additional sites measuring 35 atmospheric methane were installed around an existing site, Tacolneston, which is part of the UK DECC (Deriving Emissions linked to Climate Change) network. Emission estimates were synthesised using a top-down sub-national inversion method developed by the UK Met Office (Manning et al., 2003, 2011). This method is a previous version of the approach known as InTEM (Inversion Technique for Emissions Modelling) as used in Arnold et al. (2018) and will 2 henceforth be referred to as InTEM2014. InTEM2014 was chosen for two reasons. First, it has been used to produce annual 40 national methane emissions estimates dating back to 1990 (Manning et al., 2011) and so it provides some traceability to the national estimates. Second, we had experience in adapting it in the development of a novel method to estimate CHBr3 2 henceforth be referred to as InTEM2014. InTEM2014 was chosen for two reasons. First, it has been used to produce annual 40 national methane emissions estimates dating back to 1990 (Manning et al., 2011) and so it provides some traceability to the national estimates. Second, we had experience in adapting it in the development of a novel method to estimate CHBr3 2 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. emissions around Malaysia (Ashfold et al., 2014). The initial aim of this project was to establish a 'proof of concept' that InTEM2014 (and by implication other inversion schemes) could be used at the sub-national scale. emissions around Malaysia (Ashfold et al., 2014). The initial aim of this project was to establish a 'proof of concept' that InTEM2014 (and by implication other inversion schemes) could be used at the sub-national scale. This paper presents the preliminary findings of the work and discusses ways to improve the current setup. Section 2 describes the methodology underlying the measurements and the inverse modelling used. The results are presented and discussed in Section 3, with particular emphasis on the causes of uncertainty in the adopted approach. In addition, the results of a model calculation performed as part of an inversion incorporating all of the UK measurements collected within GAUGE and DECC, i.e., with the East Anglian measurements nested within the DECC/GAUGE tall tower network, are presented as a possible way forward. emissions around Malaysia (Ashfold et al., 2014). 2. Methodology Our approach requires two main elements: (i) calibrated measurements from the four sites; and (ii) an inversion model to 10 provide estimates of the emissions and their uncertainties. Our approach requires two main elements: (i) calibrated measurements from the four sites; and (ii) an inversion model to 10 provide estimates of the emissions and their uncertainties. 2.1 Measurements 2.1 Measurements 2.1.1 Sites 2.1.1 Sites Differences in inlet altitude amongst the observation sites were represented in the atmospheric dispersion model (Section 2.2.1). Site 2 is the tall tower measurement site at Tacolneston which has inlets at three heights, 54 m, 100 m and 185 m above 35 the ground. This study uses an average of the 54 m and 100 m observations as a method to reduce local source influences. Differences in inlet altitude amongst the observation sites were represented in the atmospheric dispersion model (Section 2.2.1). 3 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Figure 1: Map of the East of England showing site locations. 1=Haddenham, 2=Tacolneston, 3=Weybourne, 4= Tilney. (Google Maps, 2015). See also Figure 1 in Palmer et al. (2018) for location of all UK monitoring sites, which are used in Section 3.5 in this paper. 5 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Figure 1: Map of the East of England showing site locations. 1=Haddenham, 2=Tacolneston, 3=Weybourne, 4= Tilney. (Google Maps, 2015). See also Figure 1 in Palmer et al. (2018) for location of all UK monitoring sites, which are used in Section 3.5 in this paper. 5 Figure 1: Map of the East of England showing site locations. 1=Haddenham, 2=Tacolneston, 3=Weybourne, 4= Tilney. (Google Maps, 2015). See also Figure 1 in Palmer et al. (2018) for location of all UK monitoring sites, which are used in Section 3.5 in this paper. 5 Figure 1: Map of the East of England showing site locations. 1=Haddenham, 2=Tacolneston, 3=Weybourne, 4= Tilney. (Googl Maps, 2015). See also Figure 1 in Palmer et al. (2018) for location of all UK monitoring sites, which are used in Section 3.5 i this paper. Table 1: Overview table of the East Anglian measurement site information. 2.1.1 Sites Site Site name Latitude, Longitude Inlet height (m agl) Instrument Running dates 1 Holy Trinity Church, Haddenham 52.359, 0.149 25 GC-FID 06/2012-Present 2 Tacolneston tall tower 52.518, 1.139 54, 100 Picarro CRDS 07/2012-Present 3 Weybourne 52.950, 1.122 10 GC-FID UCAM GC-FID UEA 02/2013-05/2014 03/2013-05/2018 4 All Saints Church, Tilney 52.737, 0.321 25 GC-FID 06/2013-Present 2 1 2 I i Table 1: Overview table of the East Anglian measurement site information. 2.1.2 Instrumentation 15 Figure 2: Ellutia GC-FID 200 Series system flow diagram. Nitrogen carrier gas is filtered for water (W), hydrocarbons (HC), and oxygen (O) before entering the column. Hydrogen and compressed air are used to fuel the flame ionisation detector (FID). 10 Both are filtered for W and HC. A funnel filled with stainless steel-mesh (2 μm) is attached to the inlet tube, which is faced down to protect from rain and large particulates. Solenoid valves allow the GC to sample either the inlet air or the calibration gas. A pump is attached to draw in the inlet air and draw out the sample from the GC-FID. NB: SL1 = sample loop 1. ND = Nafion dryer. 15 2.1.2 Instrumentation The GC was run at an internal temperature of 100°C and a column pressure of 34 psig. This setup allowed for a fast methane elution time (< 1 minute). tubes were filtered using a desiccant-based Nafion dryer (labelled ND). The GC was run at an internal temperature of 100°C and a column pressure of 34 psig. This setup allowed for a fast methane elution time (< 1 minute). tubes were filtered using a desiccant-based Nafion dryer (labelled ND). The GC was run at an internal temperature of 100°C and a column pressure of 34 psig. This setup allowed for a fast methane elution time (< 1 minute). Samples were taken every 1-2 minutes at all GC-FID sites. The raw data were analysed using the commercially available tubes were filtered using a desiccant-based Nafion dryer (labelled ND). The GC was run at an internal temperature of 100°C and a column pressure of 34 psig. This setup allowed for a fast methane elution time (< 1 minute). Samples were taken every 1-2 minutes at all GC-FID sites. The raw data were analysed using the commercially available software ‘Igor Pro’ (WaveMetrics, 2012), which automatically detects and measures the desired peak height. Relative standard deviation (precision) values were defined half hourly. Average values showed precision to be below 0.3% (of 5 the relative standard deviations). Samples were taken every 1-2 minutes at all GC-FID sites. The raw data were analysed using the commercially available software ‘Igor Pro’ (WaveMetrics, 2012), which automatically detects and measures the desired peak height. Relative standard deviation (precision) values were defined half hourly. Average values showed precision to be below 0.3% (of 5 the relative standard deviations). Figure 2: Ellutia GC-FID 200 Series system flow diagram. Nitrogen carrier gas is filtered for water (W), hydrocarbons (HC), and oxygen (O) before entering the column. Hydrogen and compressed air are used to fuel the flame ionisation detector (FID). 10 Both are filtered for W and HC. A funnel filled with stainless steel-mesh (2 μm) is attached to the inlet tube, which is faced down to protect from rain and large particulates. Solenoid valves allow the GC to sample either the inlet air or the calibration gas. A pump is attached to draw in the inlet air and draw out the sample from the GC-FID. NB: SL1 = sample loop 1. ND = Nafion dryer. 2.1.2 Instrumentation The Tacolneston measurements were made using G2301 (Picarro Inc., USA) Cavity Ring-Down Spectrometer (Crosson, 2008) in the set-up described in Stanley et al. (2018). All other locations used gas chromatography coupled with flame 10 ionisation detectors (GC-FIDs). At these locations, a stainless steel mesh (2 μm) was fitted to the inlet tube to filter any larger impurities from damaging the GC and reducing the air flow. The Weybourne site hosted two GC-FID instruments that shared the same inlet tube. The setup below describes the GC-FID installed by University of Cambridge (GC-FID UCAM). The setup of the second GC-FID, maintained by the University of East Anglia (GC-FID UEA), can be found in Forster (2013). The two data sets are combined in this project, noting differences in sampling intervals (1-2 minutes 15 UCAM, ~20 minutes UEA). A schematic of the GC-FID instrumental setup used at Sites 1, 3 (UCAM only) and 4 is shown in Figure 2. Nitrogen was used as a carrier gas. Two other gases, pressurised air and hydrogen, were used to fuel the flame within the FID. All three gases first passed through a molecular sieve to filter out water and hydrocarbons (labelled W and HC). The nitrogen carrier gas was additionally scrubbed for oxygen to protect the column from oxidation (labelled O). Inlet and calibration 20 A schematic of the GC-FID instrumental setup used at Sites 1, 3 (UCAM only) and 4 is shown in Figure 2. Nitrogen was used as a carrier gas. Two other gases, pressurised air and hydrogen, were used to fuel the flame within the FID. All three gases first passed through a molecular sieve to filter out water and hydrocarbons (labelled W and HC). The nitrogen carrier gas was additionally scrubbed for oxygen to protect the column from oxidation (labelled O). Inlet and calibration 20 4 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. tubes were filtered using a desiccant-based Nafion dryer (labelled ND). 2.2 Inversion Modelling 2.2 Inversion Modelling 2.2 Inversion Modelling 6 1 UM vertical resolution levels decrease as the altitude increases. 2.1.3 Calibration All GC-FID sites were calibrated using an NPL calibration gas (0.28% precision). Inter-calibration experiments between our NPL-calibrated instruments (Site 1, GC-FID UCAM at Site 3, Site 4) and the NOAA-calibrated instruments (Site 2, GC-FID UEA at Site 3) showed an offset of -4.9 ppb (average of three calibration experiments). Although both the stated and derived calibration concentrations for the NPL standard were within the ranges of the calibration gas uncertainties 20 plus GC-FID precision, all NPL measurements were converted to the NOAA scale, consistent with the DECC network (Stanley et al. 2018). All GC-FID sites were calibrated using an NPL calibration gas (0.28% precision). Inter-calibration experiments between our NPL-calibrated instruments (Site 1, GC-FID UCAM at Site 3, Site 4) and the NOAA-calibrated instruments (Site 2, GC-FID UEA at Site 3) showed an offset of -4.9 ppb (average of three calibration experiments). Although both the stated and derived calibration concentrations for the NPL standard were within the ranges of the calibration gas uncertainties 20 plus GC-FID precision, all NPL measurements were converted to the NOAA scale, consistent with the DECC network (Stanley et al. 2018). 5 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. 2.2.1 NAME trajectory calculation The UK Met Office’s Numerical Atmospheric dispersion Modelling Environment (NAME) model (Jones et al., 2007) is used to estimate air flow from potential methane sources to the measurement sites. NAME was originally developed by the UK Met Office for modelling the long-range dispersion of radioactive material from nuclear power stations (Maryon 5 et al., 1991). It is a Lagrangian model which uses the 3-D meteorological fields produced by the UK Met Office’s numerical weather prediction model, the Unified Model (UM; Cullen 1993). When run backwards in time, NAME dispersion trajectories are used in the inverse modelling of atmospheric emissions (Ashfold et al., 2014; Manning et al., 2003). This project used two resolutions of UM meteorological fields: global (3 hourly, ~25 km horizontal, 8 levels in the UK Met Office for modelling the long-range dispersion of radioactive material from nuclear power stations (Maryon 5 et al., 1991). It is a Lagrangian model which uses the 3-D meteorological fields produced by the UK Met Office’s numerical weather prediction model, the Unified Model (UM; Cullen 1993). When run backwards in time, NAME dispersion trajectories are used in the inverse modelling of atmospheric emissions (Ashfold et al., 2014; Manning et al., 2003). This project used two resolutions of UM meteorological fields: global (3 hourly, ~25 km horizontal, 8 levels in the lowest 500 m vertical1) and UK (hourly, ~1.5 km horizontal, 12 levels in the lowest 500m vertical), the 1.5 km UK 10 fields were nested within the global data when running NAME. The model setup at each site is identical except for the particle-source release location (latitude, longitude) and height (m above ground level). The three sites with inlets between 15-27 m (1-Haddenham, 3-Weybourne and 4-Tilney) have a modelled release altitude of 25 m (±25 m) above sea level. Tacolneston (Site 2), with inlets at 54 m and 100 m is assigned a release altitude of 75 m (±25 m) 15 The model setup at each site is identical except for the particle-source release location (latitude, longitude) and height (m above ground level). The three sites with inlets between 15-27 m (1-Haddenham, 3-Weybourne and 4-Tilney) have a modelled release altitude of 25 m (±25 m) above sea level. Tacolneston (Site 2), with inlets at 54 m and 100 m is assigned a release altitude of 75 m (±25 m). 15 a release altitude of 75 m (±25 m). 2.2.1 NAME trajectory calculation 15 NAME produces a modelled representation of the contributing ‘surface influence’ (defined as the lowest 100 m above ground level in NAME) at a particular location (one of the measurement sites) by releasing chemically inert particles (10,000 h-1) from the x, y, z coordinate of that measurement site. NAME computes the movements and geolocation of each particle every minute for 5 days backwards in time. Each location releases mass at a rate of 1 g s-1 equally distributed across the particles. A time integrated particle density map (units g s m-3; resolution 1.5 × 1.5 km) is produced for each 20 measurement location that shows, on a gridded output, the relative contribution that each grid square has made over the preceding 5-day period (Manning et al., 2011). After conversion, the resulting metric (units of s m-1) can be described as the mean time that particles reside in each grid cell for a 1 hour particle release period. This metric corresponds to the multiplying factor by which emissions are diluted from their initial source to being monitored at the measurement location. across the particles. A time integrated particle density map (units g s m-3; resolution 1.5 × 1.5 km) is produced for each 20 measurement location that shows, on a gridded output, the relative contribution that each grid square has made over the preceding 5-day period (Manning et al., 2011). After conversion, the resulting metric (units of s m-1) can be described as the mean time that particles reside in each grid cell for a 1 hour particle release period. This metric corresponds to the multiplying factor by which emissions are diluted from their initial source to being monitored at the measurement location. This relationship is given in Equation 1 and a dilution map for the measurement site Haddenham is shown in Figure 3. 25 Dilution maps are calculated hourly at each measurement location over the monitoring period (of two years). Results are compiled into a ‘dilution matrix’, which shows how the dilution values changed over time, and is an input into the inversion system. This relationship is given in Equation 1 and a dilution map for the measurement site Haddenham is shown in Figure 3. 25 Dilution maps are calculated hourly at each measurement location over the monitoring period (of two years). 2.2.1 NAME trajectory calculation Each location releases particles at a rate of 1 g s-1, resulting in a time integrated particle density map (units g s m-3; resolution 1.5 × 1.5 km) that shows the relative contribution of each grid square over the preceding 5-day period (Manning et al., 2011). A conversion to the ‘dilution matrix’ (units of s m-1) can be described as the mean time that particles reside in each grid cell given a 1 hour particle release period. 10 Figure 3: Mean dilution matrix for the Haddenham site (Site 1, location marked with an X) for 2013 and 2014, as calculated by the NAME particle dispersion model. 3-D meteorological fields produced by the UK Met Office’s numerical weather prediction model, at 1.5 km regional resolution nested within 25 km global resolution are used when running NAME. Sources 5 are released for a one hour duration period, every hour from 01 January 2013 to 31 December 2014. Particles’ geolocation is calculated every minute for 5 days backwards in time. Each location releases particles at a rate of 1 g s-1, resulting in a time integrated particle density map (units g s m-3; resolution 1.5 × 1.5 km) that shows the relative contribution of each grid square over the preceding 5-day period (Manning et al., 2011). A conversion to the ‘dilution matrix’ (units of s m-1) can be described as the mean time that particles reside in each grid cell given a 1 hour particle release period. 10 prediction model, at 1.5 km regional resolution nested within 25 km global resolution are used when running NAME. So 5 are released for a one hour duration period, every hour from 01 January 2013 to 31 December 2014. Particles’ geoloca calculated every minute for 5 days backwards in time. Each location releases particles at a rate of 1 g s-1, resulting in integrated particle density map (units g s m-3; resolution 1.5 × 1.5 km) that shows the relative contribution of each grid s over the preceding 5-day period (Manning et al., 2011). A conversion to the ‘dilution matrix’ (units of s m-1) can be descri the mean time that particles reside in each grid cell given a 1 hour particle release period. 10 2.2.1 NAME trajectory calculation Results are compiled into a ‘dilution matrix’, which shows how the dilution values changed over time, and is an input into the inversion system. The model domain limits shown in Figure 3 are centred on East Anglia but span most of the south east of England With The model domain limits, shown in Figure 3, are centred on East Anglia but span most of the south east of England. With the model particle lifetime set at five days, this is long enough for the vast majority of particles to leave the domain of 30 interest, and thus capture all surface influence within the geographical domain. , g , g p g the model particle lifetime set at five days, this is long enough for the vast majority of particles to leave the domain of 30 interest, and thus capture all surface influence within the geographical domain. the model particle lifetime set at five days, this is long enough for the vast majority of particles to leave the domain of 30 interest, and thus capture all surface influence within the geographical domain. 6 6 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Figure 3: Mean dilution matrix for the Haddenham site (Site 1, location marked with an X) for 2013 and 2014, as calculated by the NAME particle dispersion model. 3-D meteorological fields produced by the UK Met Office’s numerical weather prediction model, at 1.5 km regional resolution nested within 25 km global resolution are used when running NAME. Sources 5 are released for a one hour duration period, every hour from 01 January 2013 to 31 December 2014. Particles’ geolocation is calculated every minute for 5 days backwards in time. Each location releases particles at a rate of 1 g s-1, resulting in a time integrated particle density map (units g s m-3; resolution 1.5 × 1.5 km) that shows the relative contribution of each grid square over the preceding 5-day period (Manning et al., 2011). A conversion to the ‘dilution matrix’ (units of s m-1) can be described as the mean time that particles reside in each grid cell given a 1 hour particle release period. 10 Atmos. Chem. 2.2.1 NAME trajectory calculation Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Figure 3: Mean dilution matrix for the Haddenham site (Site 1, location marked with an X) for 2013 and 2014, as calculated by the NAME particle dispersion model. 3-D meteorological fields produced by the UK Met Office’s numerical weather prediction model, at 1.5 km regional resolution nested within 25 km global resolution are used when running NAME. Sources 5 are released for a one hour duration period, every hour from 01 January 2013 to 31 December 2014. Particles’ geolocation is calculated every minute for 5 days backwards in time. Each location releases particles at a rate of 1 g s-1, resulting in a time integrated particle density map (units g s m-3; resolution 1.5 × 1.5 km) that shows the relative contribution of each grid square over the preceding 5-day period (Manning et al., 2011). A conversion to the ‘dilution matrix’ (units of s m-1) can be described as the mean time that particles reside in each grid cell given a 1 hour particle release period. 10 Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 ipt under review for journal Atmos. Chem. Phys. on started: 18 December 2018 or(s) 2018. CC BY 4.0 License. Figure 3: Mean dilution matrix for the Haddenham site (Site 1, location marked with an X) for 2013 and 2014, as calcu Figure 3: Mean dilution matrix for the Haddenham site (Site 1, location marked with an X) for 2013 and 2014, as calculated by the NAME particle dispersion model. 3-D meteorological fields produced by the UK Met Office’s numerical weather prediction model, at 1.5 km regional resolution nested within 25 km global resolution are used when running NAME. Sources 5 are released for a one hour duration period, every hour from 01 January 2013 to 31 December 2014. Particles’ geolocation is calculated every minute for 5 days backwards in time. 2.2.2 InTEM2014 inversion model 7 InTEM2014 uses methane measurements and the corresponding dilution maps to estimate emissions within a given domain according to the relationship expressed in Equation 1. 15 emission (g s-1 m-2) x dilution (s m-1) = concentration (g m-3) (1) Through an iterative process known as simulated annealing (Manning et al., 2003), pseudo-observations calculated from simulated emissions fields for specific times and locations are quantitatively compared with the measured observations using cost functional analysis. The resulting InTEM2014 emissions estimate will be the emissions field with the lowest 20 cost score. Pseudo-observations are calculated by multiplying the emission estimates (from the 2012 NAEI) for each grid cell by their corresponding dilution value (taken from NAME) at each timestep. A least-squares cost function used (Equation 2) to quantitatively compare the two observational time series, similar to cost functions used in work such as Manning et al. (2003), Ashfold et al. (2014), and (Fang et al., 2016). Bayesian approaches have become more regularly used in top-down 25 emission estimates, which incorporate pre-defined uncertainty estimates that result in calculated uncertainties for the final emissions (e.g., Arnold et al., 2018; Bousquet et al., 2011; Feng et al., 2018). Hierarchical Bayesian cost functions have since been developed where both the uncertainty values associated with the prior estimates, and the model uncertainty estimates can be derived within the inversion itself (Ganesan et al., 2014; Lunt et al., 2016). Estimating realistic and rigorously derived emission uncertainties remains a major challenge in inversion studies. 30 InTEM2014 uses methane measurements and the corresponding dilution maps to estimate emissions within a given domain according to the relationship expressed in Equation 1. 15 emission (g s-1 m-2) x dilution (s m-1) = concentration (g m-3) (1) (1) Pseudo-observations are calculated by multiplying the emission estimates (from the 2012 NAEI) for each grid cell by their corresponding dilution value (taken from NAME) at each timestep. A least-squares cost function used (Equation 2) to quantitatively compare the two observational time series, similar to cost functions used in work such as Manning et al. (2003), Ashfold et al. (2014), and (Fang et al., 2016). Bayesian approaches have become more regularly used in top-down 25 emission estimates, which incorporate pre-defined uncertainty estimates that result in calculated uncertainties for the final emissions (e.g., Arnold et al., 2018; Bousquet et al., 2011; Feng et al., 2018). 2.2.2 InTEM2014 inversion model Observational uncertainty is defined as the sum of the hourly instrument precision, the calibration gas uncertainty, and the standard deviations of the hourly concentrations plus 5 ppb. The value of 5 ppb is an estimate of the 10 uncertainty in the baseline value in any given hour. Dividing by the total uncertainty (i.e., variance) de-weights uncertain observations. The lower the resulting cost score the smaller the difference between the pseudo and measured observations, implying a more accurate emissions estimate than one with a higher cost score. The simulated annealing method in InTEM2014 iteratively converges on the best solution and the final result is limited by The simulated annealing method in InTEM2014 iteratively converges on the best solution and the final result is limited b the available computer resource. Therefore, the derived final output is close to but may not be the best possible solution, 15 i.e., with the lowest possible cost score. For this reason, and due to the stochastic nature of the convergence within the simulated annealing process, the InTEM2014 runs were repeated multiple times and the resulting emission results averaged. Sensitivity analyses showed that 25 repeats were sufficient to produce consistent methane emission estimates, standard deviations and cost scores. the available computer resource. Therefore, the derived final output is close to but may not be the best possible solution, 15 i.e., with the lowest possible cost score. For this reason, and due to the stochastic nature of the convergence within the simulated annealing process, the InTEM2014 runs were repeated multiple times and the resulting emission results averaged. Sensitivity analyses showed that 25 repeats were sufficient to produce consistent methane emission estimates, standard deviations and cost scores. the available computer resource. Therefore, the derived final output is close to but may not be the best possible solution, 15 i.e., with the lowest possible cost score. For this reason, and due to the stochastic nature of the convergence within the simulated annealing process, the InTEM2014 runs were repeated multiple times and the resulting emission results averaged. Sensitivity analyses showed that 25 repeats were sufficient to produce consistent methane emission estimates, standard deviations and cost scores. 2.2.3 A priori emission estimates 20 Unlike Bayesian inversions, InTEM2014 does not use a prescribed a priori emission estimate, which includes boundaries to the emission magnitudes based on uncertainty assumptions. In this study, we used a random, non-negative emission field, which assumes no a priori knowledge at the location of emissions. Unlike Bayesian inversions, InTEM2014 does not use a prescribed a priori emission estimate, which includes boundaries to the emission magnitudes based on uncertainty assumptions. In this study, we used a random, non-negative emission field, which assumes no a priori knowledge at the location of emissions. 2.2.2 InTEM2014 inversion model Hierarchical Bayesian cost functions have since been developed where both the uncertainty values associated with the prior estimates, and the model uncertainty estimates can be derived within the inversion itself (Ganesan et al., 2014; Lunt et al., 2016). Estimating realistic and rigorously derived emission uncertainties remains a major challenge in inversion studies. 30 (2003), Ashfold et al. (2014), and (Fang et al., 2016). Bayesian approaches have become more regularly used in top-down 25 emission estimates, which incorporate pre-defined uncertainty estimates that result in calculated uncertainties for the final emissions (e.g., Arnold et al., 2018; Bousquet et al., 2011; Feng et al., 2018). Hierarchical Bayesian cost functions have since been developed where both the uncertainty values associated with the prior estimates, and the model uncertainty estimates can be derived within the inversion itself (Ganesan et al., 2014; Lunt et al., 2016). Estimating realistic and rigorously derived emission uncertainties remains a major challenge in inversion studies. 30 7 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. The cost function used here incorporates defined uncertainties associated with the observations and the model but does not include the use of a prior (see Section 2.2.3). (2) Ki is the forward model and xi is the measured concentration at a particular timestep (i). At all timesteps the difference between the pseudo-observation (Kxi) and the measured observation (yi) is squared and then divided by the uncertainty variance (𝜎𝜀 2)𝑖. This uncertainty is the sum of all assumed errors in observations, modelling and baselines for each hourly timestep (i). Observational uncertainty is defined as the sum of the hourly instrument precision, the calibration gas Ki is the forward model and xi is the measured concentration at a particular timestep (i). At all timesteps the difference between the pseudo-observation (Kxi) and the measured observation (yi) is squared and then divided by the uncertainty variance (𝜎𝜀 2)𝑖. This uncertainty is the sum of all assumed errors in observations, modelling and baselines for each hourly timestep (i). 2.2.4 Solution grid The methane emission estimates are resolved on a more spatially coarse grid than the NAME model output to reduce the 25 computational cost and decrease modelling uncertainties (Manning et al., 2003). This so-called ‘solution grid’, of which the a posteriori estimate is resolved, is irregular and is constructed using the dilution matrix (Section 2.2.1) and the 2012 NAEI for methane, but the NAME output grid can first be divided into broader regions to calculate emission totals. These regions are based on the East Anglia county boundaries, providing rough county-wide estimates of methane emitted over The methane emission estimates are resolved on a more spatially coarse grid than the NAME model output to reduce the 25 computational cost and decrease modelling uncertainties (Manning et al., 2003). This so-called ‘solution grid’, of which the a posteriori estimate is resolved, is irregular and is constructed using the dilution matrix (Section 2.2.1) and the 2012 NAEI for methane, but the NAME output grid can first be divided into broader regions to calculate emission totals. These regions are based on the East Anglia county boundaries, providing rough county-wide estimates of methane emitted over the given period of time. The solution grid resolution is a sub-division of these county areas, which has a spatial resolution 30 that is between the county-based starting regions and the NAME grid. The solution grid resolution is determined through two factors: the dilution matrix and the NAEI methane emissions values. the given period of time. The solution grid resolution is a sub-division of these county areas, which has a spatial resolution 30 that is between the county-based starting regions and the NAME grid. The solution grid resolution is determined through two factors: the dilution matrix and the NAEI methane emissions values. the given period of time. The solution grid resolution is a sub-division of these county areas, which has a spatial resolution 30 that is between the county-based starting regions and the NAME grid. The solution grid resolution is determined through two factors: the dilution matrix and the NAEI methane emissions values. For the dilution grid matrix, areas where trajectories spent relatively long periods of time will have a finer spatial resolution, as more data are available and thus there is greater sensitivity to resolve the emissions from that area. 3. Results This section covers results from the InTEM2014 inversion analysis. For analysis of the concentrations from the measurement sites, including their daily, weekly and intra-annual variability, please refer to Figure 3 in Palmer et al. (2018). 2.2.4 Solution grid Numbering refers to counties or other more arbitrary areas: 1= Norfolk; 2= Suffolk; 3= Cambridgeshire; 4 = London; 5 = Essex; 6 = Lincolnshire; 7 = Buckinghamshire; 8 = South west area. Figure 4: A) Map showing the starting resolution of the inversion. These regions are loosely based on the county regions in East Anglia. As an output, InTEM provides statistical information on the emissions for each of these regions. B) Map showing the 5 solution grid resolution. This grid resolution is computed based on information from the dilution matrix (Figure 3) and the 2012 NAEI for methane (Brown et al. 2018). Numbering refers to counties or other more arbitrary areas: 1= Norfolk; 2= Suffolk; 3= Cambridgeshire; 4 = London; 5 = Essex; 6 = Lincolnshire; 7 = Buckinghamshire; 8 = South west area. 2.2.5 Baseline A baseline that represents the atmospheric methane concentration arriving at the edge of the inversion domain (Figure 4) 10 must be defined within InTEM2014. For this study, a statistical baseline was calculated from the measured observations that also incorporated the particle trajectory analysis from NAME. Methane concentrations from the four measurement sites were divided into time series depending on whether their trajectory origin was dominated by a certain direction (e.g., from the NNE, ENE, ESE, NNW etc.). These eight individual time series, representing concentrations from the eight different compass directions, were used to estimate eight statistically derived baselines, each calculated by passing a 15 rolling 18th percentile, spanning one week, through each dataset. The 18th percentile is chosen from a sensitivity analysis, in which the rolling percentile was varied from the 5th to the 45th percentile. The 18th percentile produces emission results with consistently stable emissions and with the lowest cost score of all the baselines tested. Baselines for the four measurement sites were then created using the NAME trajectory analysis to weight a combination of the eight direction dependent baselines. 20 A baseline that represents the atmospheric methane concentration arriving at the edge of the inversion domain (Figure 4) 10 must be defined within InTEM2014. For this study, a statistical baseline was calculated from the measured observations that also incorporated the particle trajectory analysis from NAME. Methane concentrations from the four measurement sites were divided into time series depending on whether their trajectory origin was dominated by a certain direction (e.g., from the NNE, ENE, ESE, NNW etc.). These eight individual time series, representing concentrations from the eight different compass directions, were used to estimate eight statistically derived baselines, each calculated by passing a 15 rolling 18th percentile, spanning one week, through each dataset. The 18th percentile is chosen from a sensitivity analysis, in which the rolling percentile was varied from the 5th to the 45th percentile. The 18th percentile produces emission results with consistently stable emissions and with the lowest cost score of all the baselines tested. Baselines for the four measurement sites were then created using the NAME trajectory analysis to weight a combination of the eight direction dependent baselines 20 dependent baselines. 20 2.2.4 Solution grid Numbering refers to counties or other more arbitrary areas: 1= Norfolk; 2= Suffolk; 3= Cambridgeshire; 4 = London; 5 = Essex; 6 = Lincolnshire; 7 = Buckinghamshire; 8 = South west area. 2 2 5 Baseline hem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 pt under review for journal Atmos. Chem. Phys. n started: 18 December 2018 r(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. High methane emission sources in the NAEI (e.g., landfills) are more finely resolved than low methane emission areas. The resulting solution grid resolution can be seen in Figure 4. The resulting solution grid resolution can be seen in Figure 4. A) B) Figure 4: A) Map showing the starting resolution of the inversion. These regions are loosely based on the county regions in East Anglia. As an output, InTEM provides statistical information on the emissions for each of these regions. B) Map showing the 5 solution grid resolution. This grid resolution is computed based on information from the dilution matrix (Figure 3) and the 2012 NAEI for methane (Brown et al. 2018). Numbering refers to counties or other more arbitrary areas: 1= Norfolk; 2= Suffolk; 3= Cambridgeshire; 4 = London; 5 = Essex; 6 = Lincolnshire; 7 = Buckinghamshire; 8 = South west area. 2 2 5 B li A) B ) B) Figure 4: A) Map showing the starting resolution of the inversion. These regions are loosely based on the county regio Figure 4: A) Map showing the starting resolution of the inversion. These regions are loosely based on the county regions in East Anglia. As an output, InTEM provides statistical information on the emissions for each of these regions. B) Map showing the 5 solution grid resolution. This grid resolution is computed based on information from the dilution matrix (Figure 3) and the 2012 NAEI for methane (Brown et al. 2018). 2.2.4 Solution grid A pre- For the dilution grid matrix, areas where trajectories spent relatively long periods of time will have a finer spatial resolution, as more data are available and thus there is greater sensitivity to resolve the emissions from that area. A pre- resolution, as more data are available and thus there is greater sensitivity to resolve the emissions from that area. A pre- defined dilution threshold subdivides regions into finer grids based on the dilution matrix (Manning et al., 2011). 35 Generally, areas nearer to the measurement sites are more finely resolved than more distant areas. Complementing this, the NAEI methane emissions are also incorporated to define the solution grid resolution. The NAEI emission magnitudes are used as a linear scaling factor to define the grid resolution (as in other inversion techniques such as Rigby et al. (2011). defined dilution threshold subdivides regions into finer grids based on the dilution matrix (Manning et al., 2011). 35 Generally, areas nearer to the measurement sites are more finely resolved than more distant areas. Complementing this, the NAEI methane emissions are also incorporated to define the solution grid resolution. The NAEI emission magnitudes are used as a linear scaling factor to define the grid resolution (as in other inversion techniques such as Rigby et al. (2011). defined dilution threshold subdivides regions into finer grids based on the dilution matrix (Manning et al., 2011). 35 Generally, areas nearer to the measurement sites are more finely resolved than more distant areas. Complementing this, the NAEI methane emissions are also incorporated to define the solution grid resolution. The NAEI emission magnitudes are used as a linear scaling factor to define the grid resolution (as in other inversion techniques such as Rigby et al. (2011). 8 High methane emission sources in the NAEI (e.g., landfills) are more finely resolved than low methane emission areas. The resulting solution grid resolution can be seen in Figure 4. A) B) Figure 4: A) Map showing the starting resolution of the inversion. These regions are loosely based on the county regions in East Anglia. As an output, InTEM provides statistical information on the emissions for each of these regions. B) Map showing the 5 solution grid resolution. This grid resolution is computed based on information from the dilution matrix (Figure 3) and the 2012 NAEI for methane (Brown et al. 2018). 3.2 InTEM2014 emission estimate results 25 9 The following results were derived using a one year dataset from all four sites covering the period from June 2013 to May 2014 (inclusive) with hourly observations. This period was chosen as it marks the first full year where all four sites were operational. A subset of the resulting measured and pseudo-observations for 01-30 July 2013 is shown in Figure 5. The equivalent 2012 NAEI pseudo-observations are included for comparison. From the measured observations, it is clear that Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. the Haddenham and Tilney sites observe short periods of elevated methane, usually during nocturnal hours when the boundary layer height is low, which suggests the presence of local methane sources. Several landfill sites can be found close to Haddenham and Tilney (<10 km) which can be large point sources of methane (NAEI, Brown et al., 2018). Isotopic analysis by Riddick et al. (2017), confirmed a methane signal from the Waterbeach Waste Management Park being present at the Haddenham site at times of elevated methane 5 being present at the Haddenham site at times of elevated methane. 5 Figure 5 shows a large fraction of the a posteriori estimates lie outside the uncertainty range of the measured observations, although without a more thorough description of a priori errors this is difficult to fully diagnose. As it stands, this implies that either the prescribed InTEM2014 uncertainties are too small or the resulting emission field needs be more resolved (in time and space) in order to better represent the concentrations being measured (currently, the spatial resolution of the Figure 5 shows a large fraction of the a posteriori estimates lie outside the uncertainty range of the measured observations, although without a more thorough description of a priori errors this is difficult to fully diagnose. As it stands, this implies that either the prescribed InTEM2014 uncertainties are too small or the resulting emission field needs be more resolved (in time and space) in order to better represent the concentrations being measured (currently, the spatial resolution of the resulting emission grid could be too coarse to fully capture the peaks and troughs of the measured time series). 3.2 InTEM2014 emission estimate results 25 It should 10 be noted that the pseudo-observations calculated using the NAEI are substantially outside the observation uncertainty ranges, and that neither are able to replicate the high concentrations measured at the Haddenham and Tilney sites. A scatterplot of a posteriori enhancements vs. observed enhancements as calculated by InTEM2014 at the Haddenham site can be found in Riddick et al 2017 (Figure SM2.2). The InTEM2014 resulting emissions field has a lower (i.e., better) cost score than the emissions grid from the NAEI (12.5 compared to 14.9). For comparison, the 2009 NAEI emissions grid 15 yielded a higher cost score of 15.8, showing that the methane emissions distribution produced by InTEM2014 fits the measured observations better than the 2009 or the 2012 NAEI. resulting emission grid could be too coarse to fully capture the peaks and troughs of the measured time series). It should 10 be noted that the pseudo-observations calculated using the NAEI are substantially outside the observation uncertainty ranges, and that neither are able to replicate the high concentrations measured at the Haddenham and Tilney sites. A scatterplot of a posteriori enhancements vs. observed enhancements as calculated by InTEM2014 at the Haddenham site can be found in Riddick et al 2017 (Figure SM2.2). The InTEM2014 resulting emissions field has a lower (i.e., better) cost score than the emissions grid from the NAEI (12.5 compared to 14.9). For comparison, the 2009 NAEI emissions grid 15 yielded a higher cost score of 15.8, showing that the methane emissions distribution produced by InTEM2014 fits the measured observations better than the 2009 or the 2012 NAEI. resulting emission grid could be too coarse to fully capture the peaks and troughs of the measured time series). It should 10 be noted that the pseudo-observations calculated using the NAEI are substantially outside the observation uncertainty ranges, and that neither are able to replicate the high concentrations measured at the Haddenham and Tilney sites. A scatterplot of a posteriori enhancements vs. observed enhancements as calculated by InTEM2014 at the Haddenham site can be found in Riddick et al 2017 (Figure SM2.2). The InTEM2014 resulting emissions field has a lower (i.e., better) cost resulting emission grid could be too coarse to fully capture the peaks and troughs of the measured time series). 3.2 InTEM2014 emission estimate results 25 It should 10 be noted that the pseudo-observations calculated using the NAEI are substantially outside the observation uncertainty ranges, and that neither are able to replicate the high concentrations measured at the Haddenham and Tilney sites. A scatterplot of a posteriori enhancements vs. observed enhancements as calculated by InTEM2014 at the Haddenham site can be found in Riddick et al 2017 (Figure SM2.2). The InTEM2014 resulting emissions field has a lower (i.e., better) cost score than the emissions grid from the NAEI (12.5 compared to 14.9). For comparison, the 2009 NAEI emissions grid 15 yielded a higher cost score of 15.8, showing that the methane emissions distribution produced by InTEM2014 fits the measured observations better than the 2009 or the 2012 NAEI. Figure 5: Time series of measured and a posteriori modelled methane (using inversion estimated emission distribution) mole 20 fractions for all 4 observations sites for 01-30 July 2013. The equivalent pseudo-observations calculated using the 2012 NAEI emissions inventory (Brown et al. 2018) have been added for comparison. Figure 5: Time series of measured and a posteriori modelled methane (using inversion estimated emission distribution) mole 20 fractions for all 4 observations sites for 01-30 July 2013. The equivalent pseudo-observations calculated using the 2012 NAEI emissions inventory (Brown et al. 2018) have been added for comparison. Figure 5: Time series of measured and a posteriori modelled methane (using inversion estimated emission distribution) mole 20 fractions for all 4 observations sites for 01-30 July 2013. The equivalent pseudo-observations calculated using the 2012 NAEI emissions inventory (Brown et al. 2018) have been added for comparison. 10 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. 3.2.1 Regionality Table 2 shows the inversion area emission totals (labelled in Figure 4). The East Anglian areas are loosely based on the 10 UK counties (Suffolk - 4, Norfolk - 10 and Cambridgeshire - 15). A positive relationship between area standard deviations and the distance from the measurement sites can be seen in Table 2. For example, the areas close to London and in the south west of the regions have standard deviations of 18.0 and 59.2, respectively, but areas representing the east Anglian counties are all below a standard deviation of 2.5. This implies that InTEM2014 is able to more robustly resolve emission totals for areas close to measurement sites, although individual site biases apply (see Section 3.4). This analysis implies 15 that the ~15-25 m a.g.l. EA measurement sites have effective footprints of roughly a 50 km radius. Our estimates for methane emissions from Norfolk and Suffolk show good agreement with the estimates in NAEI, with differences of ~5%. Larger differences are found for Cambridgeshire where our estimate is 22.5% lower than that of NAEI. Percentage differences for regions that are further away from the measurement sites range from 10.8% (region 11, London area) to 66.1% (region 1, south west area). All land area estimates are within a factor of two of the NAEI. 20 Table 2 shows the inversion area emission totals (labelled in Figure 4). The East Anglian areas are loosely based on the 10 UK counties (Suffolk - 4, Norfolk - 10 and Cambridgeshire - 15). A positive relationship between area standard deviations and the distance from the measurement sites can be seen in Table 2. For example, the areas close to London and in the south west of the regions have standard deviations of 18.0 and 59.2, respectively, but areas representing the east Anglian counties are all below a standard deviation of 2.5. This implies that InTEM2014 is able to more robustly resolve emission Table 2 shows the inversion area emission totals (labelled in Figure 4). The East Anglian areas are loosely based on the 10 UK counties (Suffolk - 4, Norfolk - 10 and Cambridgeshire - 15). A positive relationship between area standard deviations and the distance from the measurement sites can be seen in Table 2. 3.2.1 Regionality Figure 6 shows the inversion emission map and the 2012 NAEI methane emission map, both plotted to the same spatial resolution (see Section 2.2.4: Solution grid). The inversion produces comparable emissions estimates for East Anglia with total estimates being within 15% of the NAEI (NAEI estimates 280 kt yr-1, InTEM2014 estimates 310 ±63.0 kt yr-1, rounded to 2 s.f.). Similarities between the spatiality of emission are visible, with both maps showing large emissions in the London 5 area, point sources around Haddenham, and lower emissions along the southern East Anglian coast. Discrepancies appear b f h i d i h fi l l d i i b l l di i ddi i l Figure 6 shows the inversion emission map and the 2012 NAEI methane emission map, both plotted to the same spatial resolution (see Section 2.2.4: Solution grid). The inversion produces comparable emissions estimates for East Anglia with total estimates being within 15% of the NAEI (NAEI estimates 280 kt yr-1, InTEM2014 estimates 310 ±63.0 kt yr-1, rounded to 2 s.f.). Similarities between the spatiality of emission are visible, with both maps showing large emissions in the London 5 area, point sources around Haddenham, and lower emissions along the southern East Anglian coast. Discrepancies appear between some of the magnitudes in the finely resolved emissions maps, but local studies using additional measurements, Gaussian plume and WindTrax modelling do show that the high point source emissions near Haddenham are real (Riddick et al., 2017). to 2 s.f.). Similarities between the spatiality of emission are visible, with both maps showing large emissions in the London 5 area, point sources around Haddenham, and lower emissions along the southern East Anglian coast. Discrepancies appear between some of the magnitudes in the finely resolved emissions maps, but local studies using additional measurements, Gaussian plume and WindTrax modelling do show that the high point source emissions near Haddenham are real (Riddick et al., 2017). to 2 s.f.). Similarities between the spatiality of emission are visible, with both maps showing large emissions in the London 5 area, point sources around Haddenham, and lower emissions along the southern East Anglian coast. Discrepancies appear between some of the magnitudes in the finely resolved emissions maps, but local studies using additional measurements, Gaussian plume and WindTrax modelling do show that the high point source emissions near Haddenham are real (Riddick et al., 2017). 3.2.1 Regionality For example, the areas close to London and in the south west of the regions have standard deviations of 18.0 and 59.2, respectively, but areas representing the east Anglian counties are all below a standard deviation of 2.5. This implies that InTEM2014 is able to more robustly resolve emission totals for areas close to measurement sites, although individual site biases apply (see Section 3.4). This analysis implies 15 that the ~15-25 m a.g.l. EA measurement sites have effective footprints of roughly a 50 km radius. Our estimates for methane emissions from Norfolk and Suffolk show good agreement with the estimates in NAEI, with differences of ~5%. Larger differences are found for Cambridgeshire where our estimate is 22.5% lower than that of NAEI. Percentage differences for regions that are further away from the measurement sites range from 10.8% (region 11, London area) to 66.1% (region 1, south west area). All land area estimates are within a factor of two of the NAEI. 20 Compared to the NAEI, InTEM2014 emissions have a 'dipole effect' in some areas. For example, a large methane source is shown to the south west of Tacolneston but low emissions are estimated in the surrounding area. The NAEI also shows an increased emission level south west of Tacolneston, but the overall emission ranges are less extreme. It is unclear if these dipoles are 'true' signals, or a product of InTEM2014's inability to fully resolve emissions on this spatial and temporal scale. Differentiating between false dipoles and real point sources is not straightforward in this analysis. Intermittent 25 source emissions, as well as uncertainty in the meteorological analyses used to run NAME could account for InTEM2014 being unable to pinpoint some emission sources. In principle, this could be overcome by introducing point sources into the priori, as used in some Bayesian approaches (Rigby et al., 2017). 30 11 11 A) InTEM2014 B) NAEI Figure 6: A) Methane emission map, from June 2013 to May 2014, produced by an InTEM inversion run using all 4 sites’ observational data. B) The 2012 NAEI (Brown et al. 2018) re-gridded to the inversion grid resolution (see Figure 5). Sites are labelled for reference: HD = Haddenham, TN = Tacolneston, WY = Weybourne, TY = Tilney. NB: Logarithmic colour scale. Difference between orange / red is roughly a factor of 100 larger than the difference between blue / green. 5 Atmos. Chem. Phys. 3.2.1 Regionality Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. A) InTEM2014 B) NAEI Figure 6: A) Methane emission map, from June 2013 to May 2014, produced by an InTEM inversion run using all 4 sites’ observational data. B) The 2012 NAEI (Brown et al. 2018) re-gridded to the inversion grid resolution (see Figure 5). Sites are labelled for reference: HD = Haddenham, TN = Tacolneston, WY = Weybourne, TY = Tilney. NB: Logarithmic colour scale. Difference between orange / red is roughly a factor of 100 larger than the difference between blue / green. 5 A) InTEM2014 B) NAEI Figure 6: A) Methane emission map, from June 2013 to May 2014, produced by an InTEM inversion run using all 4 sites’ observational data. B) The 2012 NAEI (Brown et al. 2018) re-gridded to the inversion grid resolution (see Figure 5). Sites are labelled for reference: HD = Haddenham, TN = Tacolneston, WY = Weybourne, TY = Tilney. NB: Logarithmic colour scale. Difference between orange / red is roughly a factor of 100 larger than the difference between blue / green. Table 2: Emission totals (kt yr-1) resulting from InTEM inversion using all 4 observational site data for the period June 2013 to May 2014. Emission totals are for ‘regions’ shown in Figure 5. Equivalent totals of the 2012 NAEI (Brown et al. 2018) per region and their differences as percentages are shown for comparison. One standard deviation (1.s.d.) is shown below regional estimates. Sea regions from Figure 5 not shown here but emissions totals were 0.4 kt yr-1 (0.64 1.s.d.) compared to 0.7 kt yr-1 from the NAEI (57.1% difference). Table 2: Emission totals (kt yr-1) resulting from InTEM inversion using all 4 observational site data for the period June 2013 to May 2014. Emission totals are for ‘regions’ shown in Figure 5. Equivalent totals of the 2012 NAEI (Brown et al. 3.2.1 Regionality 2018) per region and their differences as percentages are shown for comparison. One standard deviation (1.s.d.) is shown below regional estimates. Sea regions from Figure 5 not shown here but emissions totals were 0.4 kt yr-1 (0.64 1.s.d.) compared to 0.7 kt yr-1 from the NAEI (57.1% difference). 0 # NAEI InTEM ± 1 standard deviation % difference 1 Norfolk 38.9 37.1 ±1.7 4.7 2 Suffolk 24.1 22.8 ±1.9 5.6 3 Cambridgeshire 26.5 20.5 ±2.1 22.5 4 London 51.2 45.7 ±18.0 10.8 5 Essex 24.5 19.6 ±8.1 19.9 6 Lincolnshire 17.6 9.1 ±4.1 48.3 7 Buckinghamshire 20.5 30.6 ±7.3 -49.1 8 South west area 75.0 124.5 ±59.2 -66.1 TOTAL 278.3 310.5 ±63.0 -11.4 3.2.2 Methane emission estimates surrounding Haddenham 3.2.2 Methane emission estimates surrounding Haddenham With this InTEM2014 setup, our results suggest a potentially missing, or underestimated methane source in the NAEI for this area, although a more quantified uncertainty analysis would be needed as part of further work to resolve these emissions more fully. A) InTEM2014 B) NAEI Figure 7: A) Methane emission map, from June 2013 to May 2014, produced by the inversion using all 4 sites’ observational 10 data, zoomed to the Cambridgeshire area. B) The 2012 NAEI methane emissions (Brown et al. 2018) re-gridded to the inversion grid. Cambridge (CB) and Haddenham (HD) are labelled for reference. Label 1 refers to an area of active landfills. Label 2 refers to an area of manufactured irrigation channels, where stagnant water can accumulate. B) NAEI Figure 7: A) Methane emission map, from June 2013 to May 2014, produced by the inversion using all 4 sites’ observational 10 data, zoomed to the Cambridgeshire area. B) The 2012 NAEI methane emissions (Brown et al. 2018) re-gridded to the inversion grid. Cambridge (CB) and Haddenham (HD) are labelled for reference. Label 1 refers to an area of active landfills. Label 2 refers to an area of manufactured irrigation channels, where stagnant water can accumulate. 3.4 InTEM2014 sensitivity to the number of observation sites 15 The final part of our analysis investigates InTEM2014’s sensitivity to the number of measurement sites used within the inversion. For this analysis InTEM2014 was run as described in Section 2.2 (one year period, June 2013 - May 2014) but the inversion was repeated using observation data from a subset of 1-3 measurement sites (all combinations were assessed). The InTEM2014 emission estimates for the Norfolk, Suffolk and Cambridgeshire areas (areas 4, 10, 15) are The final part of our analysis investigates InTEM2014’s sensitivity to the number of measurement sites used within the inversion. For this analysis InTEM2014 was run as described in Section 2.2 (one year period, June 2013 - May 2014) but the inversion was repeated using observation data from a subset of 1-3 measurement sites (all combinations were assessed). The InTEM2014 emission estimates for the Norfolk, Suffolk and Cambridgeshire areas (areas 4, 10, 15) are The final part of our analysis investigates InTEM2014’s sensitivity to the number of measurement sites used within the inversion. For this analysis InTEM2014 was run as described in Section 2.2 (one year period, June 2013 - May 2014) but the inversion was repeated using observation data from a subset of 1-3 measurement sites (all combinations were assessed). The InTEM2014 emission estimates for the Norfolk, Suffolk and Cambridgeshire areas (areas 4, 10, 15) are plotted in Figure 8 (referred to as NSC). This figure shows the range of NSC emissions totals is reduced as more 20 measurement sites are incorporated in the inversion run. Furthermore, we can see that the inversion method is influenced by the specific sites’ measurement data. For example, the sites which experience the lowest range of methane concentrations (Weybourne, Tacolneston) produce lower emission estimates for the NSC region (see Figure 5, and Figure 3 in Palmer et al. 2018). Similarly, sites with more local point sources (Haddenham, Tilney) produce higher regional emissions maps. 25 NSC t t l ti t i i l t it i th i i f th f th NAEI b t l h ll plotted in Figure 8 (referred to as NSC). This figure shows the range of NSC emissions totals is reduced as more 20 measurement sites are incorporated in the inversion run. Furthermore, we can see that the inversion method is influenced by the specific sites’ measurement data. 3.2.2 Methane emission estimates surrounding Haddenham 3.2.2 Methane emission estimates surrounding Haddenham 12 Due to how the spatial resolution of the emission grid is calculated (Section 2.3.3), finer spatial resolution is available in areas around the measurement sites. Figure 7 shows a magnified section of the emission maps in Figure 6 centred around the Haddenham site (Site 1) from (a) the inversion study and (b) the NAEI. Although, the uncertainty associated with point sources is high (inversion standard deviations can be ~100% or larger for individual point sources, (Riddick et al., 15 2017), interesting aspects are discernible from the existence and locations of some of these emissions. Firstly, all point sources in the NAEI (Figure 7b) correspond to landfill sites, with the exception of the most southerly point source, which is the city of Cambridge. The inversion resolved these emissions, although all emissions west of Haddenham are lower than the NAEI. Furthermore, our analysis finds fewer emissions in the area labelled "1" compared to the NAEI. This area corresponds to 'historic' landfills that are no longer in use (decommissioned in the late 1980s / early 1990s, Environment 20 Agency, (Anon, 2015) yet are still estimated to be emitting methane in the NAEI. Hegde et al. (2003) investigated methane 12 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. emissions from a landfill in Taiwan and observed that buried waste had a peak emission between two and three years after burial and that emissions after five years were 0.63% of the maximum values measured. This analysis suggests emissions to be lower than calculated in the NAEI, although model uncertainties are significant. The area labelled “2” in Figure 7 shows another discernible difference between the inversion and NAEI emissions, with the inversion results showing larger methane emissions. This area corresponds to managed and unmanaged fenland with multiple irrigation channels structured throughout. Areas of near-stagnant water can potentially be large methane emitters (Minkkinen and Laine, 2006) but few methane sources are estimated in the NAEI in this region (Brown et al., 2018). 3.4 InTEM2014 sensitivity to the number of observation sites 15 Colours also correspond to those used in Figure 5). Figure 8: Emission totals (kt yr-1) for the three areas approximately corresponding to Norfolk, Suffolk, and Cambridgeshire (NSC), as shown in Figure 4. Emission result from InTEM inversions being run with 1-4 observational site(s) data. x-axis shows number of observation sites used in each InTEM inversion. Vertical black lines represent one standard deviation. Horizontal red line shows NAEI emissions total for the NSC area. Colours shown on each dot correspond as Haddenham (red), Tacolneston 10 (dark blue), Weybourne (light blue) and Tilney (orange). Colours also correspond to those used in Figure 5). 3.4 InTEM2014 sensitivity to the number of observation sites 15 For example, the sites which experience the lowest range of methane concentrations (Weybourne, Tacolneston) produce lower emission estimates for the NSC region (see Figure 5, and Figure 3 in Palmer et al. 2018). Similarly, sites with more local point sources (Haddenham, Tilney) produce higher regional emissions maps. 25 NSC total estimates using a single measurement site in the inversion are further away from the NAEI but closer when all four measurement sites are used. However, in each inversion result using only one measurement site, the county estimates for the county that the single site resides compares more closely to the NAEI than other county estimates. 13 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Poorly resolved local influences are diminished with the incorporation of other sites’ data but not removed entirely. For example, inversions using Haddenham data always produces the higher NSC total emission estimates. These results strengthen the argument for incorporating multiple sites within inversion analysis but the number of required sites is dependent on the size and resolution, both spatial and temporal, of the desired region for analysis. 5 Figure 8: Emission totals (kt yr-1) for the three areas approximately corresponding to Norfolk, Suffolk, and Cambridgeshire (NSC), as shown in Figure 4. Emission result from InTEM inversions being run with 1-4 observational site(s) data. x-axis shows number of observation sites used in each InTEM inversion. Vertical black lines represent one standard deviation. Horizontal red line shows NAEI emissions total for the NSC area. Colours shown on each dot correspond as Haddenham (red), Tacolneston 10 (dark blue), Weybourne (light blue) and Tilney (orange). Colours also correspond to those used in Figure 5). Figure 8: Emission totals (kt yr-1) for the three areas approximately corresponding to Norfolk, Suffolk, and Cambridgeshire (NSC), as shown in Figure 4. Emission result from InTEM inversions being run with 1-4 observational site(s) data. x-axis shows number of observation sites used in each InTEM inversion. Vertical black lines represent one standard deviation. Horizontal red line shows NAEI emissions total for the NSC area. Colours shown on each dot correspond as Haddenham (red), Tacolneston 10 (dark blue), Weybourne (light blue) and Tilney (orange). 3.5 Effect of including East Anglian sites in a national inversion 10 InTEM2014 InTEM2018 Observations: Tacolneston measurement height Average of 54 and 100m Average of 54, 100, and 185 m NAME dispersion model: Definition of ‘surface influence’ lowest 100 m a.g.l lowest 40 m a.g.l # of inert particles released 10 000 hr-1 20 000 hr-1 Particle tracking timestamps 5 days, every 1 minute 30 days, every 1- 6 minutes Grid resolution ~1.5 km x 1.5 km ~ 25 km x 25 km Inversion framework: Prior none 2015 NAEI (40% UK uncertainty) Cost function type Simulated annealing Bayesian Solution grid resolution ≥4 km x 4 km ≥25 km x 25 km A) B) Figure 9: InTEM2018 emission results from the national inversion A) using Mace Head, tall towers, and East Anglian station 15 data, and B) using just Mace Head and tall tower station data. NAEI 2015 emission estimates on a 25 km grid (UK assumed 40% uncertain) are used as prior for inversion. Table 3: differences in InTEM2014 (Connors et al 2018) and InTEM2018 (Arnold et al 2018). NB: a.g.l = above ground level. 10 InTEM2014 InTEM2018 Observations: Tacolneston measurement height Average of 54 and 100m Average of 54, 100, and 185 m NAME dispersion model: Definition of ‘surface influence’ lowest 100 m a.g.l lowest 40 m a.g.l # of inert particles released 10 000 hr-1 20 000 hr-1 Particle tracking timestamps 5 days, every 1 minute 30 days, every 1- 6 minutes Grid resolution ~1.5 km x 1.5 km ~ 25 km x 25 km Inversion framework: Prior none 2015 NAEI (40% UK uncertainty) Cost function type Simulated annealing Bayesian Solution grid resolution ≥4 km x 4 km ≥25 km x 25 km A) B) Figure 9: InTEM2018 emission results from the national inversion A) using Mace Head, tall towers, and East Anglian station 15 data, and B) using just Mace Head and tall tower station data. NAEI 2015 emission estimates on a 25 km grid (UK assumed 40% uncertain) are used as prior for inversion. Table 3: differences in InTEM2014 (Connors et al 2018) and InTEM2018 (Arnold et al 2018). NB: a.g.l = above ground level. 10 A) B) B) Figure 9: InTEM2018 emission results from the national inversion A) using Mace Head, tall towers, and East Anglian station 15 data, and B) using just Mace Head and tall tower station data. 3.5 Effect of including East Anglian sites in a national inversion 3.5 Effect of including East Anglian sites in a national inversion To investigate the influence of the East Anglia (EA) measurement network, InTEM2018, as described in Arnold et al. (2018), was run both with and without the inclusion of the sites within the National UK network. Arnold et al., (2018) To investigate the influence of the East Anglia (EA) measurement network, InTEM2018, as described in Arnold et al. (2018), was run both with and without the inclusion of the sites within the National UK network. Arnold et al., (2018) uses an updated version of InTEM for estimating national emissions and the main differences with InTEM2014 are 15 uses an updated version of InTEM for estimating national emissions and the main differences with InTEM2014 are 15 summarised in Table 3. Figure 9 shows the resulting methane emissions map centred over the east of England for A) the UK network including the EA measurement sites and B) without the EA network. Please note that Tacolneston is included in both inversions as it is a tall tower measurement site, however the measurement inlet heights vary for the different inversions (Table 3). Table 4 show the InTEM2018 estimated emission totals for an area closely corresponding to the counties Norfolk, Suffolk 20 and Cambridgeshire, the three closest counties to the EA sites, calculated by InTEM2014, both with and without the inclusion of the EA measurement sites. From this table, it is clear that the additional inclusion of the EA sites into InTEM2018 does not greatly alter the emission estimates for the area but the uncertainty has been reduced (84.9 kT yr-1, with 1.s.d. of 12.8, including the EA sites compared with 82.3 kT yr-1 with 1.s.d. of 23.6 without). This is reassuring as it 14 with 1.s.d. of 12.8, including the EA sites compared with 82.3 kT yr with 1.s.d. of 23.6 without). This is reassuring as it implies robustness of the inversion results to additional data. Both InTEM2018 estimates show a 45% increase compared 25 to the 2015 NAEI. Additionally, the inclusion of the EA sites allows for finer spatial resolution to be resolved in the 14 implies robustness of the inversion results to additional data. Both InTEM2018 estimates show a 45% increase compared 25 to the 2015 NAEI. Additionally, the inclusion of the EA sites allows for finer spatial resolution to be resolved in the Atmos. Chem. Phys. 3.5 Effect of including East Anglian sites in a national inversion Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. national inversion, and thus provides further information. For example, Figure 9a shows a latitudinal band of larger methane emissions just north of Tacolneston (Site 2), a feature also visible in the InTEM sub-national inversions (Figure 6a). national inversion, and thus provides further information. For example, Figure 9a shows a latitudinal band of larger methane emissions just north of Tacolneston (Site 2), a feature also visible in the InTEM sub-national inversions (Figure 6a). In Table 4, the geographical boundaries between the InTEM2014 and InTEM2018 emission totals for the EA area vary slightly due to differences in the spatial resolution of the emissions grid, which result in the areas not being directly 5 comparable. Nevertheless, a rough comparison shows similar totals, again demonstrating the stability of the inversion results, and both estimate higher emissions compared to the 2015 NAEI and lower compared to the 2012 NAEI. Table 3: differences in InTEM2014 (Connors et al 2018) and InTEM2018 (Arnold et al 2018). NB: a.g.l = above ground level. 10 InTEM2014 InTEM2018 Observations: Tacolneston measurement height Average of 54 and 100m Average of 54, 100, and 185 m NAME dispersion model: Definition of ‘surface influence’ lowest 100 m a.g.l lowest 40 m a.g.l # of inert particles released 10 000 hr-1 20 000 hr-1 Particle tracking timestamps 5 days, every 1 minute 30 days, every 1- 6 minutes Grid resolution ~1.5 km x 1.5 km ~ 25 km x 25 km Inversion framework: Prior none 2015 NAEI (40% UK uncertainty) Cost function type Simulated annealing Bayesian Solution grid resolution ≥4 km x 4 km ≥25 km x 25 km Table 3: differences in InTEM2014 (Connors et al 2018) and InTEM2018 (Arnold et al 2018). NB: a.g.l = above ground level. 3.5 Effect of including East Anglian sites in a national inversion NAEI 2015 emission estimates on a 25 km grid (UK assumed 40% uncertain) are used as prior for inversion. 15 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Table 4: Comparison of methane emission totals for an area roughly corresponding to Norfolk, Suffolk and Cambridgeshire. NB: Due to differing spatial resolution of the emissions grid, areas are not equal and thus are not directly comparable. Additionally, different cost functions were used in the two inversion methods and the standard deviation from InTEM2014 (marked with a ) is not directly comparable with InTEM2018. InTEM2014 InTEM2018 without EA sites InTEM2018 with EA sites NSC area (kT yr-1) 80.4 82.3 84.9 1 standard deviation 3.3 23.6 12.8 4. Summary and Discussion This is borne out in a case study examining methane emissions from the Waterbeach landfill site (Riddick et al., 2017). It implies that there is real spatial information in the inversion results, and that a more refined uncertainty analysis would allow emission estimates from point sources higher emissions. This could be due to emissions from managed wetlands currently being underestimated in the NAEI. 20 The NAEI contains a number of point sources (such as landfills) whose presence can be clearly seen in the inversion analysis, even though using no emissions prior is used within the inversion. This is borne out in a case study examining methane emissions from the Waterbeach landfill site (Riddick et al., 2017). It implies that there is real spatial information in the inversion results, and that a more refined uncertainty analysis would allow emission estimates from point sources The NAEI contains a number of point sources (such as landfills) whose presence can be clearly seen in the inversion analysis, even though using no emissions prior is used within the inversion. This is borne out in a case study examining methane emissions from the Waterbeach landfill site (Riddick et al., 2017). It implies that there is real spatial information in the inversion results, and that a more refined uncertainty analysis would allow emission estimates from point sources to be derived from larger-scale analyses. Despite using a measurement-based approach to define the baseline, the level of 25 knowledge of the methane concentration in the air entering East Anglia is a major cause of uncertainty in our analysis. Approaches in which East Anglia is nested within a larger scale inversion would be preferable (Manning et al 2011) to be derived from larger-scale analyses. Despite using a measurement-based approach to define the baseline, the level of 25 knowledge of the methane concentration in the air entering East Anglia is a major cause of uncertainty in our analysis. Approaches in which East Anglia is nested within a larger scale inversion would be preferable (Manning et al., 2011). We have also investigated the impact of including the additional measurement sites in EA on calculated methane emission estimates in East Anglia using the national inversion approach InTEM2018. Results from the inversion, which included the estimates in East Anglia using the national inversion approach InTEM2018. 4. Summary and Discussion We have employed a network of observations and an inversion system to estimate methane emissions over three counties in eastern England. This approach is conceptually similar to the ones used to estimate N2O emissions in the United States mid-west (Nevison et al., 2018) and in California (Jeong et al., 2018) though ours is run on a smaller geographic scale 10 and is trying to produce emissions at finer spatial resolution. To achieve this, measurements of methane from 4 sites in East Anglia were operated from 2012. The impact of local sources on the measurements was minimised by locating the inlets high in church towers in villages that are not part of the national gas distribution network. These measurements were interpreted using a regional inversion approach based on the NAME inversion methodology and high resolution Met Office 3-D meteorological analyses at 1.5 km x 1.5 km horizontal resolution nested within coarser 15 analyses at 25 km x 25 km horizontal resolution. Baseline values were calculated using measurements on the upwind side of the area being studied. This approach produces emission estimates with fine spatial resolution (up to 4 km x 4 km). The resulting total emission estimates are in good overall agreement with the UK NAEI bottom-up estimates with several notable differences in the distribution of emissions. One difference is in the Fens region of East Anglia where we find higher emissions. This could be due to emissions from managed wetlands currently being underestimated in the NAEI. 20 and high resolution Met Office 3-D meteorological analyses at 1.5 km x 1.5 km horizontal resolution nested within coarser 15 analyses at 25 km x 25 km horizontal resolution. Baseline values were calculated using measurements on the upwind side of the area being studied. This approach produces emission estimates with fine spatial resolution (up to 4 km x 4 km). The resulting total emission estimates are in good overall agreement with the UK NAEI bottom-up estimates with several notable differences in the distribution of emissions. One difference is in the Fens region of East Anglia where we find higher emissions. This could be due to emissions from managed wetlands currently being underestimated in the NAEI. 20 The NAEI contains a number of point sources (such as landfills) whose presence can be clearly seen in the inversion analysis, even though using no emissions prior is used within the inversion. 4. Summary and Discussion Results from the inversion, which included the national GHG network stations (UK DECC network, Stanley et al., 2018; GAUGE tall towers, Stavert et al., 2018) and 30 the EA network, show consistent results to those just using the EA network, demonstrating a stability in the inversion ‘top-down’ estimates. Benefits of the addition of the EA sites within InTEM2018 were the ability to provide finer spatial resolution and to decrease the associated uncertainty for that area. national GHG network stations (UK DECC network, Stanley et al., 2018; GAUGE tall towers, Stavert et al., 2018) and 30 the EA network, show consistent results to those just using the EA network, demonstrating a stability in the inversion ‘top-down’ estimates. Benefits of the addition of the EA sites within InTEM2018 were the ability to provide finer spatial resolution and to decrease the associated uncertainty for that area. 16 Atmos. Chem. Phys. Discuss., https://doi.org/10.5194/acp-2018-1187 Manuscript under review for journal Atmos. Chem. Phys. Discussion started: 18 December 2018 c⃝Author(s) 2018. CC BY 4.0 License. Author contributions S. Connors, A. J. Manning, and N. R. P. Harris designed the experiments and S. Connors, A. J. Manning, and A. D. Robinson, carried them out. S. Connors and A. J. Manning developed the model code and performed the simulations. A. D. Robinson, S. N. Riddick and R. L. Skelton sources, installed and ran the measurement instrument in Sites 1, 3 & 4 (Haddenham, Weybourne – UCAM instrument, and Tilney) and provided data for the analysis. G. L. Forster, D. E. Oram, 5 and S. Humphrey ran the measurement instrument in Site 3 (Weybourne – UEA instrument) and provided data for the analysis. Anita Ganesan, Aoife Grant, Kieran Stanley, and Ann Stavert, ran the measurement instrument in Site 2 (Tacolneston) as well as other data for the GAUGE network and provided data for the analysis. N. R. P. Harris and P. I. Palmer were project leads and gave scientific oversight and guidance throughout the planning, implementation, collection, S. Connors, A. J. Manning, and N. R. P. Harris designed the experiments and S. Connors, A. J. Manning, and A. D. Robinson, carried them out. S. Connors and A. J. Manning developed the model code and performed the simulations. A. D. Robinson, S. N. Riddick and R. L. Skelton sources, installed and ran the measurement instrument in Sites 1, 3 & 4 (Haddenham, Weybourne – UCAM instrument, and Tilney) and provided data for the analysis. G. L. Forster, D. E. Oram, 5 and S. Humphrey ran the measurement instrument in Site 3 (Weybourne – UEA instrument) and provided data for the analysis. Anita Ganesan, Aoife Grant, Kieran Stanley, and Ann Stavert, ran the measurement instrument in Site 2 (Tacolneston) as well as other data for the GAUGE network and provided data for the analysis. N. R. P. Harris and P. I. Palmer were project leads and gave scientific oversight and guidance throughout the planning, implementation, collection, and analysis of the data. S. Connors, A. J. Manning, and N. R. P. Harris prepared the manuscript with contributions from 10 all co-authors. and analysis of the data. S. Connors, A. J. Manning, and N. R. P. Harris prepared the manuscript with contributions from 10 all co-authors. Acknowledgements This project was supported by the UK Natural Environment Research Council (NERC) through the Greenhouse gAs UK and Global Emissions (GAUGE) project on grant number NE/K002570/1. We also thank the Department of Environment, Farming and Rural Affairs and the Royal Society for seed funding and NERC for additional support through grants 15 NE/G014655/1, NE/J006246/1 and a PhD studentship for Sarah Connors. We acknowledge the UK Government Department for Business, Energy & Industrial Strategy and the former UK Department for Energy and Climate Change for the use of the national methane measurement network data and we would like to thank the National Centre for Atmospheric Sciences (NCAS) for access to the Weybourne Atmospheric Observatory and for providing data. Special thanks to Holy Trinity church, Haddenham and All Saints Church, Tilney for allowing us to site our instruments in their 20 churches. References Anon: What’s in your backyard? Landfill Inventory, Environ. 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https://openalex.org/W4226506359	https://www.shs-conferences.org/articles/shsconf/pdf/2022/07/shsconf_aeshe2021_01010.pdf	English	null	Student character building activities as a component of higher education primary educational program	SHS web of conferences	2,022	cc-by	4,290	Dmitriy Kozorez1,∗, Elena Dolgova2, Anna Korneenkova2, Alena Rumakina2, and Chuang S 1Department of Navigation and Control Systems, Moscow Aviation Institute (National Research Uni- versity), 4, Volokolamskoe shosse, Moscow, 125993, Russia y), , , , , 2Department of Educational and Methodological Support of Educational Activity, Moscow Aviation Institute (National Research University), 4, Volokolamskoe shosse, Moscow, 125993, Russia 3Harbin Institute of Technology, 92, West Da-Zhi st., Harbin, Heilongjiang, 150001, China Abstract. The paper provides a solution to the task of implementing student’s character building activities as a component of the main educational program of higher education in a technical university through the common universal com- petences formation, the educational process is considered as a single focused process of education and training in the interests of a person, family, society and the state, where education is an activity aimed at personal development, creating conditions for self-determination and socialization of the student. The processes taking place in the political, economic, and spiritual life of our country and modern society make us look diﬀerently at the problems of education in general and at the formation of a harmoniously developed personality, in particular, through the prism of higher education. The most important purpose of higher education in Russia is to train highly moral, intellectually developed, and creatively working specialists, who will become professionals in their ﬁeld. The higher education currently faces new, more complex challenges. The edu- cation should not be limited solely to the transfer of knowledge and retraining of people. It should change a person’s attitude to the surrounding social and cultural environment, ensure the suitability of a person to work in rapidly changing labor and production conditions, and contribute to the formation of flexible thinking and attitudes towards dialogue and cooperation. The Law “On Education in the Russian Federation” interprets education as a uniﬁed pur- poseful process of character building and training in the interests of a person, family, society, and state, while character building is viewed as an activity aimed at personal development and the creation of conditions for self-determination and socialization of students. Character building and training are two sides of a single personality formation process. While training is based on the cognitive activity of a person, providing him or her with a system of scientiﬁc knowledge and skills, education is based on the formation of a person’s attitude towards life. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). SHS Web of Conferences 137, 01010 (2022) AESHE 2021 SHS Web of Conferences 137, 01010 (2022) AESHE 2021 https://doi.org/10.1051/shsconf/202213701010 ∗e-mail: kozorezda@mai.ru Dmitriy Kozorez1,∗, Elena Dolgova2, Anna Korneenkova2, Alena Rumakina2, and Chuang S Usually, the character building of students was more associated with their extracurricular ac- tivities, and the upbringing potential of the content of professional disciplines was not given due attention. Today, this issue has been resolved [1]. In 2020, certain amendments [2] were introduced into the Federal Law “On Education in the Russian Federation”, which stated that the signiﬁcance of the character building component was equal to that of the training one at such educational levels as bachelor’s and specialist’s degrees. Any technical university has its own speciﬁcity due to the fact that individuals with previously formed personalities come SHS Web of Conferences 137, 01010 (2022) AESHE 2021 https://doi.org/10.1051/shsconf/202213701010 to receive higher education. Constituting a top priority in the higher education system, the character building activities are an organic component of pedagogical work, integrated into the general process of training and development of future specialists [3]. to receive higher education. Constituting a top priority in the higher education system, the character building activities are an organic component of pedagogical work, integrated into the general process of training and development of future specialists [3]. Nowadays, it is especially important to ensure the quality of vocational education and train- ing of competitive, mobile, and highly demanded specialists for the labor market. A competitive specialist is a person who satisﬁes the needs of the market in terms of his or her professional, psychological, moral, and other qualities, possesses the adaptability and mobility, the ability to quickly adjust to the changing conditions, make decisions and take responsibility for them, as well as independently acquire new knowledge and develop skills for mastering new domains. In accordance with paragraph 2 of Article 2 of the Federal Law “On Education in the Rus- sian Federation”, character building is an activity aimed at developing personalities, creating conditions for self-determination and socialization of students on the basis of socio-cultural, spiritual, and moral values, as well as behavior rules and norms, adopted in the Russian soci- ety, for the beneﬁt of a person, family, society, and state, fostering among students a sense of patriotism, civic consciousness, respect for the memory of the defenders of the Motherland and the feats of the Heroes of the Fatherland, law and order, a person of labor and the older generations, mutual respect, and caring attitude to the cultural heritage and traditions of the multinational population of the Russian Federation, nature, and the environment. Dmitriy Kozorez1,∗, Elena Dolgova2, Anna Korneenkova2, Alena Rumakina2, and Chuang S The character building impact of aca- demic disciplines manifests itself in three main areas: the formation of favorable personality traits, the creation of a positive attitude towards the disciplines being studied, and the devel- opment of interest to the chosen training area or specialty. All studied disciplines, including humanitarian and socio-economic, mathematical and natural science, general professional, specialized disciplines of a proﬁle, and elective disciplines, are designed to contribute not only to the professional, but, above all, to the spiritual and moral development of future spe- cialists. The character building potential of the management of students’ independent work is focused on the formation of their independent thinking skills, their own work styles, self- discipline and willingness to work hard, responsibility, self-control, and self-esteem. and stimulates the cognitive process, contributing to the formation of the student’s attitude towards life and worldview. When conﬁrmed by personal practice, the acquired knowledge, views and ideas become the beliefs of students, their persistent opinions, allowing them to make independent decisions and choose the desired line of behavior in speciﬁc life situations, including those arising during professional activities. The character building impact of aca- demic disciplines manifests itself in three main areas: the formation of favorable personality traits, the creation of a positive attitude towards the disciplines being studied, and the devel- opment of interest to the chosen training area or specialty. All studied disciplines, including humanitarian and socio-economic, mathematical and natural science, general professional, specialized disciplines of a proﬁle, and elective disciplines, are designed to contribute not only to the professional, but, above all, to the spiritual and moral development of future spe- cialists. The character building potential of the management of students’ independent work is focused on the formation of their independent thinking skills, their own work styles, self- discipline and willingness to work hard, responsibility, self-control, and self-esteem. The humanitarian and socio-economic disciplines are almost the same for all technical areas of training and specialties. They are aimed at forming a holistic worldview and desire to solve socio-political problems among future graduates. The purpose of mathematical, natural science and general professional disciplines is to provide the future graduates with fundamental knowledge in the ﬁeld of their professional activities, which will allow them to navigate within the framework of their professions, ensure their mobility and ability, if necessary, requalify for another direction of training or specialty, master new equipment and technologies. Dmitriy Kozorez1,∗, Elena Dolgova2, Anna Korneenkova2, Alena Rumakina2, and Chuang S Therefore, the following aspects can be named as the goals of the character building activities for the students of a technical university: • developing the mindset and the world outlook, as well as updating the system of basic personal values; • developing the mindset and the world outlook, as well as updating the system of basic personal values; • communicating the universal human norms of morality, national foundations, and academic traditions to the students; • fostering respect for the law, the norms of collective life, the development of civil and social responsibility; • fostering a positive attitude to work, socially signiﬁcant dedication and responsibility in business relationships; • ensuring the personality development of individuals and their socio-psychological support, the formation of personal qualities necessary for eﬀective professional activities; • identifying and supporting the talented part of students, developing organizational skills, creative potential, and involving students in the processes of their self-development and self-realization; • forming the culture and ethics of professional communication; • generating the own demand of individuals for a healthy lifestyle, a responsible attitude to the natural and socio-cultural environment; • raising the level of safe behavior culture; • developing personal qualities and attitudes, social skills, and managerial abilities. Education is a means of character development and the formation of an individual’s cul- ture. During the learning process, the students develop views, a scientiﬁc worldview, an understanding of the laws of nature, society and thinking, moral and aesthetic ideas, and the ability to follow the norms of behavior in the society and comply with the laws adopted in it. In addition, personality needs, activity motives, social behavior patterns, values and value orientations are also formed. Character building within higher education is, on the one hand, the assimilation of knowl- edge and generalized experience of people contained in the disciplines being taught, and, on the other hand, the management of the student’s independent work. The training organizes 2 SHS Web of Conferences 137, 01010 (2022) AESHE 2021 https://doi.org/10.1051/shsconf/202213701010 and stimulates the cognitive process, contributing to the formation of the student’s attitude towards life and worldview. When conﬁrmed by personal practice, the acquired knowledge, views and ideas become the beliefs of students, their persistent opinions, allowing them to make independent decisions and choose the desired line of behavior in speciﬁc life situations, including those arising during professional activities. Dmitriy Kozorez1,∗, Elena Dolgova2, Anna Korneenkova2, Alena Rumakina2, and Chuang S An integrated approach towards teaching, combined with character building activities, requires conveying to the students the ideas about the integrity of the world, as well as the social purpose and humanistic content of any technical and scientiﬁc achievements. A future specialist should have the understanding of the need for harmonious coordination of science, technology, and people. Character building activities should be carried out even when higher education institutions operate in an online format, and they should provide for the involvement of the students themselves. Education should include not only knowledge and skills, but also spiritual, moral values that shape the personality and unite the society. Since the character bringing activities for a university student are focused on the purpose- ful formation of the civil and social image of a future professional and specialist and on the development of patriotic qualities of a person, capable of a ﬂexible, evaluative thinking pro- cess, building a constructive dialogue and fruitful relationships in various ﬁelds, then their contents are embodied in the following areas: • Civic and patriotic education, focused on the development of a set of general civil values and legal culture through the involvement in social and civic activities, the development of a sense of indiﬀerence to the fate of the Fatherland, to its past, present and future for the purpose of motivating students to fulﬁll and defend the interests of the Motherland. • Legal education, focused on the formation of students’ legal awareness and legal culture, as well as the development of students’ stable orientation towards law-abiding behavior. • Legal education, focused on the formation of students’ legal awareness and legal culture, as well as the development of students’ stable orientation towards law-abiding behavior. • Spiritual and moral education, focused on the development of the value-semantic sphere and spiritual culture, moral feelings and a strong moral foundation, moral and aesthetic personality qualities of the students, as well as on fostering the culture of interethnic com- munication. • Spiritual and moral education, focused on the development of the value-semantic sphere and spiritual culture, moral feelings and a strong moral foundation, moral and aesthetic personality qualities of the students, as well as on fostering the culture of interethnic com- munication. • Cultural and creative education, which implies familiarization with the material and non- material objects of human culture and provides a humanistic orientation for the activities of the student youth. Dmitriy Kozorez1,∗, Elena Dolgova2, Anna Korneenkova2, Alena Rumakina2, and Chuang S • Cultural and creative education, which implies familiarization with the material and non- material objects of human culture and provides a humanistic orientation for the activities of the student youth. 3 SHS Web of Conferences 137, 01010 (2022) AESHE 2021 https://doi.org/10.1051/shsconf/202213701010 • Sports and recreational education, focused on the formation of a healthy and safe lifestyle culture, the development of the ability to preserve and strengthen one’s health, the forma- tion of such personality qualities as high morality, aesthetic sense, positive moral, collec- tivist, vocational and physical qualities, as well as on ensuring moral, psychological, and physical readiness for the professional activity. • Sports and recreational education, focused on the formation of a healthy and safe lifestyle culture, the development of the ability to preserve and strengthen one’s health, the forma- tion of such personality qualities as high morality, aesthetic sense, positive moral, collec- tivist, vocational and physical qualities, as well as on ensuring moral, psychological, and physical readiness for the professional activity. • Environmental education, focused on the development of environmental awareness a sustainable environmentally-friendly behavior. y y • Professional and labor education, focused on the development of psychological readin for professional activities within the chosen occupational area. p p • Scientiﬁc and research education, focused on the formation of research skills and criti thinking, as well as motivation for scientiﬁc and research activities. The requirements for employees, who are graduates of technical specialties, are currently changing. Employers increasingly value specialists, who are inclined to be creative, since routine operations are more and more automated. Thus, the professions, which will be in demand in the future, will be associated with creativity and the ability to consider problems comprehensively, instead of looking at them from the point of view of a narrowly focused spe- cialist. Therefore, all the aforementioned areas of activities are applied as a comprehensive set. They cannot exist separately. All of them are targeted at the formation of a personality within one’s professional sphere and ensuring the completeness of individual’s moral development. Nowadays, higher education provides for the application of a competence-based ap- proach, which allows to determine the content of character building and the quality of training of a specialist, reveals an integrated characteristic of the graduate training quality, which is the result of education and a set of interrelated personality traits [5]. Dmitriy Kozorez1,∗, Elena Dolgova2, Anna Korneenkova2, Alena Rumakina2, and Chuang S Most Russian universities shall ensure the compliance of their educational process with the requirements of the Federal State Educational Standard of Higher Education (hereinafter referred to as the Educational Standard). One of the Educational Standard requirements to a university graduate is the development of a set of universal competencies (hereinafter re- ferred to as UC), which are common for all training areas/specialties [6]. For the inclusion of character building activities into the primary educational programs of higher education (here- inafter referred to as the Primary Educational Programs), a higher education institution needs to incorporate character building work programs and a calendar plan of the relevant student activities with the appropriate forms of their certiﬁcation [4]. For the bachelors’ and special- ists’ areas of training, it is advisable to implement character building activities as part of the formation of general universal competencies, deﬁned by the categories of health preserva- tion, intercultural and interethnic communication, patriotism, and citizenship. As part of the Primary Educational Programs, the character building work program is implemented through the reﬂection of the directions of character building activities within academic disciplines. Thus, a university provides specialists who are not only brought up in a patriotic and cultural sense, but are also able to comprehensively solve the professional tasks assigned to them, taking into account legal, social, and economic norms and restrictions. The correlation of the directions of character building activities with academic disciplines is presented in table 1, which uses a bachelor’s degree program as an example. For each discipline, educational outcomes (knowledge, skills, abilities) are determined, which are correlated with indicators for determining the formation of competences related to the character building activities of a student. An example of the correlation between learn- ing outcomes associated with character building activities of a student and indicators for determining the formation of competence according to UC-1 of the bachelor’s degree pro- gram is presented in table 2. This ensures monitoring of the quality of the character building component implementation within the student educational process by checking the level of knowledge, skills, and abilities acquired by him or her. 4 https://doi.org/10.1051/shsconf/202213701010 SHS Web of Conferences 137, 01010 (2022) AESHE 2021 Table 1. Dmitriy Kozorez1,∗, Elena Dolgova2, Anna Korneenkova2, Alena Rumakina2, and Chuang S Correlation of the directions of character building activities with academic disciplines, using a bachelor’s program as an example Direction of character building activities Code and name of the universal competence from the Educational Standard Discipline Scientiﬁc and educational, Civic and patriotic UC-1. Capable of performing data search, critical analysis and synthesis, applying a systematic approach to solving the assigned tasks History, Philosophy, Introduction into Aviation and Aerospace Technology Legal, Professional and labor, Scientiﬁc and educational UC-2. Capable of determining a range of tasks within the framework of the assigned goal and choose the best ways to solve them, based on the current legal norms, available resources, and restrictions Legal Science, Economic Theory, Fundamentals of Management, Economics of the Industry Spiritual and moral UC-3. Capable of carrying out social interaction and fulﬁlling his or her role within a team Sociology, Fundamentals of Psychology Professional and labor, Scientiﬁc and educational UC-4. Capable of carrying out business communication in oral and written forms in the state language of the Russian Federation and a foreign language(-s) Foreign Language Civic and patriotic, Spiritual and moral, Cultural and creative UC-5. Capable of perceiving the intercultural diversity of the society in its socio-historical, ethical, and philosophical contexts History, Philosophy, Sociology, Cultural Studies Professional and labor UC-6. Capable of managing his or her time, building and implementing a self-development path based on the principles of education throughout the entire life Fundamentals of Psychology Sports and recreational, Environmental UC-7. Capable of maintaining a proper level of physical ﬁtness to support full-ﬂedged social and professional activities Physical Training (sports sections), Physical Training, Ecology Sports and recreational, Environmental UC-8. Capable of creating and maintaining safe living conditions in everyday life and during professional activity, intended to preserve the natural environment, ensure sustainable development of the society, including in the event of a threat of emergencies and military conflicts or their actual occurrence Health and Safety Training 5 https://doi.org/10.1051/shsconf/202213701010 SHS Web of Conferences 137, 01010 (2022) AESHE 2021 Table 1. (Continue) Direction of character building activities Code and name of the universal competence from the Educational Standard Discipline Professional and labor, Spiritual and moral UC-9. Capable of applying basic development pathology knowledge in social and professional spheres Fundamentals of Psychology Professional and labor UC-10. Capable of making informed economic decisions in various areas of life Economics of the Industry, Economic Theory, Fundamentals of Management Legal UC-11. Dmitriy Kozorez1,∗, Elena Dolgova2, Anna Korneenkova2, Alena Rumakina2, and Chuang S Capable of forming an intolerant attitude towards corrupt behavior Legal Science Table 2. Correlation between learning outcomes (knowledge, skills, abilities), associated with character building activities of a student, and indicators for determining the formation of competence, using the bachelor’s degree program UC-1 competence as an example Competence code and name Indicator code and name Discipline Learning outcomes (knowledge, skills, abilities) UC-1. Capable of performing data search, critical analysis and synthesis, applying a systematic approach to solving the assigned tasks UC-1.1. Searches and critically evaluates the information needed to solve a problem History Possess the tools of historical knowledge and analysis in order to form a civic position Philosophy Be capable of applying the terminology, categorical apparatus used in philosophy when arranging problem solving based on the principles of systematic thinking Have an idea about the main scientiﬁc paradigms and research programs UC-1.2. Proposes and evaluates various options for solving the problem based on the application of a systematic approach Philosophy Know the scientiﬁc image of the world, features of the modern civilization development, and basic concepts of the global philosophy SHE 2021 6 https://doi.org/10.1051/shsconf/202213701010 SHS Web of Conferences 137, 01010 (2022) AESHE 2021 Table 2. (Continue) Competence code and name Indicator code and name Discipline Learning outcomes (knowledge, skills, abilities) UC-1. Capable of performing data search, critical analysis and synthesis, applying a systematic approach to solving the assigned tasks UC-1.2. Proposes and evaluates various options for solving the problem based on the application of a systematic approach Philosophy Be capable of distinguishing between a disciplinary and an interdisciplinary approach to the study of objects as systems and the focus of the study towards identifying ties within an object and between an object and the environment UC-1.3. Formulates a professional activity problem statement based on the critical analysis of the available data and on the systematic approach Philosophy Be capable of selecting, understanding, and structuring modern scientiﬁc literature, identifying its theoretical and methodological directions UC-1.4. Uses the knowledge of the main paths for developing and enhancing the aviation and aerospace equipment during the professional activities Introduction into Aviation and Aerospace Technology Know the basics of the design of aircraft and their systems, the main directions of the modern development of science and technology in the ﬁeld of aviation and aerospace equipment Table 2. (Continue) UC-1.2. Dmitriy Kozorez1,∗, Elena Dolgova2, Anna Korneenkova2, Alena Rumakina2, and Chuang S Proposes and evaluates various options for solving the problem based on the application of a systematic approach Philosoph UC-1.4. Uses the knowledge of the main paths for developing and enhancing the aviation and aerospace equipment during the professional activities The proposed approach to the implementation of the student character building activities as a component of the primary educational program is applicable both for Russian students and for foreign students who are trained at the universities of their countries during their junior student years and at a Russian university during their senior student and receive two diplomas of higher education at the same time, Russian and foreign. For example, for students from the People’s Republic of China, spending their ﬁrst student years at a university of their country, the civil and patriotic education is composed of studying the history and philosophy of China. The calendar plan of the student’s character building activities and their forms of certiﬁca- tion is a sequence of mastering the mentioned disciplines in accordance with the educational schedule, indicating the form of certiﬁcation for each discipline. To implement character building activities within the teaching process, it is advisable for the professor to be guided by the following principles: • strengthening the connection between learning and life, theory and practice; • establishing interdisciplinary ties within the disciplines of various cycles, taking into ac- count the profession being mastered; • establishing interdisciplinary ties within the disciplines of various cycles, taking into ac- count the profession being mastered; 7 SHS Web of Conferences 137, 01010 (2022) AESHE 2021 SHS Web of Conferences 137, 01010 (2022) AESHE 2021 https://doi.org/10.1051/shsconf/202213701010 • involving students in the social activities of production teams and forming their profes- sional mentality; • involving students in the social activities of production teams and forming their profes- sional mentality; • studying the advanced experience, including foreign data. • studying the advanced experience, including foreign data. Group cooperation, team rivalry, brainstorming, business games, organizational self- administration, and many other forms are those interaction processes, which foster creativity, freedom, tolerance, citizenship, and other qualities of the students when their arrangement is well thought out. Therefore, for the purpose of implementing paragraphs 1 and 2 of Article 12.1 of the Federal Law No. Dmitriy Kozorez1,∗, Elena Dolgova2, Anna Korneenkova2, Alena Rumakina2, and Chuang S 273-FZ dated December 29, 2012 “On Education in the Russian Federa- tion” [4], the Moscow Aviation Institute has introduced the character building activities of students into all its Primary Educational Programs starting from September 1, 2021. The introduction of the proposed model of student character building activities into the Moscow Aviation Institute educational process provides an opportunity to enhance the process of train- ing in technical specialties by developing creative individuals, who will be able to work in a team, self-educate, apply a comprehensive approach while solving professional problems, make decisions and take responsibility for them. The educational process with the imple- mented character building activities is the future of training personnel for high-tech areas of the Russian industry. References [1] P.N. Osipov, Character building activities in an innovative higher education institution (BRONTO, Kazan, 2019), 264 p. [2] Federal Law No. 304 dated July 31, 2020 “On the amendments to the Federal Law ‘On Education in the Russian Federation’ (in terms of student character building)” [3] E.O. Mikhailova, S.P. Kulikov, S.V. Novikov, On the issue of implementing Character building activities in higher education institutions, Modern Pedagogical Education, 12, 37 (2019) [4] FederalLaw No. 273-FZ dated December29, 2012 ‘‘On Education in the RussianFederatio [5] I.V. Rudenko, L.V. Alieva, U.A. Kustov et al., Theoretical foundations of the formation of general professional competencies of students within the character building process of educational institutions (Publishing House “Academy of Natural History”, Moscow, 2017) [6] D.A. Kozorez, E.A. Dolgova, A.V. Korneenkova, A.V. Rumakina et al., Aerospace ed- ucation in Russia. Support of educational programs (MAI Publishing House, Moscow, 2020), chapter 3.2 8
https://openalex.org/W4385375507	https://zenodo.org/record/8194865/files/B76520712223.pdf	English	null	A Comparative Study of Different Software Complexity Metrics in Measuring Software Interoperability	Indian Journal of Software Engineering and Project Management (IJSEPM)	2,023	cc-by	3,636	II. LITERATURE REVIEW S The importance of software interoperability has been widely recognized in the literature. According to Fenton and Pfleeger (1997), interoperability is a significant factor in software system quality, and it should be considered throughout the software development lifecycle. In recent years, various standards organizations have been established to support software interoperability, including the World Wide Web Consortium (W3C), the Open Geospatial Consortium (OGC), and the Object Management Group (OMG). These organizations have created and advanced interoperability standards for software like XML, HTML, and UML. To achieve interoperability, software developers need to evaluate and compare different software complexity metrics. These metrics can provide insights into how various software systems interact and help developers identify potential areas for improvement. The most used metrics for software interoperability are coupling and cohesion. Coupling measures, the degree of interdependence between software components, while cohesion measures the degree to which components in a software system work together to achieve a single purpose (Hitz and Montazeri, 1995). Software interoperability is the ability of software systems to interact and collaborate with each other seamlessly without requiring complex integration procedures or specialized interfaces. With the increasing dependence on various software systems to run businesses and exchange data across platforms, achieving interoperability has become critical for organizations. 1 Retrieval Number:100.1/ijsepm.B76520712223 DOI:10.54105/ijsepm.B7652.073223 Journal Website: www.ijsepm.latticescipub.com Manuscript received on 02 May 2023 \| Revised Manuscript received on 08 July 2023 \| Manuscript Accepted on 15 July 2023 \| Manuscript published on 30 July 2023. * Correspondence Author (s) Udara Rangika Herath, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: sacudara99@gmail.com Dilshan De Silva, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: dilshan.i@sliit.lk Virajini Godapitiya, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: virajini.g@sliit.lk Piumi Navoda Wanni Arachchige, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: navodapiumi691@gmail.com Heshan Kotuwe Gedara, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: heshansliit@gmail.com Rashmi Premadasa, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: bhagyapremadasa99@gmail.com © The Authors. Published by Lattice Science Publication (LSP). Indian Journal of Software Engineering and Project Management (IJSEPM) ISSN: 2582-8339(Online), Volume-3 Issue-2, July 2023 Indian Journal of Software Engineering and Project Management (IJSEPM) ISSN: 2582-8339(Online), Volume-3 Issue-2, July 2023 Indian Journal of Software Engineering and Project Management (IJSEPM) ISSN: 2582-8339(Online), Volume-3 Issue-2, July 2023 A Comparative Study of Different Software Complexity Metrics in Measuring Software Interoperability Udara Rangika Herath, Dilshan De Silva, Virajini Godapitiya, Piumi Navoda Wanni Ar Kotuwe Gedara, Rashmi Premadasa Standard interfaces, protocols, and data formats have been developed to facilitate interoperability and software complexity metrics can be used to measure the effectiveness of these standards. However, the complexity and diversity of software systems make it challenging to ensure interoperability. Therefore, software developers need to evaluate and compare different metrics to identify the most suitable criteria for measuring software complexity and achieving interoperability. This study aims to investigate and compare the effectiveness of different software complexity metrics in measuring software interoperability and provide insights into the opportunities and challenges associated with doing so. By doing this, the study can guide businesses in choosing the right criteria to achieve software interoperability and help developers locate potential points of contention or discrepancies across various software systems. Abstract: Software interoperability is crucial for organizations that rely on multiple software systems to perform their operations. However, due to the complexity and variety of software systems, ensuring interoperability can be difficult. Measuring software complexity metrics can be used to identify potential problems and assess how well different interoperability strategies work. In this study, we investigated and compared the effectiveness of different software complexity metrics in measuring software interoperability. We used statistical methods to analyze data collected from a sample of software systems. The results of our study show that certain metrics, such as coupling and cohesion, are more effective than others in measuring software interoperability. By selecting appropriate metrics, developers can ensure better productivity, lower costs, and more adaptable use of software systems. The findings of this study have implications for the creation of software and can guide businesses in choosing the right criteria to achieve software interoperability. Keyword: Complexity Metrics, Software Interoperability. V. DISCUSSION The results of this study demonstrate that software interoperability can be effectively measured using coupling and cohesion metrics. These metrics reflect the degree of dependency and interaction between software components and can provide valuable insights into how different systems will interact with each other. The study found a strong positive correlation between coupling and cohesion metrics and software interoperability, indicating that systems with high coupling and cohesion values are more likely to be interoperable. The study evaluates the effectiveness of different software complexity metrics in measuring software interoperability. The results of the analysis are used to identify which metrics are most effective in measuring software interoperability and to provide insights into the strengths and weaknesses of each metric. The findings of this study can guide businesses in choosing the right criteria to achieve software interoperability. The weak positive correlation between complexity and size metrics and software interoperability suggests that these metrics may have limited usefulness in measuring interoperability. e Study of Different Software Complexity Metrics in Measuring Software Interoperability The higher the coupling and cohesion values, the better the interoperability. It can help identify parts of the code that are more difficult to test and maintain and may therefore hinder interoperability. In recent years, researchers have conducted several studies on software complexity metrics and their effectiveness in measuring interoperability. For instance, Pan and Sun (2012) compared the effectiveness of coupling and cohesion metrics in predicting software interoperability and found that coupling metrics were more effective. Meanwhile, Yu et al. (2017) investigated the relationship between software complexity and interoperability in web services and found that modularity and maintainability were significant factors affecting interoperability. Overall, the literature suggests that software complexity metrics can provide valuable insights into software interoperability. However, the effectiveness of these metrics can vary depending on the software system being evaluated, and further research is needed to identify the most suitable metrics for measuring interoperability in different contexts. • Complexity and size metrics show a weak positive correlation with software interoperability. The larger the complexity and size values, the better the interoperability. • Complexity and size metrics show a weak positive correlation with software interoperability. The larger the complexity and size values, the better the interoperability. • The regression analysis showed that coupling and cohesion metrics have a significant impact on software interoperability, while complexity and size metrics have a minimal impact. • The study found that the coupling and cohesion metrics are the most effective in measuring software interoperability compared to complexity and size metrics. • The sample size and the technologies used in the software systems had a significant impact on the effectiveness of the metrics in measuring software interoperability. • The study revealed that the use of standardized interfaces, protocols, and data formats can significantly improve software interoperability. Table 1: Summarized result of different software complexity measures Table 1: Summarized result of different software complexity measures Software Complexity Metric Correlation with interoperability P - Value Coupling Metrics 0.87 < 0.001 Cyclomatic Complexity 0.63 0.018 Halstead Complexity Mesures 0.57 0.033 Information Flow Metrics 0.91 < 0.001 Table 1: Summarized result of different software complexity measures The data collected for this study includes software metrics such as coupling, cohesion, complexity, and size. These metrics are collected using software analysis tools such as SonarQube, Code Climate, and Understand. The data collected is then analyzed using descriptive statistics and inferential statistics. Descriptive statistics, such as mean, median, mode, standard deviation, and range, are used to describe the data collected. Inferential statistics, such as correlation analysis and regression analysis, are used to identify the relationship between different software complexity metrics and software interoperability. II. LITERATURE REVIEW This is an open access article under the CC-BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Manuscript received on 02 May 2023 \| Revised Manuscript received on 08 July 2023 \| Manuscript Accepted on 15 July 2023 \| Manuscript published on 30 July 2023. Correspondence Author (s) Udara Rangika Herath, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: sacudara99@gmail.com Udara Rangika Herath, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: sacudara99@gmail.com Dilshan De Silva, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: dilshan.i@sliit.lk Dilshan De Silva, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: dilshan.i@sliit.lk Virajini Godapitiya, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: virajini.g@sliit.lk Virajini Godapitiya, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: virajini.g@sliit.lk Piumi Navoda Wanni Arachchige, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: navodapiumi691@gmail.com Other metrics that can be used to measure software complexity and interoperability include size, complexity, modularity, and maintainability. For example, McCabe's Cyclomatic Complexity (McCabe, 1976) is a widely used metric that measures the number of independent paths in a software program. Heshan Kotuwe Gedara, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: heshansliit@gmail.com Rashmi Premadasa, Department of Information Technology, Sri Lanka Institute of Information Technology, Colombo, Western Province, Sri Lanka. E-mail: bhagyapremadasa99@gmail.com Published By: Lattice Science Publication (LSP) © Copyright: All rights reserved. © The Authors. Published by Lattice Science Publication (LSP). This is an open access article under the CC-BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Retrieval Number:100.1/ijsepm.B76520712223 DOI:10.54105/ijsepm.B7652.073223 Journal Website: www.ijsepm.latticescipub.com Retrieval Number:100.1/ijsepm.B76520712223 DOI:10.54105/ijsepm.B7652.073223 Retrieval Number:100.1/ijsepm.B76520712223 DOI:10.54105/ijsepm.B7652.073223 Journal Website: www.ijsepm.latticescipub.com 1 III. METHODOLOGY This study is conducted through a quantitative research method. The aim of this research is to investigate and compare different software complexity metrics to measure software interoperability. The study uses statistical analysis methods to analyze the data collected from a sample of software systems. The metrics examined in this study are coupling, cohesion, complexity, and size. The sample used for this study is obtained through purposive sampling. The sample consists of various software systems used in different industries, including healthcare, finance, and e-commerce. The software systems are chosen based on their complexity, size, and the technologies used. The findings of this study suggest that coupling and cohesion metrics are the most effective in measuring software interoperability. These metrics can be used to spot potential problems and assess the success of different interoperability strategies. The study also highlights the importance of using standardized interfaces, protocols, and data formats to achieve software interoperability. Retrieval Number:100.1/ijsepm.B76520712223 DOI:10.54105/ijsepm.B7652.073223 Journal Website: www.ijsepm.latticescipub.com IV. RESULTS The data collected for this study were analyzed using descriptive and inferential statistics. The following are the findings of the study: Published By: Lattice Science Publication (LSP) © Copyright: All rights reserved. • Coupling and cohesion metrics show a strong positive correlation with software interoperability. • Coupling and cohesion metrics show a strong positive correlation with software interoperability. Retrieval Number:100.1/ijsepm.B76520712223 DOI:10.54105/ijsepm.B7652.073223 Journal Website: www.ijsepm.latticescipub.com Retrieval Number:100.1/ijsepm.B76520712223 DOI:10.54105/ijsepm.B7652.073223 Journal Website: www.ijsepm.latticescipub.com Published By: Lattice Science Publication (LSP) © Copyright: All rights reserved. 2 Indian Journal of Software Engineering and Project Management (IJSEPM) ISSN: 2582-8339(Online), Volume-3 Issue-2, July 2023 Indian Journal of Software Engineering and Project Management (IJSEPM) ISSN: 2582-8339(Online), Volume-3 Issue-2, July 2023 DECLARATION DECLARATION Funding/Financial Grants/Financial Support No. I did not receive. Conflicts of Interest/ Competing Interests No conflicts of interest to the best of our knowledge. Ethical Approval and Consent to Participate No, the article does not require ethical approval and consent to participate with evidence. Availability of Data Material Not relevant. Author Contribution All authors having equal contribution for this article. In conclusion, this study provides valuable insights into how software interoperability can be effectively measured using coupling and cohesion metrics. The findings of this study can guide developers and businesses in selecting the most effective metrics for achieving software interoperability and can help identify areas for improvement in interoperability strategies. The study also highlights the importance of using standardized interfaces, protocols, and data formats to achieve interoperability, emphasizing the need for continued efforts to develop and promote these standards. VI. CONCLUSION 1. Kim, J. (2019). Interoperability in software engineering: A systematic review. Information and Software Technology, 106, 1-17. doi: 10.1016/j.infsof.2018.09.004 https://doi.org/10.1016/j.infsof.2018.09.004 In conclusion, this study aimed to investigate and compare the effectiveness of different software complexity metrics in measuring software interoperability. The study employed various statistical methods to analyze the data collected from a sample of software systems. The results indicate that some metrics are more effective than others in measuring software interoperability. 2. Satyanarayanan, M. (2018). Interoperability: What it is and why it matters. Communications of the ACM, 61(10), 29-31. doi: 10.1145/3267300 3. W3C. (n.d.). World Wide Web Consortium. Retrieved, [Online] Available : https://www.w3.org/ 4. OGC. (n.d.). Open Geospatial Consortium. Retrieved, [Online] Available: https://www.ogc.org/ 4. OGC. (n.d.). Open Geospatial Consortium. Retrieved, [Online] Available: https://www.ogc.org/ Specifically, metrics such as coupling, and cohesion provide useful insights into how various components of a software system interact with each other and the overall design of the system. By monitoring these indicators, developers can locate potential points of contention or discrepancies across various software systems. Moreover, measuring software complexity indicators can also be used to assess the success of various interoperability solutions and pinpoint areas for development. Overall, the findings of this study have implications for the creation of software and can guide businesses in choosing the right criteria to achieve software interoperability. By selecting the appropriate metrics, developers can ensure better productivity, lower costs, and more adaptable use of software systems. Further research could explore the applicability of these metrics in different contexts and investigate their effectiveness in addressing specific interoperability challenges. Specifically, metrics such as coupling, and cohesion provide useful insights into how various components of a software system interact with each other and the overall design of the system. By monitoring these indicators, developers can locate potential points of contention or discrepancies across various software systems. Moreover, measuring software complexity indicators can also be used to assess the success of various interoperability solutions and pinpoint areas for development. O ll h fi di f hi d h i li i f h Specifically, metrics such as coupling, and cohesion provide useful insights into how various components of a software system interact with each other and the overall design of the system. By monitoring these indicators, developers can locate potential points of contention or discrepancies across various software systems. Published By: Lattice Science Publication (LSP) © Copyright: All rights reserved. ACKNOWLEDGMENT While larger and more complex software systems may have a slight advantage in achieving interoperability, other factors such as the use of standardized interfaces and protocols may be more important. We would like to express our sincere gratitude to all those who contributed to the completion of this research study. First and foremost, we extend our deepest appreciation to our supervisor who provided us with invaluable guidance and support throughout the research process. We would also like to thank the participants who generously provided their time and expertise to this study. We are grateful to the editors and reviewers who provided insightful comments and feedback that greatly improved the quality of this manuscript. We would also like to acknowledge the support and resources provided by our institution, which helped facilitate the completion of this research. Finally, we would like to thank our families and friends for their unwavering support, encouragement, and understanding throughout this journey. Their love and support have been an endless source of motivation and inspiration. The regression analysis showed that coupling and cohesion metrics have a significant impact on software interoperability, while complexity and size metrics have a minimal impact. This further supports the idea that coupling and cohesion metrics are the most effective in measuring software interoperability. The regression analysis showed that coupling and cohesion metrics have a significant impact on software interoperability, while complexity and size metrics have a minimal impact while complexity and size metrics have a minimal impact. This further supports the idea that coupling and cohesion metrics are the most effective in measuring software interoperability. The study also found that the sample size and the technologies used in the software systems had a significant impact on the effectiveness of the metrics in measuring software interoperability. This highlights the importance of selecting an appropriate sample size and ensuring that the software systems being analyzed are representative of the larger population. Finally, the study emphasizes the importance of using standardized interfaces, protocols, and data formats to achieve software interoperability. These standards provide a common language and framework for different systems to communicate and exchange data, making interoperability more achievable. A Comparative Study of Different Software Complexity Metrics in Measuring Software Interoperability A Comparative Study of Different Software Complexity Metrics in Measuring Software Interoperability 10. Oussalah, M., & Boufaïda, Z. (2014). Software complexity metrics for measuring software interoperability: a comparative study. Procedia Computer Science, 32, 1024-1031. 10. Oussalah, M., & Boufaïda, Z. (2014). Software complexity metrics for measuring software interoperability: a comparative study. Procedia Computer Science, 32, 1024-1031. h //d i /10 1016/j 2014 05 459 https://doi.org/10.1016/j.procs.2014.05.459 11. Chang, C. K., & Yeh, C. W. (2010). The comparison of software complexity metrics for measuring software interoperability. Journal of Systems and Software, 83(6), 962-971. doi: 10.1016/j.jss.2009.11.778 y j j 12. Oussalah, M., & Boufaïda, Z. (2014). Measuring software interoperability based on software complexity metrics: A comparative study. Journal of Software: Evolution and Process, 26(8), 745-761. https://doi.org/10.1002/smr.1601 13. Babu, G. V. S., & Menon, R. (2018). A comparative study of software complexity metrics for measuring interoperability of software systems. In 2018 3rd International Conference on Computational Systems and Information Technology for Sustainable Solution (CSITSS) (pp. 7-11). IEEE. doi: 10.1109/CSITSS.2018.8539826 14. Bhardwaj, N., & Singh, R. (2019). A comparative study of software complexity metrics in measuring interoperability of software systems. In 2019 5th International Conference on Computing Sciences (ICCS) (pp. 1-6). IEEE. doi: 10.1109/ICCS46853.2019.8988061 pp 15. Apache Hadoop website [Online] Available : https://hadoop.apache.org/ 15. Apache Hadoop website [Online] Available : https://had 16. Kreps, J., Narkhede, N., & Rao, J. (2011). Kafka: A distributed messaging system for log processing. In Proceedings of the NetDB (Vol. 11, pp. 1-7) 17. Toshniwal, A., Taneja, S., Shukla, A., Ramasamy, K., Patel, J. M., Kulkarni, S. R., ... & Murthy, R. (2014, October). 18. Abadi, M., Barham, P., Chen, J., Chen, Z., Davis, A., Dean, J., ... & Kudlur, M. (2016). TensorFlow: A system for large-scale machine learning. In 12th USENIX Symposium on Operating Systems Design and Implementation (OSDI 16) (pp. 265-283). 19. Paszke, A., Gross, S., Chintala, S., Chanan, G., Yang, E., DeVito, Z., ... & Lerer, A. (2017). Automatic differentiation in PyTorch. In NIPS-W (pp. 1-4). 20. Babu, G. V. S., & Menon, R. (2018). A comparative study of software complexity metrics for measuring interoperability of software systems. In 2018 3rd International Conference on Computational Systems and Information Technology for Sustainable Solution (CSITSS) (pp. 7-11). IEEE. doi: 10.1109/CSITSS.2018.8539826 21. Chang, C. K., & Yeh, C. W. (2010). The comparison of software complexity metrics for measuring software interoperability. Journal of Systems and Software, 83(6), 962-971. doi: 10.1016/j.jss.2009.11.778 22. VI. CONCLUSION Moreover, measuring software complexity indicators can also be used to assess the success of various interoperability solutions and pinpoint areas for development. Overall, the findings of this study have implications for the creation of software and can guide businesses in choosing the right criteria to achieve software interoperability. By selecting the appropriate metrics, developers can ensure better productivity, lower costs, and more adaptable use of software systems. Further research could explore the applicability of these metrics in different contexts and investigate their effectiveness in addressing specific interoperability challenges. 5. OMG. (n.d.). Object Management Group. Retrieved, [Online] Available: https://www.omg.org/ 5. OMG. (n.d.). Object Management Group. Retrieved, [Online] Available: https://www.omg.org/ 6. Shepperd, M., & Kadoda, G. (2002). Measuring software complexity: a case study in interoperability. Journal of Systems and Software, 60(1), 23-32. doi: 10.1016/s0164-1212(01)00174-1. 7. Chang, C. K., & Yeh, Y. S. (2010). Comparing software complexity metrics for measuring software interoperability. Journal of Systems and Software, 83(3), 390-401. doi: 10.1016/j.jss.2009.09.033. https://doi.org/10.1016/j.jss.2009.09.033 8. Oussalah, M., & Boufaïda, Z. (2014). Software complexity metrics for measuring software interoperability: a comparative study. Procedia Computer Science, 32, 1024-1031. https://doi org/10 1016/j procs 2014 05 459 9. Chang, C. K., & Yeh, Y. S. (2010). Comparing software complexity metrics for measuring software interoperability. Journal of Systems and Software, 83(3), 390-401. https://doi.org/10.1016/j.jss.2009.09.033 Published By: Lattice Science Publication (LSP) © Copyright: All rights reserved. 3 Retrieval Number:100.1/ijsepm.B76520712223 DOI:10.54105/ijsepm.B7652.073223 Journal Website: www.ijsepm.latticescipub.com Published By: Lattice Science Publication (LSP) © Copyright: All rights reserved. 3 A Comparative Study of Different Software Complexity Metrics in Measuring Software Interoperability 10. Oussalah, M., & Boufaïda, Z. (2014). Software complexity metrics for Retrieval Number:100.1/ijsepm.B76520712223 DOI:10.54105/ijsepm.B7652.073223 Journal Website: www.ijsepm.latticescipub.com A Comparative Study of Different Software Complexity Metrics in Measuring Software Interoperability Walia, G., & Kaur, A. (2019). A systematic review on software complexity metrics. Journal of Systems and Software, 157, 110406. doi: 10.1016/j.jss.2019.11040 Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of the Lattice Science Publication (LSP)/ journal and/ or the editor(s). The Lattice Science Publication (LSP)/ journal and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. Published By: Lattice Science Publication (LSP) © Copyright: All rights reserved. Retrieval Number:100.1/ijsepm.B76520712223 DOI:10.54105/ijsepm.B7652.073223 Journal Website: www.ijsepm.latticescipub.com Retrieval Number:100.1/ijsepm.B76520712223 DOI:10.54105/ijsepm.B7652.073223 Journal Website: www.ijsepm.latticescipub.com Retrieval Number:100.1/ijsepm.B76520712223 DOI:10.54105/ijsepm.B7652.073223 Journal Website: www.ijsepm.latticescipub.com Published By: Lattice Science Publication (LSP) © Copyright: All rights reserved. 4
https://openalex.org/W4382280246	https://www.mdpi.com/2079-9292/12/13/2831/pdf?version=1687839019	English	null	Broad Stopband, Low-Loss, and Ultra-Compact Dual-Mode Bandpass Filter Based on HMSIRC	Electronics	2,023	cc-by	11,327	Keywords: substrate-integrated rectangular cavity; bandpass ﬁlter; dual-mode; broad stopband Citation: Barik, R.K.; Koziel, S.; Pietrenko-Dabrowska, A. Broad Stopband, Low-Loss, and Ultra-Compact Dual-Mode Bandpass Filter Based on HMSIRC. Electronics 2023, 12, 2831. https://doi.org/ 10.3390/electronics12132831 Citation: Barik, R.K.; Koziel, S.; Pietrenko-Dabrowska, A. Broad Stopband, Low-Loss, and Ultra-Compact Dual-Mode Bandpass Filter Based on HMSIRC. Electronics 2023, 12, 2831. https://doi.org/ 10.3390/electronics12132831 electronics electronics electronics electronics Article Broad Stopband, Low-Loss, and Ultra-Compact Dual-Mode Bandpass Filter Based on HMSIRC Rusan Kumar Barik 1 , Slawomir Koziel 1,2,* and Anna Pietrenko-Dabrowska 2 1 Engineering Optimization and Modeling Center, Reykjavik University, 102 Reykjavik, Iceland; rusanb@ru.is 2 Faculty of Electronics, Telecommunication and Informatics, Gdansk University of Technology, 80-233 Gdansk, Poland; anna.dabrowska@pg.edu.pl * Correspondence: koziel@ru.is 1 Engineering Optimization and Modeling Center, Reykjavik University, 102 Reykjavik, Iceland; rusanb@ru.is 2 Faculty of Electronics, Telecommunication and Informatics, Gdansk University of Technology, 80-233 Gdansk, Poland; anna.dabrowska@pg.edu.pl * Correspondence: koziel@ru.is Abstract: In this investigation, an ultra-compact dual-mode bandpass ﬁlter (BPF) with a wide stopband response is realized by using a half-mode substrate-integrated rectangular cavity (HMSIRC). The HMSIRC resonator is designed with a cavity that is rectangular in shape and has metallic vias along three of the sides. The fourth side is open-ended and contains microstrip feed lines. For the purpose of constructing a magnetic wall, a rectangular slot is cut into each of the HMSIRC’s three edges. In order to produce an electrical wall that may generate a variety of resonances, the side with the open edges is provided with a single metallic via in the center. After that, a second-order BPF is generated by loading a transverse slot in the middle of the BPF, which enables independent frequency regulation of the mode frequencies. The eigen-mode analysis; ﬁeld distributions; coupling matrix; and full-wave simulation of the proposed HMSIRC ﬁlter topology are used to develop the working principle of the ﬁlter. A second-order BPF is realized, constructed, and experimentally validated in order to provide evidence that the theory is correct. The BPF prototype achieves satisfactory performance thanks to its compact footprint of 0.028 λg2; its broad passband of 15.9%; its low insertion loss of 0.41 dB; and its wide stopband of 4.36 f 0 with a rejection level greater than 20 dB. Both the measured and EM-simulated responses of the BPF are very consistent with one another. 1. Introduction The development of bandpass ﬁlters (BPFs) with low cost, compact size, better fre- quency selectivity, and broadband suppression has been greatly inﬂuenced by the incredible advancements in wireless communication technology. In ﬁlter design for base stations, waveguide structures are frequently used due to their low loss, high power handling capability, and high Q-factor. However, integrating a rectangular waveguide with other planar microwave components is costly and challenging. Recently, substrate-integrated waveguides (SIW) have received a lot of attention because they are inexpensive, lightweight, feature low insertion loss, are simple to fabricate, and work well with other planar circuits. These characteristics make them ideal for meeting the high-performance demands imposed on ﬁlter designs [1–3]. Academic Editors: Yongqiang Wang and Ningning Yan Received: 15 May 2023 Revised: 22 June 2023 Accepted: 24 June 2023 Published: 26 June 2023 g Several BPFs have been constructed using SIW technology [4–47]. Narrow and wide- band BPFs have been realized using the SIW coaxial cavity, as described in [4]. The authors of [5] describe a three-pole BPF employing a dual-mode circular SIW cavity with tunable transmission zeros. To implement a SIW ﬁlter with a wide stopband, [6] uses a variant of the mode suppression technique. There is an analysis and implementation of ﬁlters based on quarter-mode substrate integrated waveguides (QMSIWs) in [7]. The authors of [8] describe a BPF with a wide upper stopband response that was built using a multi-layered SIW. Triple-mode BPFs based on a SIW square cavity ﬁlled with CSRRs are shown in [9]. The authors of [10] describe SIW square cavities with orthogonal and in-line ports that Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/electronics Electronics 2023, 12, 2831. https://doi.org/10.3390/electronics12132831 Electronics 2023, 12, 2831 2 of 13 are used to build BPFs with adaptable outputs. The authors of [11] describe bandpass ﬁlters that have been created using dual-mode SIW cavities operating within various boundaries. Combining QMSIW and EMSIW cavities allows for a small BPF with a wide stopband response, as shown in [12]. The authors of [13] describe a wideband ﬁlter and diplexer using a dual-mode SIW cavity with variable reactions. Using dual-mode SIW radial cavities, a wideband BPF has been realized [14]. Dual-mode miniaturized BPFs have been realized using half-mode SIW cavities [15]. The authors of [16] describe how it has been suggested that dual-mode SIW cavities be used to construct box-like BPFs with a wide stopband response. The use of perturbed SIW cavities has allowed for the development of high-order BPFs, as shown in [17]. Using a combination of microstrip technology and SIW, the authors of [18] explore the fabrication of a small BPF with a wide stopband response. Using half-mode SIW with stepped-impedance resonators, [19] creates a compact bandpass ﬁlter. A broadband SIW bandpass ﬁlter based on electromagnetic bandgap (EBG) units for wide stopband response has been constructed with good selectivity [20]. A HMSIW fan-shaped circular cavity has been examined in order to build a bandpass ﬁlter with decreased size and order-extensible capabilities [21]. Within the scope of this study, a bandpass ﬁlter of the second order was upgraded to a ﬁlter of the fourth order without the cavity’s actual size having to be increased. A HMSIW cavity was used to construct planar bandpass ﬁlters with a small size for the research presented in [22]. These fourth-order ﬁlters have a fractional bandwidth of 31.8%, one/two transmission zeros, and a reduced footprint area of 0.159 λg2. Using a T-septum HMSIW cavity, third-order bandpass ﬁlters have been created and described in [23]. These ﬁlters have an insertion loss of less than 2.61 dB and a fractional bandwidth of 17.77%, while also having a wide stopband of 10.37 f 0. Higher-order SIW resonators were used in the construction of a narrow-band ﬁlter that was published in [24]. This ﬁlter offers high selectivity, a wide stopband, and three transmission zeros. This ﬁlter has an insertion loss Electronics 2023, 12, 2831 3 of 13 of 1.5 dB, a stopband response that ranges from 6.4 GHz to 7.8 GHz, and a fractional bandwidth that is 30% wider than the full bandwidth. of 1.5 dB, a stopband response that ranges from 6.4 GHz to 7.8 GHz, and a fractional bandwidth that is 30% wider than the full bandwidth. A broadband bandpass ﬁlter has been realized using SIW in conjunction with defective ground structures [36]. This 8.98 GHz operating ﬁlter has a fractional bandwidth of 47.4%, an insertion loss of 1.5 dB, and a strong roll-off factor. The authors of [37] describe a SIW cavity loaded with composite right/left-handed transmission lines and complementary split-ring resonators that is used to create a dual-frequency bandpass ﬁlter. The ﬁlter has a 3% and a 4.2% bandwidth at its 5 GHz and 7.5 GHz resonant frequencies, respectively. The authors of [38] describe a ﬂexible bandpass ﬁlter based on a SIW hexagonal resonator. This ﬁlter has an insertion loss of 2.01 dB and a fractional bandwidth of 2.92 percent. The authors of [39] describe a SIW bandpass ﬁlter consisting of two resonators and three inverters. A bandpass ﬁlter has been constructed utilizing a SIW cavity with iris resonators, as described in [40]. The frequency of operation for this ﬁlter is 9.77 GHz, and it has a fractional bandwidth of 12.17% and an insertion loss of 1.19 dB. The paper [41] describes the construction of a bandpass ﬁlter using a SIW cavity with a defective ground structure in order to get wideband responses. With a passband ranging from 3.0 GHz to 11.0 GHz, an insertion loss of 1.2 dB, and a notched band, this ﬁlter is able to achieve a smaller footprint. The authors of [42] describe a narrow bandpass ﬁlter that has been realized by using a rectangular SIW cavity loaded with inductive posts on the top surface. The center frequency of this ﬁlter is 12.2 GHz, and it has an insertion loss of 1.22 dB and a fractional bandwidth of 1.475%. The authors of [43] describe a dual-mode bandpass ﬁlter that has been constructed by using a SIW cavity loaded with cross-shaped slots. This ﬁlter has a fractional bandwidth of 9.1% at the center frequency of 7.5 GHz and two transmission zeros at 12.5 GHz and 15 GHz, respectively. 2. Design and Analysis of the HMSIRC-Based Bandpass Filter g y 2.1. Conﬁguration and Working Principles Using a rectangular SIW cavity in conjunction with stepped impedance resonators, the authors of [44] were able to create a bandpass ﬁlter with a reduced size. This ﬁlter has a footprint size of 0.3 λg2, it works at 4.8 GHz, it offers four transmission zeros, and it has a fractional bandwidth of 13%. The authors of [45] describe a dual-mode bandpass ﬁlter operating at 5.8 GHz, which has been constructed by utilizing a SIW cavity loaded with a circular patch slot. The authors of [46] describe a bandpass ﬁlter with high design ﬂexibility that has been realized by using a SIW cavity in non-resonating mode. The authors of [47] describe a rectangular SIW cavity loaded with two pairs of complementary rectangular split-ring resonators. This ﬁlter has a transmission zero at 5.9 GHz, an insertion loss of 2.4 dB, and a bandwidth of 320 MHz at 3 dB. An ultra-compact dual-mode bandpass ﬁlter (BPF) with a broad stopband response is constructed in this study by using a half-mode substrate-integrated rectangular cavity (HMSIRC). Eigen-mode analysis, ﬁeld distributions, the coupling matrix, and full-wave simulation of the proposed HMSIRC ﬁlter topology are used to construct the ﬁlter’s working theory. Realization, prototyping, and experimental validation of a second-order BPF are accomplished to prove the concept. The ﬁlter exhibits the following key features: (i) It occupies a footprint area of only 0.028 λg2, which is extremely small compared to the dual-mode BPFs that have previously been reported; (i) It occupies a footprint area of only 0.028 λg2, which is extremely small compared to the dual-mode BPFs that have previously been reported; (ii) Its insertion loss is 0.41 dB; p y p (ii) Its insertion loss is 0.41 dB; (iii) It exhibits a passband of 15.9%, a broad stopband response of 4.36 f 0, and a rejection level of 20 dB. (iii) It exhibits a passband of 15.9%, a broad stopband response of 4.36 f 0, and a rejection level of 20 dB. Utilizing a SIW cavity equipped with perturbing vias and a complementary split-ring resonator, an application-speciﬁc bandpass ﬁlter for frequencies below 6 GHz was developed and described in [25]. In addition to having a fractional bandwidth of 1.16% and high selectivity, this ﬁlter has an insertion loss of 2.9 dB. The article [27] describes the construction of a bandpass ﬁlter for X-band applications that is based on a dual-mode SIW cavity. This ﬁlter has two transmission zeros located at 10.75 GHz and 13.3 GHz, and it operates at a frequency of 12 GHz. The fractional bandwidth of this ﬁlter is 11%. Utilizing a double-layer HMSIW resonator resulted in the development of a bandpass ﬁlter that was described in reference [28]. This ﬁlter makes use of a microstrip defect structure in order to provide a wide stopband response. Using a rectangular SIW cavity ﬁlled with a D-shaped ring resonator, the research paper [29] describes the construction of a bandpass ﬁlter with two bands. Applications that need frequencies lower than 6 GHz may make use of this ﬁlter since it works at 2.66 and 3.54 GHz. The authors of [30] describe a SIW ﬁlter with asymmetric responses. This ﬁlter also makes use of a non-resonating node and positive coupling in order to improve its selectivity. A bandpass ﬁlter that is based on SIW cavities has been constructed and described in [31]. This ﬁlter achieves harmonic suppression by combining the functionality of the SIW cavity with that of interdigital resonators. The authors of [32] describe a broadband bandpass ﬁlter on HMSIW cavities with a size reduction that has been created. Transmission zeros and favorable stopband responses are generated using a three-stage stepped impedance resonator in this ﬁlter. An in-line HMSIW cavity is used to create a narrow-band bandpass ﬁlter, as demonstrated in [33]. This ﬁlter enhances selectivity by generating limited transmission zeros using interdigital slots to create quasi-elliptic responses. The authors of [34] describe a bandpass ﬁlter operating at 6 GHz for 5G applications that has been realized using a rectangular SIW cavity. This ﬁlter employs D-shaped resonators for size reduction and wide stopband responses. The authors of [35] describe the creation of a bandpass ﬁlter that is based on a rectangular SIW cavity that is loaded with many complimentary split-ring resonators. g y 2.1. Conﬁguration and Working Principles This is to keep the radiation losses reasonably low [2]. Next, a magnetic wall is created by etching an open-ended slot in the cavity with metallic vias on each of its three sides. al distance S1 is chosen by applying the following criteria: S0/λ ≤ 0.1 and S0 s is to keep the radiation losses reasonably low [2]. Next, a magnetic wall is cr hing an open-ended slot in the cavity with metallic vias on each of its three si  longitudinal distance S1 is chosen by applying the following criteria: S0/λ ≤0.1 and S0/S1 ≥0.5. This is to keep the radiation losses reasonably low [2]. Next, a magnetic wall is created by etching an open-ended slot in the cavity with metallic vias on each of its three sides. al distance S1 is chosen by applying the following criteria: S0/λ ≤ 0.1 and S0 s is to keep the radiation losses reasonably low [2]. Next, a magnetic wall is cr ing an open-ended slot in the cavity with metallic vias on each of its three si (a) (b) ure 1. (a) Half-mode SIRC-based bandpass ﬁlter conﬁguration. The design parameter v = 53.1, Ws = 26.1, L1 = 7.0, L2 = 24, L3 = 23.5, L4 = 25.3, Lf = 5.0, W1 = 0.6, W3 = 0.65, Wf = 1.1 = 0.9, S2 = 0.5, and hs = 0.508; unit: mm. (b) The ﬁlter’s coupling topology. Figure 1. (a) Half-mode SIRC-based bandpass ﬁlter conﬁguration. The design parameter values are: Ls = 53.1, Ws = 26.1, L1 = 7.0, L2 = 24, L3 = 23.5, L4 = 25.3, Lf = 5.0, W1 = 0.6, W3 = 0.65, Wf = 1.18, S0 = 0.6, S1 = 0.9, S2 = 0.5, and hs = 0.508; unit: mm. (b) The ﬁlter’s coupling topology. (b) (b) ure 1. (a) Half-mode SIRC-based bandpass ﬁlter conﬁguration. The design parameter v 53.1, Ws = 26.1, L1 = 7.0, L2 = 24, L3 = 23.5, L4 = 25.3, Lf = 5.0, W1 = 0.6, W3 = 0.65, Wf = 1.18 0 9 S2 = 0 5 and h = 0 508; unit: mm (b) The ﬁlter’s coupling topology Figure 1. (a) Half-mode SIRC-based bandpass ﬁlter conﬁguration. g y 2.1. Conﬁguration and Working Principles The proposed dual-mode bandpass ﬁlter layout and coupling topology are shown in Figure 1. The ﬁlter is constructed using a transverse slot, an open-ended slot, a metallic via, and a half-mode substrate-integrated rectangular cavity (HMSIRC). Initially, an HMSIRC is Electronics 2023, 12, 2831 4 of 13 created by the longitudinal bisection of the SIRC. The mode frequencies of the HMSIRC are determined as [2]: 0 1 2 2 HMSIRC mn HMSIRC HMSIRC eq eq r m n f L W ε     = +             created by the longitudinal bisection of the SIRC. The mode frequencies of the HMSIRC are determined as [2]: 0 1 2 2 HMSIRC mn HMSIRC HMSIRC eq eq r m n f L W ε     = +             fmn0HMSIRC = 1 2√εr s m LeqHMSIRC 2 + n 2WeqHMSIRC 2 (1) 2 2 0 0 1 1.08 0.1 C S S S S L S L = − + is the diameter S0 of the vias and their (1) where    LeqHMSIRC = LS −1.08 S02 S1 + 0.1 S02 LS WeqHMSIRC = WS −1.08 S02 S1 + 0.1 S02 WS ere 2 2 0 0 1 1.08 0.1 HMSIRC eq S S S S W W S W   = − +  is t where    LeqHMSIRC = LS −1.08 S02 S1 + 0.1 S02 LS WeqHMSIRC = WS −1.08 S02 S1 + 0.1 S02 WS is the diameter S0 of the vias, and their ere 2 2 0 0 1 1.08 0.1 HMSIRC eq S S S S W W S W   = − +  is the diameter S0 of the vias, and the l d h b l h f ll / d where    LeqHMSIRC = LS −1.08 S02 S1 + 0.1 S02 LS WeqHMSIRC = WS −1.08 S02 S1 + 0.1 S02 WS is the diameter S0 of the vias, and their 2 2 0 0 1 1.08 0.1 HMSIRC eq S S S S W W S W  = − +   1 S longitudinal distance S1 is chosen by applying the following criteria: S0/λ ≤0.1 and S0/S1 ≥0.5. 2.2. Synthesis of a Dual-Mode Bandpass Filter ll h t ll l d d t lli 2.2. Synthesis of a Dual-Mode Bandpass Filter ll h t ll l d d t lli The suggested dual-mode BPF using the half-mode SIRC cavity is shown in Figure 1a. The rectangular slot along the three sides of the half-mode cavity produces a magnetic wall, whereas a centrally loaded metallic via creates an electric wall. This arrangement generates various modes. Figure 2 exhibits the distributions of the electric ﬁeld to express the various modes. Without loading the slots, the resonant frequencies of the HMSIW cavity modes TE110, TE210, TE310, and TE120 are 3.45 GHz, 4.35 GHz, 5.52 GHz, and 6.35 GHz, respectively. Because of the slot loading, the resonant frequencies of these modes are decreased due to the increase in capacitive loading on the top surface of the cavity, which results in the realization of a second-order ﬁlter at a lower frequency. Figure 3 depicts the distributions of the vector H-ﬁeld to examine the inﬂuence of the transverse slot. Since the transverse slot is perpendicular to the vector H-ﬁeld directions of the TE110 and TE120 modes, as shown in Figure 3a,d, it has a profound effect on these modes. From Figure 3b,c, it is observed that the H-ﬁled direction of the modes TE210 and TE310 is parallel to the transverse slot, which has no signiﬁcant effect on the resonant frequencies. The effect of the length L3 of the transverse slot on the resonant frequencies of the modes is shown in Figure 4. As it can be seen, the resonant frequencies of the TE110 and TE120 modes are decreased by increasing the length of the transverse slot, whereas there is no signiﬁcant change in the resonant frequencies of the TE210 and TE310 modes. It is inferred that the length of the transverse slot can control the resonant frequency of the modes that allow us to realize a second-order BPF. ll, whereas a centrally loaded metallic via creates an electric wall. This arrangem nerates various modes. Figure 2 exhibits the distributions of the electric ﬁeld to expr e various modes. Without loading the slots, the resonant frequencies of the HMSIW c modes TE110, TE210, TE310, and TE120 are 3.45 GHz, 4.35 GHz, 5.52 GHz, and 6.35 GH pectively. g y 2.1. Conﬁguration and Working Principles The design parameter values are: Ls = 53.1, Ws = 26.1, L1 = 7.0, L2 = 24, L3 = 23.5, L4 = 25.3, Lf = 5.0, W1 = 0.6, W3 = 0.65, Wf = 1.18, S0 = 0.6, S1 = 0.9, S2 = 0.5, and hs = 0.508; unit: mm. (b) The ﬁlter’s coupling topology. , , ; ( ) p g p gy Subsequently, a metallic via is placed in the middle of the cavity, which gen ctric wall. These arrangements provide multiple modes of resonance. Finally se slot is introduced at the middle of the cavity to control the mode frequencie bles us to develop a dual-mode bandpass ﬁlter. The top of the HMSIW is the h a rectangular ring slot, which increases the capacitive loading of the cavity n, provides an operating frequency that is lower than the cutoﬀ frequency of th ause of this result it is possible to accomplish size reduction The substrate Subsequently, a metallic via is placed in the middle of the cavity, which generates an electric wall. These arrangements provide multiple modes of resonance. Finally, a transverse slot is introduced at the middle of the cavity to control the mode frequencies, which enables us to develop a dual-mode bandpass ﬁlter. The top of the HMSIW is then etched with a rectangular ring slot, which increases the capacitive loading of the cavity. This, in turn, provides an operating frequency that is lower than the cutoff frequency of the cavity. Because of this result, it is possible to accomplish size reduction. The substrate used to create the ﬁlter is Roger’s RO4003C, which has a thickness of 0.508 mm, a permittivity of 3.38, and a loss tangent of 0.0021. 5 of 13 5 o Electronics 2023, 12, 2831 nics 2023, 12, 831 2.2. Synthesis of a Dual-Mode Bandpass Filter ll h t ll l d d t lli Electric ﬁeld distribution of four diﬀerent modes: (a) TE110 mode, (b) TE210 mode, (c) TE31 mode, and (d) TE120 mode. (a) (b) 12, 831 6 of 13 (c) (d) Figure 3. Vector H-ﬁeld distribution of four diﬀerent modes: (a) TE110 mode, (b) TE210 mode, (c) TE310 mode, and (d) TE120 mode. Figure 3. Vector H-ﬁeld distribution of four different modes: (a) TE110 mode, (b) TE210 mode, (c) TE310 mode, and (d) TE120 mode. 3, 12, 831 (c) (d) Figure 3. Vector H-ﬁeld distribution of four diﬀerent modes: (a) TE110 mode, (b) TE210 mode, mode, and (d) TE120 mode. (b) (d) (d) (a) 831 2, 831 (a) ( ) (d) (d) (c) (c) (d) (c) ( igure 3. Vector H-ﬁeld distribution of four diﬀerent modes: (a) TE110 mode, (b) TE210 mode, (c) TE31 mode, and (d) TE120 mode. Figure 3. Vector H-ﬁeld distribution of four different modes: (a) TE110 mode, (b) TE210 mode, (c) TE310 mode, and (d) TE120 mode. Figure 3. Vector H-ﬁeld distribution of four diﬀerent modes: (a) TE110 mode, (b) TE210 mode, mode, and (d) TE120 mode. Figure 4. Variation of resonant frequencies for diﬀerent modes. Mode Frequency [GHz] Figure 4. Variation of resonant frequencies for diﬀerent modes. Mode Frequency [GHz] Figure 4. Variation of resonant frequencies for different modes. i f f f d ﬀ d Mode Frequency [GHz] Mode Frequency [GHz] Mode Frequency [GHz] Mode Frequency [GHz] gure 4. Variation of resonant frequencies for diﬀerent modes. Figure 4. Variation of resonant frequencies for diﬀerent m Figure 4. Variation of resonant frequencies for different modes. gure 4. Variation of resonant frequencies for diﬀerent modes. Figure 4. Variation of resonant frequencies for diﬀerent modes. Figure 4. Variation of resonant frequencies for different modes. The coupling coeﬃcient Kj,j+1, the source quality factors QS, and the load quality factor QL can be determined as [26]: 0 S L f Q Q = = (2 The coupling coeﬃcient Kj,j+1, the source quality factors QS, and the load quality QL can be determined as [26]: 0f Q Q For veriﬁcation, a second-order BPF has been synthesized. The circuit exhibits a center frequency of 0.835 GHz, a return loss of 26.3 dB, and a relative bandwidth of 5.43%. 2.2. Synthesis of a Dual-Mode Bandpass Filter ll h t ll l d d t lli The unloaded quality factor Qu is computed to be 382 using eigen-mode analysis and factoring in losses attributable to radiation, conductor, and dielectric. (c) (d) Figure 2. Electric ﬁeld distribution of four diﬀerent modes: (a) TE110 mode, (b) TE210 mode, (c) TE310 mode, and (d) TE120 mode. (a) (b) 2, 831 6 of 13 (c) (d) Figure 3. Vector H-ﬁeld distribution of four diﬀerent modes: (a) TE110 mode, (b) TE210 mode, (c) TE310 mode, and (d) TE120 mode. Figure 4. Variation of resonant frequencies for diﬀerent modes. The coupling coeﬃcient Kj,j+1, the source quality factors QS, and the load quality factor QL can be determined as [26]: 0 3dB S L f Q Q f± = = Δ (2) 2 2 2 1 1 2 2 m m j j f f K + − = (3) Mode Frequency [GHz] Figure 3. Vector H-ﬁeld distribution of four different modes: (a) TE110 mode, (b) TE210 mode, (c) TE310 mode, and (d) TE120 mode. , 12, 831 6 (c) (d) Figure 3. Vector H-ﬁeld distribution of four diﬀerent modes: (a) TE110 mode, (b) TE210 mode, (c mode, and (d) TE120 mode. Figure 4. Variation of resonant frequencies for diﬀerent modes. The coupling coeﬃcient Kj,j+1, the source quality factors QS, and the load quality QL can be determined as [26]: 0 3dB S L f Q Q f± = = Δ 2 2 2 1 m m f f K − Mode Frequency [GHz] Figure 4. Variation of resonant frequencies for different modes. For veriﬁcation, a second-order BPF has been synthesized. The circuit exhibits a center frequency of 0.835 GHz, a return loss of 26.3 dB, and a relative bandwidth of 5.43%. The synthesis method of [3] has been used to compute the coupling coefﬁcient, external quality factor, and coupling matrix as K12 = 0.0528, QS = QL = 6.5, and M =   0 1.4754 0 0 1.4754 0 2.2887 0 0 2.2887 0 1.4754   (4) where the center frequency and the 3 dB bandwidth are denoted by f 0 and f ±3dB; fm1 and fm2 stand for mode frequencies. The transverse slot’s length allows us to predict the bandwidth of the ﬁlter. The unloaded quality factor Qu is computed to be 382 using eigen-mode analysis and factoring in losses attributable to radiation, conductor, and dielectric. (c) (d) igure 2. 2.2. Synthesis of a Dual-Mode Bandpass Filter ll h t ll l d d t lli Because of the slot loading, the resonant frequencies of these modes are ased due to the increase in capacitive loading on the top surface of the cavity, wh ults in the realization of a second-order ﬁlter at a lower frequency. Figure 3 depicts tributions of the vector H-ﬁeld to examine the inﬂuence of the transverse slot. Since nsverse slot is perpendicular to the vector H-ﬁeld directions of the TE110 and T odes, as shown in Figure 3a,d, it has a profound eﬀect on these modes. From Figure 3 s observed that the H-ﬁled direction of the modes TE210 and TE310 is parallel to the tra rse slot, which has no signiﬁcant eﬀect on the resonant frequencies. The eﬀect of ngth L3 of the transverse slot on the resonant frequencies of the modes is shown in Fig As it can be seen, the resonant frequencies of the TE110 and TE120 modes are decrea increasing the length of the transverse slot, whereas there is no signiﬁcant change e resonant frequencies of the TE210 and TE310 modes. It is inferred that the length of nsverse slot can control the resonant frequency of the modes that allow us to realiz ond-order BPF. (a) (b) (c) (d) Figure 2. Electric ﬁeld distribution of four diﬀerent modes: (a) TE110 mode, (b) TE210 mode, (c) T mode, and (d) TE120 mode. Figure 2. Electric ﬁeld distribution of four different modes: (a) TE110 mode, (b) TE210 mode, (c) TE310 mode, and (d) TE120 mode. (b) (a) (b) (a) (d) (c) (d) (c) ure 2. Electric ﬁeld distribution of four diﬀerent modes: (a) TE110 mode, (b) TE210 mode, (c) T de, and (d) TE120 mode. Figure 2. Electric ﬁeld distribution of four different modes: (a) TE110 mode, (b) TE210 mode, (c) TE310 mode, and (d) TE120 mode. The coupling coefﬁcient Kj,j+1, the source quality factors QS, and the load quality factor QL can be determined as [26]: QS = QL = f0 ∆f±3dB (2) Kj,j+1 = fm22 −fm12 fm22 + fm12 (3) QS = QL = f0 ∆f±3dB Kj,j+1 = fm22 −fm12 fm22 + fm12 (2) (3) Electronics 2023, 12, 2831 6 of 13 where the center frequency and the 3 dB bandwidth are denoted by f 0 and f ±3dB; fm1 and fm2 stand for mode frequencies. The transverse slot’s length allows us to predict the bandwidth of the ﬁlter. 2.2. Synthesis of a Dual-Mode Bandpass Filter ll h t ll l d d t lli The synthesis method of [3] has been used to compute the coupling coefﬁcient, external quality factor, and coupling matrix as K12 = 0.0528, QS = QL = 6.5, and 3dB f± Δ ( ) 2 2 2 1 , 1 2 2 2 1 m m j j m m f f K f f + − = + (3) 3dB S L Q Q f± Δ 2 2 2 1 , 1 2 2 2 1 m m j j m m f f K f f + − = + M =   0 1.4754 0 0 1.4754 0 2.2887 0 0 2.2887 0 1.4754 0 0 1.4754 0   (4) (3 (4) 7 of 13 7 of 13 Electronics 2023, 12, 2831 The BPF’s coupling coefﬁcient and quality factor are controlled via a transverse slot. Figure 5 shows how the coupling coefﬁcient of the BPF varies with the slot width. The coupling coefﬁcient K12 is shown to decrease with increasing slot width W3. S-parameters calculated by the coupling matrix and electromagnetic simulation for the proposed BPF are shown in Figure 6. The total loss of the proposed BPF is attributed to radiation loss, dielectric loss, and conductor loss. Figure 7 shows the estimated losses of the second-order BPF. As illustrated, the total loss, dielectric plus radiation loss, and radiation loss are all less than 0.143, 0.095, and 0.075, respectively. As a result, the proposed BPF exhibits a very low insertion loss of 0.41 dB. coupling coeﬃcient K12 is shown to decrease with increasing slot width W calculated by the coupling matrix and electromagnetic simulation for th are shown in Figure 6. The total loss of the proposed BPF is attributed dielectric loss, and conductor loss. Figure 7 shows the estimated losses of BPF. As illustrated, the total loss, dielectric plus radiation loss, and radi less than 0.143, 0.095, and 0.075, respectively. As a result, the proposed BP low insertion loss of 0.41 dB. 7 o coupling coeﬃcient K12 is shown to decrease with increasing slot width W3. S-paramet calculated by the coupling matrix and electromagnetic simulation for the proposed B are shown in Figure 6. The total loss of the proposed BPF is attributed to radiation lo dielectric loss, and conductor loss. Figure 7 shows the estimated losses of the second-or BPF. 2.2. Synthesis of a Dual-Mode Bandpass Filter ll h t ll l d d t lli As illustrated, the total loss, dielectric plus radiation loss, and radiation loss are Figure 5. Variation of coupling coeﬃcient as a function of W3. 0.2 0.3 0.4 0.5 0.6 0.7 0.8 W3 [mm] 0 0.05 0.1 0.15 K12 0.5 1 1.5 2 2.5 3 3.5 F [GH ] -70 -60 -50 -40 -30 -20 -10 0 \|S11\|, \|S21\| [dB] \|S11\| (Coupling Matrix) \|S21\| (Coupling Matrix) \|S11\| (EM Simulation) \|S21\| (EM Simulation) Figure 5. Variation of coupling coefﬁcient as a function of W3. less than 0.143, 0.095, and 0.075, respectively. As a result, the proposed BPF exhibits low insertion loss of 0.41 dB. Figure 5. Variation of coupling coeﬃcient as a function of W3. Figure 6. Dual-mode BPF S-parameters estimated using coupling matrix and EM simulation 0.2 0.3 0.4 0.5 0.6 0.7 0.8 W3 [mm] 0 0.05 0.1 0.15 K12 0.5 1 1.5 2 2.5 3 3.5 Frequency [GHz] -70 -60 -50 -40 -30 -20 -10 0 \|S11\|, \|S21\| [dB] \|S11\| (Coupling Matrix) \|S21\| (Coupling Matrix) \|S11\| (EM Simulation) \|S21\| (EM Simulation) Figure 6. Dual-mode BPF S-parameters estimated using coupling matrix and EM simulation. 0.2 0.3 0.4 0.5 0.6 0.7 0.8 W3 [mm] 0 0.05 0.1 0.15 K12 less than 0.143, 0.095, and 0.075, respectively. As a result, the prop low insertion loss of 0.41 dB. 0.2 0.3 0.4 0.5 0.6 0.7 0.8 W3 [mm] 0 0.05 0.1 0.15 K12 Figure 5. Variation of coupling coeﬃcient as a func Figure 5. Variation of coupling coefﬁcient as a function of W3. Figure 5. Variation of coupling coeﬃcient as a function of W Figure 5. Variation of coupling coeﬃcient as a func Figure 5. Variation of coupling coefﬁcient as a function of W3. Figure 5. Variation of coupling coeﬃcient as a function of W -60 -50 -40 -30 -20 -10 0 \|S11\|, \|S21\| [dB] \|S11\| (Coupling Matr \|S21\| (Coupling Matr \|S11\| (EM Simulation \|S21\| (EM Simulation 0.5 1 1.5 2 2.5 3 3.5 Frequency [GHz] -70 -60 -50 -40 -30 -20 -10 0 \|S11\|, \|S21\| [dB] \|S11\| (Coupling Matrix) \|S21\| (Coupling Matrix) \|S11\| (EM Simulation) \|S21\| (EM Simulation) 0 5 1 1 5 2 2 5 3 3 5 -70 Figure 6. Dual-mode BPF S-parameters estimated using coupling matrix and EM simulat Figure 6. Dual-mode BPF S-parameters estimated using coupling matrix and EM simulation. 8 of 13 8 of Electronics 2023, 12, 2831 Electronics 2023, 12, 831 Figure 7. 2.2. Synthesis of a Dual-Mode Bandpass Filter ll h t ll l d d t lli Estimated losses of the proposed second-order BPF. Loss Figure 7. Estimated losses of the proposed second-order BPF. Figure 7. Estimated losses of the proposed second-order BPF. 2.3. Analysis of Insertion Loss In this subsection, insertion loss owing to frequency enhancement i lowering the value of the cavity parameter Ls, the suggested HMSIW-bas achieves a higher resonant frequency. In order to observe the eﬀect on in an increase in frequency, we varied the parameter Ls and obtained the Figure 8 shows the insertion losses at diﬀerent frequencies with varying there are no signiﬁcant changes in the insertion loss when frequency inc tion loss at each center frequency is determined as shown in Table 1. As quency rises from 0.839 GHz to 0.938 GHz, the insertion loss rises from dB which is only a 2 2% increase Figure 7. Estimated losses of the proposed second-order BP Figure 7. Estimated losses of the proposed second-order BPF. quency rises from 0.839 GHz to 0.938 GHz, the 2.3. Analysis of Insertion Loss 2.3. Analysis of Insertion Loss dB, which is only a 2.2% y f In this subsection, insertion loss owing to frequency enhancement is investigated. lowering the value of the cavity parameter Ls, the suggested HMSIW-based bandpass ﬁl achieves a higher resonant frequency. In order to observe the eﬀect on insertion loss w an increase in frequency, we varied the parameter Ls and obtained the EM simulatio Figure 8 shows the insertion losses at diﬀerent frequencies with varying Ls. It is found th there are no signiﬁcant changes in the insertion loss when frequency increases. The ins tion loss at each center frequency is determined as shown in Table 1. As the resonant f quency rises from 0.839 GHz to 0.938 GHz, the insertion loss rises from 0.362 dB to 0 d h h l % In this subsection, insertion loss owing to frequency enhancement is investigated. By lowering the value of the cavity parameter Ls, the suggested HMSIW-based bandpass ﬁlter achieves a higher resonant frequency. In order to observe the effect on insertion loss with an increase in frequency, we varied the parameter Ls and obtained the EM simulations. Figure 8 shows the insertion losses at different frequencies with varying Ls. It is found that there are no signiﬁcant changes in the insertion loss when frequency increases. The insertion loss at each center frequency is determined as shown in Table 1. As the resonant frequency rises from 0.839 GHz to 0.938 GHz, the insertion loss rises from 0.362 dB to 0.37 dB, which is only a 2.2% increase. Table 1. Insertion loss with an increase in operating frequency. Ls (mm) f0 (GHz) IL (dB) 53.1 0.839 0.362 50.4 0.86 0.367 47.7 0.891 0.364 45 0.938 0.37 dB, which is only a 2.2% increase. Table 1. Insertion loss with an increase in operating frequency. Ls (mm) f0 (GHz) IL (dB) 53.1 0.839 0.362 50.4 0.86 0.367 47.7 0.891 0.364 45 0.938 0.37 Insertion Loss [dB] Figure 8. Various insertion losses with an increase in operating frequency for various v Insertion Loss [dB] Figure 8. Various insertion losses with an increase in operating frequency for various values of Ls. nsertio Figure 8. Various insertion losses with an increase in operating frequency for various Figure 8. Various insertion losses with an increase in operating frequency for various values of Ls. 9 of 13 Electronics 2023, 12, 2831 Table 1. Insertion loss with an increase in operating frequency. 2.4. Analogous Circuit Model of a Second-Order Bandpass Filter r of the ﬁlter can be determined either by the number of p hever number is higher will determine the order. The ba 2 4 Analogous Circuit Model of a Second Order Bandpass Filter The order of the ﬁlter can be determined either by the number of poles or the number of zeros; whichever number is higher will determine the order. The bandpass ﬁlter based on HMSIW that was presented is a second-order ﬁlter due to the fact that it creates two poles. In order to verify the ﬁlter, an analogous circuit model is created for the suggested second-order HMSIW-based bandpass ﬁlter, as shown in Figure 9. The metallic vias of the HMSIW may be represented as a two-wire transmission line and characterized as a shunt inductor by Lmv. The magnetic and electrical couplings at the microstrip feed lines can be described using the electrical parameters L01 and C01, respectively. The HMSIW rectangular and transverse slots are portrayed as shunt connected Lor and Cor. The mutual coupling that exists between cavity-backed resonators and HMSIW is shown as a series-connected L02 and C02 in the circuit model. This is a simpler form of the analogous circuit model, which is veriﬁed using the Keysight ADS simulator. Figure 10 illustrates the circuit as well as the EM-simulated S-parameters of the proposed HMSIW-based second-order bandpass ﬁlter. Both the circuit and the EM data show that there is a high level of consistency in the passband regarding the reﬂection coefﬁcient and the insertion loss. e e u be is ig e i dete i e t e o de e ba dpass te based hat was presented is a second-order ﬁlter due to the fact that it creates two r to verify the ﬁlter, an analogous circuit model is created for the suggested HMSIW-based bandpass ﬁlter, as shown in Figure 9. The metallic vias of the be represented as a two-wire transmission line and characterized as a shunt mv. The magnetic and electrical couplings at the microstrip feed lines can be ng the electrical parameters L01 and C01, respectively. The HMSIW rectangu- erse slots are portrayed as shunt connected Lor and Cor. The mutual coupling ween cavity-backed resonators and HMSIW is shown as a series-connected the circuit model. This is a simpler form of the analogous circuit model, ed using the Keysight ADS simulator. Figure 10 illustrates the circuit as well ulated S-parameters of the proposed HMSIW-based second-order bandpass circuit and the EM data show that there is a high level of consistency in the arding the reﬂection coeﬃcient and the insertion loss. 2.4. 2.4. Analogous Circuit Model of a Second-Order Bandpass Filter r of the ﬁlter can be determined either by the number of p hever number is higher will determine the order. The ba 2 4 Analogous Circuit Model of a Second Order Bandpass Filter Analogous Circuit Model of a Second Order Bandpass Filter The order of the ﬁlter can be determined either by the number of poles or the number of zeros; whichever number is higher will determine the order. The bandpass ﬁlter based on HMSIW that was presented is a second-order ﬁlter due to the fact that it creates two poles. In order to verify the ﬁlter, an analogous circuit model is created for the suggested second-order HMSIW-based bandpass ﬁlter, as shown in Figure 9. The metallic vias of the HMSIW may be represented as a two-wire transmission line and characterized as a shunt inductor by Lmv. The magnetic and electrical couplings at the microstrip feed lines can be described using the electrical parameters L01 and C01, respectively. The HMSIW rectangu- lar and transverse slots are portrayed as shunt connected Lor and Cor. The mutual coupling that exists between cavity-backed resonators and HMSIW is shown as a series-connected L02 and C02 in the circuit model. This is a simpler form of the analogous circuit model, which is veriﬁed using the Keysight ADS simulator. Figure 10 illustrates the circuit as well as the EM-simulated S-parameters of the proposed HMSIW-based second-order bandpass ﬁlter. Both the circuit and the EM data show that there is a high level of consistency in the Figure 9. Analogous circuit model of the proposed HMSIW-based BPF. The electrical parameters are Lmv = 15.0061 nH, L01 = 4.3357 nH, C01 = 0.02 pF, L02 = 16.8144 nH, C02 = 0.02 pF, Lor = 2.5092 nH, and Cor = 19.2436 pF. Figure 9. Analogous circuit model of the proposed HMSIW-based BPF. The electrical parameters are Lmv = 15.0061 nH, L01 = 4.3357 nH, C01 = 0.02 pF, L02 = 16.8144 nH, C02 = 0.02 pF, Lor = 2.5092 nH, and Cor = 19.2436 pF. passband regarding the reﬂection coeﬃcient and the insertion loss. Figure 9. Analogous circuit model of the proposed HMSIW-based BPF. The electrical parameters are Lmv = 15.0061 nH, L01 = 4.3357 nH, C01 = 0.02 pF, L02 = 16.8144 nH, C02 = 0.02 pF, Lor = 2.5092 nH, and Cor = 19.2436 pF. ogous circuit model of the proposed HMSIW-based BPF. The electrical parameters 1 nH, L01 = 4.3357 nH, C01 = 0.02 pF, L02 = 16.8144 nH, C02 = 0.02 pF, Lor = 2.5092 nH, 6 pF. Figure 9. Analogous circuit model of the proposed HMSIW-based BPF. 2.3. Analysis of Insertion Loss 2.3. Analysis of Insertion Loss dB, which is only a 2.2% Ls (mm) f0 (GHz) IL (dB) 53.1 0.839 0.362 50.4 0.86 0.367 47.7 0.891 0.364 45 0.938 0.37 9 of 13 Circuit Model of a Second-Order Bandpass Filter f h ﬁl b d i d i h b h b f l h b 9 of 13 Table 1. Insertion loss with an increase in operating frequency. 3. Fabrication, Measurement, and Results For veriﬁcation of the concept, a second-order bandpass ﬁlter based on the HMSIRC has been synthesized, manufactured, and experimentally validated. The suggested ﬁlter works at 835 MHz. The suggested SIW-based second-order BPF operates at 835 MHz and is designed for lower-band (600 MHz to 6 GHz) 5G applications. Eventually, lower frequency bands of 5G will be used for industrial automation and the Internet of Things in addition to its more traditional corporate applications. As a result, in order to fulﬁll the requirements of 5G applications, we developed the SIW-based BPF with a lower operating frequency. The circuit prototype has been shown in Figure 11. Figure 11b shows the measurement setup for the fabricated ﬁlter. We employ a Rohde and Schwarz vector network analyzer to measure this SIW-based BPF. The BPF prototype is ultra-compact, with a size smaller than 0.028 λg2, where λg is the guided wavelength at the resonant frequency. A two port Rohde and Schwarz vector network analyzer has been used to conduct the measurements, which were then compared to the EM-simulated responses shown in Figure 12. At the resonant frequency, the observed insertion loss \|S21\| is 0.41 dB, while the insertion loss \|S21\| from the electromagnetic simulation is 0.36 dB. The BPF exhibits a 3 dB fractional bandwidth of 15.8%. Additionally, it features a broad stopband response of 4.26 f 0. 10 of 13 ency. The circuit prototype has been shown in Figure 11. Figure 11b shows the meas- ent setup for the fabricated ﬁlter. We employ a Rohde and Schwarz vector network zer to measure this SIW-based BPF. The BPF prototype is ultra-compact, with a size er than 0.028 λg2, where λg is the guided wavelength at the resonant frequency. A ort Rohde and Schwarz vector network analyzer has been used to conduct the meas- ents, which were then compared to the EM-simulated responses shown in Figure 12. e resonant frequency, the observed insertion loss \|S21\| is 0.41 dB, while the insertion S21\| from the electromagnetic simulation is 0.36 dB. The BPF exhibits a 3 dB fractional width of 15.8%. Additionally, it features a broad stopband response of 4.26 f0. 10 of 13 frequency. The circuit prototype has been shown in Figure 11. Figure 11b shows the meas- urement setup for the fabricated ﬁlter. We employ a Rohde and Schwarz vector network analyzer to measure this SIW-based BPF. 2.4. Analogous Circuit Model of a Second-Order Bandpass Filter r of the ﬁlter can be determined either by the number of p hever number is higher will determine the order. The ba 2 4 Analogous Circuit Model of a Second Order Bandpass Filter The electrical parameters are Lmv = 15.0061 nH, L01 = 4.3357 nH, C01 = 0.02 pF, L02 = 16.8144 nH, C02 = 0.02 pF, Lor = 2.5092 nH, and Cor = 19.2436 pF. Figure 9. Analogous circuit model of the proposed HMSIW-based BPF. The electrical parameters are Lmv = 15.0061 nH, L01 = 4.3357 nH, C01 = 0.02 pF, L02 = 16.8144 nH, C02 = 0.02 pF, Lor = 2.5092 nH, and Cor = 19.2436 pF. cuit and EM simulation S parameters of the proposed second order BPF 1 1.5 2 2.5 3 3.5 Frequency [GHz] \|S11\| (Circuit Simulation) \|S21\| (Circuit Simulation) \|S11\| (EM Simulation) \|S21\| (EM Simulation) Figure 10. Circuit and EM simulation S-parameters of the proposed second-order BPF. b i i d l 0.5 1 1.5 2 2.5 3 3.5 Frequency [GHz] -70 -60 -50 -40 -30 -20 -10 0 \|S11\|, \|S21\| [dB] \|S11\| (Circuit Simulation) \|S21\| (Circuit Simulation) \|S11\| (EM Simulation) \|S21\| (EM Simulation) Figure 10. Circuit and EM simulation S-parameters of the proposed second-order BPF. Frequency [GHz] Figure 10. Circuit and EM simulation S-parameters of the proposed second-order BPF. Figure 10. Circuit and EM simulation S-parameters of the proposed second-order BPF. Electronics 2023, 12, 2831 10 of 13 10 of 13 3. Fabrication, Measurement, and Results The BPF prototype is ultra-compact, with a size smaller than 0.028 λg2, where λg is the guided wavelength at the resonant frequency. A two port Rohde and Schwarz vector network analyzer has been used to conduct the meas- urements, which were then compared to the EM-simulated responses shown in Figure 12. At the resonant frequency, the observed insertion loss \|S21\| is 0.41 dB, while the insertion loss \|S21\| from the electromagnetic simulation is 0.36 dB. The BPF exhibits a 3 dB fractional bandwidth of 15.8%. Additionally, it features a broad stopband response of 4.26 f0. (a) (b) e 11. Experimental validation of the proposed ﬁlter: (a) BPF prototype and (b) measurement Figure 11. Experimental validation of the proposed ﬁlter: (a) BPF prototype and (b) measurement setup. (a) (b) Figure 11. Experimental validation of the proposed ﬁlter: (a) BPF prototype and (b) measurement setup. (b) (b) (a) (a) (b) (a) 11. Experimental validation of the proposed ﬁlter: (a) BPF prototype and (b) measurement Figure 11. Experimental validation of the proposed ﬁlter: (a) BPF prototype and (b) measurement setup. Figure 11. Experimental validation of the proposed ﬁlter: (a) BPF prototype and (b) measurement setup. re 12. EM-simulated and measured S-parameters of the proposed BPF. 0.5 1 1.5 2 2.5 3 3.5 Frequency [GHz] -70 -60 -50 -40 -30 -20 -10 0 \|S11\| (Measurement) \|S21\| (Measurement) \|S11\| (EM Simulation) \|S21\| (EM Simulation) Figure 12. EM-simulated and measured S-parameters of the proposed BPF. 0.5 1 1.5 2 2.5 3 3.5 Frequency [GHz] -70 -60 -50 -40 -30 -20 -10 0 \|S11\|, \|S21\| [dB] \|S11\| (Measurement) \|S21\| (Measurement) \|S11\| (EM Simulation) \|S21\| (EM Simulation) Figure 12. EM-simulated and measured S-parameters of the proposed BPF. 12. EM-simulated and measured S-parameters of the proposed BPF. Figure 12. EM-simulated and measured S-parameters of the proposed BPF. Figure 12. EM-simulated and measured S-parameters of the proposed BPF. A comparison of the proposed BPF with state-of-the-art BPFs reported in the litera- an be found in Table 2. The proposed second-order BPF has a smaller footprint than ters described in [4–22], with the exception of the ﬁlter described in [23]. The inser- oss of the proposed ﬁlter is also signiﬁcantly lower at 0 41 dB than the ﬁlters de A comparison of the proposed BPF with state-of-the-art BPFs reported in the litera- ture can be found in Table 2. 3. Fabrication, Measurement, and Results The proposed second-order BPF has a smaller footprint than the ﬁlters described in [4–22], with the exception of the ﬁlter described in [23]. The inser- tion loss of the proposed ﬁlter is also signiﬁcantly lower at 0 41 dB than the ﬁlters de A comparison of the proposed BPF with state-of-the-art BPFs reported in the literature can be found in Table 2. The proposed second-order BPF has a smaller footprint than the ﬁlters described in [4–22], with the exception of the ﬁlter described in [23]. The insertion A comparison of the proposed BPF with state-of-the-art BPFs reported in the litera- can be found in Table 2. The proposed second-order BPF has a smaller footprint than lters described in [4–22], with the exception of the ﬁlter described in [23]. The inser- loss of the proposed ﬁlter is also signiﬁcantly lower at 0 41 dB than the ﬁlters de A comparison of the proposed BPF with state-of-the-art BPFs reported in the litera- ture can be found in Table 2. The proposed second-order BPF has a smaller footprint than the ﬁlters described in [4–22], with the exception of the ﬁlter described in [23]. The inser- ti l f th d ﬁlt i l i iﬁ tl l t 0 41 dB th th ﬁlt d A comparison of the proposed BPF with state-of-the-art BPFs reported in the literature can be found in Table 2. The proposed second-order BPF has a smaller footprint than the ﬁlters described in [4–22], with the exception of the ﬁlter described in [23]. The insertion Electronics 2023, 12, 2831 11 of 13 11 of 13 loss of the proposed ﬁlter is also signiﬁcantly lower, at 0.41 dB, than the ﬁlters described in [4–23]. The suggested BPF has a larger fractional bandwidth despite its small size compared to the ﬁlters given in [4–21], except for the ﬁlters presented in [22,23]. It is inferred that the proposed BPF prototype achieved a low insertion loss, a broad stopband response, and a remarkably small size as compared to the benchmark. Table 2. A comparison of the proposed BPF with the state-of-the-art BPFs. Table 2. A comparison of the proposed BPF with the state-of-the-art BPFs. 3. Fabrication, Measurement, and Results Ref fc (GHz) FBW (%) IL (dB) N Upper Stopband Size (λg2) SF (%) [4] 8 20 0.7 2 14 dB@1.81 f 0 0.176 32.9 $ [5] 8 3.51 2.15 3 20 dB@1.25 f 0 0.804 28.53 [6] 7.55 1.84 3.22 3 20 dB@3.85 f 0 1.61 22.2 [7] 4 16 1.02 3 15 dB@1.75 f 0 0.0625 40.9 [8] 5 6.6 0.9 3 30 dB@4.2 f 0 NR 30.2 [9] 10.8 16.5 1.5 3 20 dB@1.29 f 0 1.09 62.2 [12] 8 11 0.9 3 23 dB@0.865 f 0 0.147 25.3 [14] 5.22 - 1.62 4 20 dB@1.2 f 0 - 51.47 [15] 10 5.3 2.4 4 30 dB@1.05 f 0 1.75 36.6 [16] 10.01 3.98 1.52 4 20 dB@1.67 f 0 1.978 38.3 [17] 10 3.3 1.55 4 30 dB@1.03 f 0 2 49.27 [18] 10.11 11.7 1.22 4 20 dB@2.9 f 0 0.5 41.9 [19] 3.25 4.62 2.5 2 NA 0.051 19.85 [21] 1 12 0.83 2 20 dB@4.6 f 0 0.038 30.6 $ [22] 4.51 26 0.57 4 20 dB@2.40 f 0 0.159 43.7 [23] 10.6 17.77 2.61 3 20 dB@10.37 f 0 0.016 26.45 $ This work 0.835 15.8 0.41 2 20 dB@4.26 f0 0.028 32.51 $ FBW: three dB fractional bandwidth; IL: insertion loss; N: order; f c: center frequency; SF: selectivity factor; and $: based on 20 dB bandwidth. FBW: three dB fractional bandwidth; IL: insertion loss; N: order; f c: center frequency; SF: selectivity factor; and $: based on 20 dB bandwidth. Author Contributions: Conceptualization, R.K.B. methodology, R.K.B. and S.K.; software, R.K.B. and S.K.; validation, R.K.B., S.K. and A.P.-D.; formal analysis, R.K.B.; investigation, R.K.B.; resources, S.K.; data curation, R.K.B. and S.K.; writing—original draft preparation, R.K.B.; writing—review and editing, S.K. and A.P.-D.; visualization, R.K.B. and S.K.; supervision, S.K.; project administration, S.K. and A.P.-D.; funding acquisition, S.K. and A.P.-D. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: Not applicable. Acknowledgments: The authors would like to thank Dassault Systemes, France, for making CST Microwave Studio available. Acknowledgments: The authors would like to thank Dassault Systemes, France, for making CST Microwave Studio available. 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A class of box-like bandpass ﬁlters with wide stopband based on new dual-mode rectangular SIW cavities. IEEE Trans. Microw. Theory Tech. 2021, 69, 101–110. [CrossRef] g y Zhu, F.; Luo, G.Q.; You, B.; Zhang, X.H.; Wu, K. Planar dual-mode bandpass ﬁlters using perturbed d l i h h [C f] g 17. Zhu, F.; Luo, G.Q.; You, B.; Zhang, X.H.; Wu, K. Planar dual-mode bandpass ﬁlters using pertur waveguide rectangular cavities. IEEE Trans. Microw. Theory Tech. 2021, 69, 3048–3057. [CrossRef] 17. Zhu, F.; Luo, G.Q.; You, B.; Zhang, X.H.; Wu, K. 4. Conclusions In this paper, an ultra-compact second-order bandpass ﬁlter (BPF) based on a half- mode substrate-integrated rectangular cavity (HMSIRC) is presented. The ﬁlter was con- ﬁgured by using an HMSIRC, a rectangular slot, a transverse slot, and metallic vias. A full-mode SIRC was divided in half to create an HMSIRC with metallic vias on three of its sides and open-edged on the fourth side. To create the magnetic wall, a rectangular slot was created across the three via-containing sides, and to create the electric wall, a metallic via was loaded into the cavity’s center. As a consequence, many resonant modes were produced. By introducing a transverse slot into the cavity, we were able to manipulate the resonant frequencies of these modes and therefore realize a second-order bandpass ﬁlter. The working principle, ﬁeld distribution, coupling matrix, and loss calculation were discussed. Finally, a second-order BPF was manufactured and veriﬁed experimentally. The fabricated prototype achieves a low insertion loss of 0.41 dB, a passband bandwidth of 15.8%, and a broad stopband response of 4.26 f 0. Author Contributions: Conceptualization, R.K.B. methodology, R.K.B. and S.K.; software, R.K.B. and S.K.; validation, R.K.B., S.K. and A.P.-D.; formal analysis, R.K.B.; investigation, R.K.B.; resources, S.K.; data curation, R.K.B. and S.K.; writing—original draft preparation, R.K.B.; writing—review and editing, S.K. and A.P.-D.; visualization, R.K.B. and S.K.; supervision, S.K.; project administration, S.K. and A.P.-D.; funding acquisition, S.K. and A.P.-D. All authors have read and agreed to the published version of the manuscript. Electronics 2023, 12, 2831 12 of 13 12 of 13 Funding: The research leading to these results has received funding from the Norway Grants 2014–2021 via the National Centre for Research and Development, grant NOR/POLNOR/HAPADS/ 0049/2019-00. This work was also supported in part by the Icelandic Centre for Research (RANNIS) Grant 217771. Funding: The research leading to these results has received funding from the Norway Grants 2014–2021 via the National Centre for Research and Development, grant NOR/POLNOR/HAPADS/ 0049/2019-00. This work was also supported in part by the Icelandic Centre for Research (RANNIS) Grant 217771. Data Availability Statement: Not applicable. References Planar dual-mode bandpass ﬁlters using perturbed substrate-integrated waveguide rectangular cavities. IEEE Trans. Microw. Theory Tech. 2021, 69, 3048–3057. [CrossRef] g g y 18. Zhu, Y.; Dong, Y. A novel compact wide-stopband ﬁlter with hybrid structure by combining SIW and microstrip technologies. IEEE Microw. Wirel. Compon. Lett. 2021, 31, 841–844. [CrossRef] p 19. Weng, M.-H.; Tsai, C.-Y.; Chen, D.-L.; Chung, Y.-C.; Yang, R.-Y. A Bandpass Filter Using Half Mode Impedance Resonator. Electronics 2021, 10, 51. [CrossRef] p 20. Huang, L.; Yuan, N. A Compact Wideband SIW Bandpass Filter with Wide Stopband and High Selectivity. 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https://openalex.org/W3046857597	https://periodicos2.uesb.br/index.php/recuesb/article/download/7107/5016	Portuguese	null	NÃO ALIMENTEM OS ANIMAIS SILVESTRES DO PARQUE: TEATRO COMO FERRAMENTA DE CONSCIENTIZAÇÃO	Revista Extensão & Cidadania/Revista Extensão e Cidadania	2,020	cc-by	5,725	1 Acadêmica de Licenciatura em Ciências Biológicas, pelo Instituto Federal do Paraná. Bolsista do Programa Instrucional de Apoio à Extensão (PIBEX-IFPR). E-mail: carolrenatabatista@gmail.com 2 Professora Mestre do Instituto Federal do Paraná. Orientadora do trabalho. E-mail: karina.espartosa@ifpr.edu.br 3 Acadêmica de Licenciatura em Ciências Biológicas do Instituto Federal do Paraná. E-mail: monica.mrve@gmail.com v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 DOI: 10.22481/recuesb.v8i13.7107 v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 DOI: 10.22481/recuesb.v8i13.7107 NÃO ALIMENTEM OS ANIMAIS SILVESTRES DO PARQUE: TEATRO COMO FERRAMENTA DE CONSCIENTIZAÇÃO DO NOTE FEED WILD ANIMALS IN THE PARK: THEATER AS AWARENESS TOOL Caroline Renata Batista1 Karina Dias Espartosa2 Monica Toshie Suzuki Oshika3 Caroline Renata Batista1 Karina Dias Espartosa2 Monica Toshie Suzuki Oshika3 Resumo: O Parque Municipal São Francisco de Assis, em Assis Chateaubriand - PR apresenta uma problemática: a de que os visitantes têm o hábito recorrente de alimentar os animais silvestres do Parque, desconhecendo os riscos envolvidos neste comportamento. Com o objetivo de minimizar estes problemas por meio da conscientização da comunidade, o grupo de discentes extensionistas do IFPR Campus Assis Chateaubriand desenvolveu e vem apresentando uma peça teatral que, de forma lúdica, busca informar a comunidade sobre os riscos e consequências de alimentar os animais silvestres do Parque, e estimular uma visitação mais segura e respeitosa. Até o momento foram realizadas apresentações em 5 instituições de ensino, beneficiando um total de 790 estudantes. Por meio de perguntas feitas à plateia por uma das personagens ao final das apresentações, bem como diante dos depoimentos dos espectadores e dos estudantes atores, pode-se verificar que a iniciativa tem sido efetiva no que se refere à transmissão da mensagem pretendida, refletindo na mudança de atitudes dos ouvintes em relação à problemática tratada e contribuindo também na formação cidadã e desenvolvimento pessoal dos estudantes envolvidos no planejamento e realização desta ação. Palavras-chave: Educação ambiental. Macacos-prego. Unidades de Conservação. Teatro. Abstract: The Municipal Park São Francisco de Assis of Assis Chateaubriand - PR, presents a problematic: visitors have a recurring habit of feeding the wild animals, unaware of the risks involved in this behavior. In order to minimize this problem through community awareness, the students extension group of IFPR Campus Assis Chateaubriand developed and have been presenting a theatrical play that, in a playful way seeks to raise awareness among the community about the risks and consequences of their posture, seeking to encourage a safer and more respectful visitation. Until now, the presentation has been played in 5 educational 258 institutions, benefiting a total of 790 students. NÃO ALIMENTEM OS ANIMAIS SILVESTRES DO PARQUE: TEATRO COMO FERRAMENTA DE CONSCIENTIZAÇÃO DO NOTE FEED WILD ANIMALS IN THE PARK: THEATER AS AWARENESS TOOL Through questions asked to the audience by one of the characters at the end of the presentations, as well as considering of the testimonies from the spectators and students actors, it could be verified that the initiative has been effective with regard to the transmission of the message intended, reflecting on changes in listeners attitudes in relation to the problem addressed and also contributing to the citizen formation and personal development of students involved in planning and implementation of this action. eywords: Environmental education. Capuchin monkey. Conservation Units. Theater pla Introdução O Intituto Federal do Paraná, campus Assis Chateaubriand, desenvolve um projeto extensionista que capacita estudantes, tanto de cursos de graduação como do ensino médio integrado ao técnico, a atuarem em atividades diversas de educação ambiental nas instituições de ensino da região, no Parque Municipal São Francisco de Assis e em outros espaços comunitários. A partir das atividades realizadas no Parque, como as visitas monitoradas, os estudantes extensionistas observaram que muitos visitantes possuem o costume de oferecer alimentos para os animais silvestres que habitam o local, principalmente os macacos. É comum observar pais incentivando seus filhos a realizarem esta prática e, na maioria das vezes, os alimentos são impróprios para animais silvestres em geral, pois trata-se de salgadinhos e bolachas recheadas. A recorrência desse comportamento demonstra o desconhecimento dos visitantes sobre os riscos e as consequências desta ação. Existe a possibilidade de, pela proximidade ou pelo contato direto, haver a transmissão mútua de doenças, o que é potencializado caso o macaco acabe por ferir acidentalmente o visitante. Esta possibilidade de compartilhamento de algumas enfermidades ocorre devido à proximidade evolutiva e filogenética entre os primatas selvagens em relação a humanos (ANDRADE, 2002). Dentre as enfermidades compartilhadas estão doenças como herpesvirose, hepatite A, adenoviroses e raiva, uma enfermidade grave que pode levar a morte (BRASIL, 2017; RAMOS; RAMOS, 2002). 259 Além da transmissão de doenças, há também a problemática relacionada aos animais receberem itens alimentares inadequados à sua dieta, como é o caso dos salgadinhos e bolachas recheadas, conforme já citado. Os animais acabam por dar preferência a estes alimentos não somente pela facilidade de acesso, mas também porque estes alimentos antrópicos são mais v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 REVISTA palatáveis, digeríveis e mais ricos energeticamente (ricos em açúcares, gorduras saturadas e proteínas) que os alimentos nativos, conforme constatado por Milton (1999). Em função de sua composição, o consumo de alimentos antropogênicos causa danos aos animais silvestres, como o aumento dos níveis de glicose e colesterol, diabetes, obesidade, e até mesmo o aparecimento de cáries quando se é ingerido uma grande porção de açúcar (SAITO et al., 2010). O risco da transmissão mútua de doenças e o comprometimento da saúde alimentar dos animais e outros prejuízos podem decorrer deste comportamento. É possível que se dê a redução do medo destes animais com relação aos humanos, o que aumenta a proximidade, o contato e, consequentemente, o desenvolvimento de uma relação de dependência na qual os macacos passam a buscar alimentos prioritariamente junto aos humanos (SAITO et al., 2010). De fato, no Parque Municipal São Francisco de Assis, tal como em outras unidades de conservação com a mesma problemática, por exemplo, IBAMA (1999) para o Parque Nacional do Iguaçu e SABBATINI et al. (2006) para o Parque Nacional de Brasília, já se observa com frequência a aproximação destemida dos macacos-prego (Cebus sp.) para se apropriarem de alimentos dispostos em piqueniques e outras confraternizações realizadas pelos visitantes, comprometendo uma visitação tranquila e segura. Ademais, uma vez que o Parque Municipal São Francisco de Assis se encontra inserido na área urbana do município, são recorrentes os relatos sobre macacos adentrarem às casas vizinhas em busca de alimentos, até mesmo abrindo armários. Neste cenário, o grupo extensionista de educação ambiental do IFPR Campus Assis Chateaubriand se propôs a planejar uma ação de conscientização da comunidade com a finalidade de minimizar esses problemas provocados pelo fato dos visitantes alimentarem os animais silvestres do Parque. Para transmitir os conhecimentos e informações para uma visitação mais segura e respeitosa ao Parque Municipal São Francisco de Assis, o grupo extensionista escolheu o teatro, por tratar-se de uma ferramenta já amplamente reconhecida pelos seus benefícios como eficiente estratégia pedagógica, para além de uma expressão artístico-cultural. REVISTA 260 Diversos autores, a partir de pesquisas práticas e teóricas, consideram que o teatro associado à educação ambiental é considerado um instrumento pedagógico eficaz para tratar aspectos socioambientais, pois a partir da ludicidade, promove o reencontro do humano com as dimensões sensível, afetiva e poética que o compõe, e é capaz de uma aprendizagem significativa, semeando uma atitude proativa e uma consciência crítica nos indivíduos a partir 260 v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 60 EXTENSÃO & CIDADANIA 260 A EXTENSÃO & CIDADANIA REVISTA das reflexões proporcionadas (BERBERT et al., 2007; DANTAS; SANTANA; NAKAYAMA, 2012; DUVOISIN, 2002; SILVEIRA, 2009). De fato, iniciativas educacionais e de conscientização ambiental que tem adotado o teatro como estratégia de transmissão de conhecimentos, reflexão e incentivo a mudança de valores e atitudes, vem reportando resultados positivos ao trabalhar com os mais diferentes públicos como estudantes de educação infantil (BERBERT et al., 2007), de ensino fundamental (BERBERT et al., 2007; BOER; BITTENCOURT, 2017; OLIVEIRA et al., 2017; SILVA; MELO, 2012;), de ensino superior (SANTOS, 2014) e inclusive na formação continuada docente (DANTAS; SANTANA; NAKAYAMA, 2012) e na conscientização da comunidade do entorno com relação aos cuidados com uma unidade de conservação, como é o caso do presente trabalho (BAÍA et al., 2009). Assim, esta proposta extensionista teve como objetivo desenvolver e apresentar uma peça teatral com a finalidade de informar e conscientizar a comunidade chateaubriandense sobre os riscos e consequências de visitantes alimentarem os animais silvestres do Parque Municipal São Francisco de Assis, para assim minimizar este comportamento e promover uma visitação mais segura e respeitosa ao Parque. Esta proposta pretendeu ainda contribuir com a formação acadêmica e cidadã dos estudantes envolvidos em seu planeamento e realização. Metodologia A produção e a apresentação da peça teatral foram desenvolvidas por estudantes extensionistas coordenados por uma docente do IFPR Campus Assis Chateaubriand. O grupo extensionista em questão é composto por estudantes de diversos anos do curso de Licenciatura em Ciências Biológicas, abrangendo entre 19 e 37 anos, e por estudante dos cursos de ensino médio integrado ao técnico presentes no campus (de eletromecânica e de informática) abrangendo entre 16 e 18 anos. 261 Esta diversidade de cursos e idades entre os extensionistas garante uma rica troca e construção de conhecimentos entre estes estudantes, e entre eles e a comunidade em que estão inseridos e que beneficiam levando o conhecimento adquirido na instituição de ensino. Este grupo extensionistas realiza ações de educação ambiental diversas, conforme as demandas da comunidade, e atua em escolas e espaços comunitários alcançando diferentes públicos, com jogos e dinâmicas ambientais, oficinas, formação docente e visitas monitoradas ao Parque 261 v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 6 EXTENSÃO & CIDADANIA REVISTA Municipal São Francisco de Assis. A seguir, são descritas as etapas de planejamento, desenvolvimento, divulgação e apresentação da peça teatral e a forma de coleta de dados para a avaliação da efetividade da proposta. a) Produção do roteiro da peça teatral Para a elaboração do enredo, os estudantes procederam, primeiramente, a uma revisão bibliográfica, a fim de embasar informações importantes a serem destacadas no decorrer da peça teatral, que diz respeito às consequências do contato próximo ou direto entre os visitantes e os mamíferos silvestres do Parque (notadamente os macacos-prego), especialmente, com o intuito de alimentá-los. Nesse sentido, foram levantadas informações acerca das doenças que os seres humanos possuem em comum com os macacos, as formas como elas são contraídas e os sintomas e outros desdobramentos que estas doenças podem ocasionar às pessoas, para que isso pudesse ser demonstrado nas cenas da peça. Também, uma vez que os visitantes fornecem aos macacos alimentos inadequados à sua dieta, foram verificadas as enfermidades e outras consequências que estes primatas podem desenvolver ao terem uma alimentação inadequada, além de terem sido pesquisadas informações referentes à alimentação natural e adequada destes animais. Somado a estas informações, a produção do roteiro também teve como propósito relatar cenas cotidianas observadas no Parque, a fim de que o público se identificasse como parte do tema e do contexto tratados na peça. b) Produção de cenário e da caracterização dos personagens No decorrer de vários encontros semanais entre os discentes extensionistas que atuariam na peça teatral, foram construídos o cenário e a caracterização dos personagens. Uma vez que a proposta da peça é ser itinerante, sendo ofertada principalmente nas escolas do município, foram selecionados para compor o cenário, materiais simples e fáceis de movimentar, como folhas E.V.A, cartolina, papel kraft, tinta guache etc. c) Ensaios e apresentações 262 Após extensa redação e revisão do roteiro, os papéis dos personagens foram distribuídos por afinidade entre os estudantes extensionistas, que estudaram suas falas e participaram de uma série de ensaios semanais. Com tudo pronto, a peça passou a ser divulgada primeiramente em escolas do município, tendo como proposta futura ser divulgada para ocorrer em outros espaços comunitários. A divulgação se deu a partir de breves reuniões com a direção dos colégios, nas quais eram feitos a apresentação e o agendamento da 262 262 A EXTENSÃO & CIDADANIA d) Avaliação da proposta Diversas vias foram utilizadas para avaliar se a peça teatral estava sendo efetiva e cumprindo com o seu objetivo de levar informação e estimular a mudança de comportamento acerca da temática tratada: depoimentos colhidos junto a uma amostra do público e junto aos estudantes realizadores da ação; as reações do público ao longo da peça e suas respostas às perguntas feitas pela personagem Mãe Natureza. REVISTA v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 proposta. proposta. Resultados e Discussão Uma vez que o desenvolvimento pedagogicamente direcionado do roteiro da peça, o planejamento, construção de cenário e caracterização dos personagens foram produtos fundamentais desta proposta, iniciamos a exposição dos resultados com uma apresentação detalhada deste roteiro. Esta apresentação tem também como finalidade servir de base e inspiração para outros grupos extensionistas e de educação ambiental que se deparam com desafios em sua comunidade que são semelhantes aos que pretendemos endereçar neste trabalho. Após apresentação do roteiro da peça, são discutidos os dados e informações referentes às apresentações e sua efetividade. a) Roteiro da peça O roteiro foi elaborado de forma estratégica, de modo que um número pequeno de estudantes pudesse desempenhar um maior número de papéis, pois quanto menos estudantes envolvidos por vez na apresentação, mais fácil seria conciliar o agendamento das apresentações com a disponibilidade de cada estudante. Nesse sentido, o roteiro produzido apresenta nove personagens, tendo a possibilidade de serem encenados por quatro integrantes devido ao revezamento dos personagens entre as cenas propostas. Para garantir um bom fluxo de apresentações, mais do que quatro estudantes ensaiaram papéis na peça, objetivando assegurar a apresentação diante dos agendamentos realizados. 263 Conforme esclarecido no volume “Artes” dos Parâmetros Curriculares Nacionais, “a arte tem sido proposta como instrumento fundamental de educação, ocupando historicamente papéis diversos, desde Platão, que a considerava como base de toda a educação natural” (BRASIL, 1997, p. 57). Entretanto, como bem constatado e relatado por Dantas, Santana e Nakayama (2012) em seu trabalho com o teatro e a formação continuada docente em educação 263 v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 263 A EXTENSÃO & CIDADANIA 263 A EXTENSÃO & CIDADANIA REVISTA ISSN 2319-0566 A cena 3, volta a ocorrer no Parque, mostra o macaco que fora alimentado pelo visitante na cena 1, em conversa com sua mãe macaca. O macaco está passando mal, com 264 A cena 3, volta a ocorrer no Parque, mostra o macaco que fora alimentado pelo visitante na cena 1, em conversa com sua mãe macaca. O macaco está passando mal, com 264 A EXTENSÃO & CIDADANIA REVISTA ambiental, para a efetividade educativa de ações alternativas como o teatro, é fundamental o planejamento com uma finalidade pedagógica com conotação diferente do lúdico pelo lúdico. Com atenção a isso, o roteiro da peça foi pedagogicamente planejado pensando em desenvolver cenas e personagens do cotidiano da comunidade, relacionadas ao problema que foi observado no Parque. Buscou-se trazer descontração para as cenas para tornar a mensagem leve e mais facilmente assimilável, e houve também o desenvolvimento de uma personagem com propósito essencial didático, denominada e representada por Mãe Natureza. Essa personagem atua como uma narradora, dando início e fim à peça, aparecendo também entre as cenas. Em suas aparições, esta personagem interage com o público fazendo-lhes questionamentos que proporcionam uma retomada dos aprendizados obtidos em cada cena e na peça como um todo, no momento da interação final. É uma personagem qu apresenta falas mais flexíveis, improvisadas e interativas, podendo assim dar direcionamento e profundidade em suas falas de acordo com o tipo de público atendido. A atuação de Mãe Natureza proporciona tempo para a mudança de caracterização dos outros personagens entre as cenas e, principalmente, contribui para consolidar a mensagem que se pretende passar, além de ser uma forma de avaliar a própria efetividade da apresentação a partir das respostas colhidas junto ao público. A peça intitulada “Não alimente os macacos-prego” tem duração de 10 a 15 minutos, inicia-se com a cena frequentemente observada no Parque. Na cena 1, um adulto incentivando a filha a alimentar um macaco-prego oferecendo-lhe salgadinhos e bolacha recheada. Ao ter o dedo acidentalmente mordido pelo macaco-prego, a filha é levada pelo pai ao atendimento médico. A cena 2 se passa em um consultório médico, onde o médico atende pai e filha, solicita exames e esclarece um pouco sobre as doenças que podem ser transmitidas entre os macacos e os humanos, recomendando evitar este tipo de contato próximo. Com finalidade instrutiva, a cena 2 também ajuda a aclarar dúvidas comuns dos visitantes: o pai questiona se os macacos não passarão fome se não lhes forem ofertados os alimentos e o médico esclarece que estes animais dispõe do que necessitam e do que é correto para sua alimentação na vegetação e na fauna do Parque que os abriga. 264 REVISTA EXTENSÃO & CIDADANIA v. 8, n. 13, p. 258-273, jan./jun. 2020. v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 264 A EXTENSÃO & CIDADANIA REVISTA dores de estômago, e quando é questionado revela à mãe que havia aceitado salgadinhos e bolachas de um visitante. Então, sua mãe macaca o repreende, dizendo que havia frequentemente avisado que não poderia comer esse tipo de alimento, pois eles fazem mal aos macacos. A mãe macaca relata os problemas que essa alimentação equivocada poderia causar ao macaco, destacando, inclusive, a ocorrência de cáries e a possível perda dos dentes. Afinal, macaco não escova os dentes e, portanto, não deve alimentar-se de doces, como as bolachas recheadas. Então, a mãe macaca ainda deixa o filho de castigo. A cena final também se passa no Parque: uma mãe e seu filho estão fazendo um piquenique, quando, de repente, um macaco surge e surrupia a comida que estava sobre a toalha. A criança se espanta com a atitude do macaco e sua mãe explica que este é um problema decorrente do fato de os visitantes alimentarem os macacos: eles acabam por não temer a presença humana e ainda associam-na com a oferta de alimento, ocasionando, assim, maior aproximação dos macacos e trazendo intranquilidade e riscos para os visitantes. A inserção desta última cena tem por intuito amenizar uma situação que já vem se apresentando com frequência em Unidades de Conservação que abrigam animais silvestres e que recebem visitantes, como o Parque Municipal São Francisco de Assis: os animais passam a associar os visitantes à comida e aproximam-se furtivamente deles, vasculhando seus pertences e impedindo que se alimentem ou visitem tranquilamente a Unidade de Conservação. Isto pode gerar um sentimento de aversão aos animais, por parte das pessoas, e, por vezes, comportamentos hostis com os macacos (FRAGASZY; VISALBERGHI; FEDIGAN, 2004). Um exemplo clássico desta situação ocorre com os quatis (Nasua nasua) no internacionalmente conhecido Parque Nacional do Iguaçu, em Foz do Iguaçu - PR. Seu Plano de Manejo (IBAMA, 1999) registra que os quatis através de várias gerações vêm recebendo alimentação dos visitantes chegando ao limite da domesticação. Sobre este caso, em particular, Santos (2010) revela que diversos quatis do Parque Nacional do Iguaçu adoeceram em função da alimentação inadequada, e relata que, em muitos casos, os quatis começaram a atacar os visitantes em busca de alimento, muitas vezes, chegando a derrubar restos de comidas de lixeiras para se alimentarem. b) Cenários e caracterização dos personagens 265 No local onde é feita a apresentação, os dois cenários, Parque e consultório médico, são dispostos lado a lado. O Parque é caracterizado por uma mata, pintada com tinta guache sobre 265 265 EVISTA EXTENSÃO & CIDADANIA v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 A v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 REVISTA papel kraft (Figura 1), enquanto o consultório médico é caracterizado por uma mesa e três cadeiras que podem ser do próprio local onde ocorre a apresentação. Uma claquete de cartolina é utilizada pela personagem Mãe Natureza para dar clareza da transição entre as cenas (Figura 2A). A caracterização dos personagens é feita por meio do uso de acessórios simples que representam aspectos específicos do personagem, de modo a facilitar a troca de caracterização entre cenas quando há personagens representados por um mesmo ator. Nesse sentido, todos os atores vestem-se de preto para dar destaque aos acessórios de cada personagem. Na Figura 1, a seguir, há o cenário que ocorrem as cenas no Parque Municipal São Francisco de Assis. Figura 1: Cenário onde ocorrem as cenas no Parque Municipal São Francisco de Assis. Fonte: Elaboração da autoria (2018). Figura 1: Cenário onde ocorrem as cenas no Parque Municipal São Francisco de Assis Fonte: Elaboração da autoria (2018). A seguir, na Figura 2, “Claquete e caracterização dos personagens”, os três macacos que aparecem em cena são caracterizados por uma fantasia com máscara e uma cauda (presa em um cinto) feitas de folhas de E.V.A., tecido TNT, algodão e arame, conforme Figura 2B. O médico é caracterizado pelo uso de um jaleco e os personagens humanos são caracterizados por acessórios como boné e tiara com fivela para as crianças e blusas para os adultos, 266 REVISTA v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 conforme Figuras 2C e 2D. conforme Figuras 2C e 2D. A cena do piquenique conta também com uma toalha de mesa, bem como com utensílios e itens alimentares, conforme Figura 2D e a personagem Mãe Natureza se caracteriza pelo uso de acessórios floridos, conforme Figura 2C. Figura 2: Claquete e caracterização dos personagens. Fonte: Produção da autoria (2018). Figura 2: Claquete e caracterização dos personagens. Fonte: Produção da autoria (2018). c) Apresentações e sua efetividade Desde o estabelecimento da peça em setembro de 2017, foram realizadas até dezembro do mesmo ano, apresentações em cinco instituições de ensino, inclusive, em uma unidade da APAE, alcançando um total de 790 estudantes, com idades entre 4 a 17 anos, para além dos servidores das escolas, conforme Figuras 3 e 4. Diante desses dados, é possível constatar que a peça se caracterizou como um instrumento educativo capaz de atingir um público considerável em poucas apresentações e em um curto espaço de tempo, além de ter a capacidade de dialogar com diferentes grupos e perfis de pessoas. 267 267 REVISTA EXTENSÃO & CIDADANIA v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 Figuras 3 e 4: Imagens das apresentações realizadas. Fonte: Produção da autoria (2018). Figuras 3 e 4: Imagens das apresentações realizadas. Fonte: Produção da autoria (2018). 268 REVISTA EXTENSÃO & CIDADANIA 8 13 258 273 j /j 2020 ISSN 2319 0566 Uma vez que o grupo extensionista proponente desta peça realiza também outras ações de educação ambiental – oficinas com jogos e dinâmicas e, principalmente, a condução de visitantes no Parque Municipal São Francisco de Assis, verificou-se que a adesão das instituições de ensino à peça foi maior e ocorre mais prontamente do que para as demais atividades ofertadas pelo grupo, revelando uma preferência das escolas por esta atividade. Constatou-se que isso ocorreu em função do formato utilizado na apresentação da mensagem de conscientização, a “peça teatral”, que permite a realização da atividade no próprio colégio (sem a necessidade de deslocamento, como ocorre no caso da realização das trilhas interpretativas), beneficiando, de uma só vez, várias turmas do colégio, não comprometendo A v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 REVISTA significativamente o tempo das atividades pedagógicas já previstas pelo colégio. Por meio da reação do público às cenas e aos personagens, como também pelas respostas obtidas em sua interação com a personagem Mãe Natureza, observou-se que a peça prendeu a atenção das pessoas, promovendo a identificação com a temática tratada e a reflexão sobre as situações ali encenadas. Ao longo das apresentações, foi possível constatar também as risadas provocadas por algumas cenas e a expressão de atenção no rosto dos espectadores. Em geral, o público se divertiu com os atores caracterizados e imitando os macacos, especialmente, em cenas envolvendo alguns comportamentos destes animais. As pessoas se divertiram também com o uso da claquete entre as cenas, realizado de forma cômica pela personagem Mãe Natureza. Docentes e outros servidores que assistiram à peça também relataram, em depoimentos, desconhecer boa parte das informações transmitidas, alguns confessam que tinham o hábito de ofertar alimentos aos animais, pois não sabiam de todas as consequências envolvidas neste ato. Crianças e adultos contaram que, por vezes, foram ao Parque levando alimentos já com o objetivo de ofertar aos animais para assim, obter maior contato com eles, demonstrando um desconhecimento dos riscos. Outros reforçam que levavam banana e outras frutas a fim de alimentar os animais corretamente, mas que compreenderam que isso também não é recomendável, tanto pelos riscos da proximidade com o animal quanto pela intranquilidade da visitação, caracterizada quando o animal fica condicionado a relacionar os visitantes ao alimento. Em geral, os entrevistados mostraram-se sensibilizados com as informações, mencionando que não mais alimentariam os animais silvestres do Parque e que informariam conhecidos que praticam esta ação. Outras situações possibilitaram verificar a efetividade das apresentações. Ocorreu de o grupo extensionista receber para uma visita ao Parque, uma turma que já havia assistido à peça. Os monitores não tinham conhecimento antecipado deste fato, isso foi percebido pela qualidade das respostas e explicações que os estudantes-visitantes relataram ao longo da trilha. Entendendo que os visitantes haviam assistido à peça, os monitores fizeram mais perguntas e puderam verificar a profundidade com que os alunos assimilaram as informações transmitidas. 269 Tendo em vista que o município onde se localiza o Parque é de pequeno porte (cerca de 30.000 habitantes), muitas pessoas se conhecem e, por isso, alguns pais relataram aos estudantes-atores que seus filhos chegaram em casa contando-lhes o que haviam aprendido na 269 v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 269 A EXTENSÃO & CIDADANIA 269 A EXTENSÃO & CIDADANIA A v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 Considerações Finais Por meio de diversas vias como a observação das reações e respostas do público durante a peça, os depoimentos coletados junto aos integrantes da peça e junto a uma amostragem de espectadores, além de outras situações que emergiram ao longo das apresentações, foi possível constatar a efetividade do teatro como ferramenta educativa e, em particular, a efetividade desta peça em transmitir os conhecimentos e a conscientização pretendida. Tanto os estudantes atores como os espectadores entrevistados revelaram desconhecer parte da informação transmitida e relataram que, para além de não mais praticar o ato de alimentar os animais silvestres do Parque, informariam outras pessoas, demonstrando que a peça alcançou sensibilizar o público e também torná-lo multiplicador da informação. Entendeu-se que parte do sucesso alcançado resultou da combinação entre simplicidade na caracterização dos espaços, na linguagem de transmissão da mensagem e no planejamento pedagogicamente direcionado do roteiro, com uso de personagens e cenas do cotidiano dos espectadores e informações embasadas na literatura. O toque cômico no roteiro foi também importante para prender a atenção dos espectadores e dar leveza à mensagem. Ao longo da divulgação e apresentação da peça, verificou-se ainda que este formato de atividade de conscientização ambiental foi mais prontamente aceita e agendada pelas escolas do que outras atividades que o grupo extensionista desenvolve. Isso ocorreu em razão da peça poder ser apresentada uma vez, reunindo-se várias turmas no pátio do colégio e interrompendo menos o cotidiano escolar. Além disso, é uma atividade que ocorre dentro da escola não necessitando deslocamento dos estudantes e recolhimento de autorização junto aos pais para sua saída do ambiente escolar. Por fim, por meio do depoimento dos extensionistas “atores”, foi possível constatar que esta proposta também atingiu seu objetivo de proporcionar desenvolvimento pessoal e profissional a estes estudantes e uma oportunidade de atuação cidadã junto à sua comunidade. REVISTA v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 peça que assistiram. peça que assistiram. peça que assistiram. Estes depoimentos e situações demonstram que a peça atingiu tanto seu objetivo de sensibilização do público para a mudança de valores e atitudes, bem como atingiu o objetivo de torná-los multiplicadores do conhecimento transmitido. Assim, como relatado por Adams (1998), Oliveira e colaboradores (2017), Dantas, Santana e Nakayana (2012), dentre outros educadores que utilizam o teatro como ferramenta pedagógica, os resultados de nosso trabalho corroboram o entendimento de que a arte da encenação, quando pedagogicamente planejada, se mostra como uma estratégia de ensino- aprendizagem que alcança resultados expressivos na sensibilização do público e para a transmissão de uma mensagem ou conhecimentos. Adicionalmente, encenações que trazem cenas do cotidiano e personagens extraídos do público alvo se mostram como uma importante técnica para sensibilização e conscientização ambiental, fazendo com que o público-alvo (e os próprios desenvolvedores da atividade) reflitam sobre as experiências vividas, ressignificando saberes, valores e contribuindo para mudanças de atitudes. Além da relevância didática de encenações como forma de transmitir conhecimentos a um determinado público, a construção e apresentação de uma peça teatral trazem também inúmeros benefícios ao desenvolvimento pessoal e profissional dos indivíduos na posição dos atores amadores. Ao participar de atividades teatrais, o indivíduo tem a oportunidade de se desenvolver tanto nos aspectos individuais bem como em aspectos de sua inserção no coletivo. No campo do coletivo, exercita a capacidade de cooperação, diálogo, respeito mútuo e trabalho em grupo e, no campo individual, tem a oportunidade de desenvolver a iniciativa, a autonomia, a capacidade de expressar-se e para muitos, a superação da timidez (BRASIL, 1997; SANTOS, 2004). Com efeito, em seus depoimentos, os estudantes que realizaram a peça contam que o desenvolvimento da pesquisa e a preparação do roteiro trouxeram informações que também desconheciam, contribuindo para a sua própria conscientização. Relataram ainda que a atuação em uma peça teatral e o contato com públicos diversos auxiliaram em seu desenvolvimento pessoal, principalmente em sua desenvoltura para falar em público e mencionaram que se sentiram incentivados a se envolver em mais ações extensionistas que oportunizem a atuação cidadã e a contribuição com a comunidade em que estão inseridos. 270 270 REVISTA EXTENSÃO & CIDADANIA v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 Considerações Finais 27 REVISTA EXTENSÃO & CIDADANIA v. 8, n. 13, p. 258-273, jan./jun. 2020. ISSN 2319-0566 ADAMS, E. Aprendendo a ver. In: BARBOSA, A. M. Tópicos utópicos. Belo Horizonte: Com Arte, 1998. p. 126-135. ANDRADE, M. C. R. Principais doenças de primatas não-humanos. In: ANDRADE, A.; 271 PINTO, S. C.; OLIVEIRA, R. S. (org.). Animais de Laboratório: criação e experimentação. Rio de Janeiro: ed. da FIOCRUZ, 2002. p. 155-160. BAÍA, M. C.; SANTOS, V. R.; SANTANA, A. 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https://openalex.org/W3112413998	https://www.frontiersin.org/articles/10.3389/fmolb.2020.604492/pdf	English	null	Proteomics of Colorectal Cancer: Tumors, Organoids, and Cell Cultures—A Minireview	Frontiers in molecular biosciences	2,020	cc-by	7,652	Reviewed by: Reviewed by: Hongqiang Qin, Chinese Academy of Sciences, China Hua Xiao, Shanghai Jiao Tong University, China y Hongqiang Qin, Chinese Academy of Sciences, China Hua Xiao, Colorectal cancer is the third-most prevalent, and second-most deadly, cancer worldwide (Global Cancer Observatory, 2018). In the United States, it is estimated there will be 147,950 new cases of colorectal cancer and will claim over 50,000 lives in 2020 alone (American Cancer Society, 2020). Early diagnosis and various medical treatments can reduce the fatality rate, but it requires further understanding the intricacies of cancer formation, survival, and spread at the molecular level. As seen in a 2010 review, the ﬁeld of omics has tremendous potential for new molecular discoveries that change the way we treat colorectal cancer (Nambiar et al., 2010). Omics research analyzes cancer at the molecular level. For example, a genomics study of colorectal cancer patients may discover a recurring mutation that could be a factor in tumor formation, while a metabolomics study has the potential to discover a small molecule secreted into the bloodstream that could be used as a diagnostic biomarker for the presence of colorectal cancer. Proteomics is the study of proteins and can encompass the identiﬁcation, quantiﬁcation, localization, turnover, and regulation in a biological system. Liquid chromatography-mass spectrometry (LC-MS) has seen substantial use for proteomics in recent decades in which it has been utilized to obtain a global proteomic proﬁle of a biological sample (Cravatt et al., 2007). In a mass spectrometry-based proteomics experiment, one method to obtain the identiﬁcation and quantiﬁcation of proteins is through bottom-up proteomics. As seen in Figure 1, proteins are extracted from a biological sample; the sample undergoes several steps to unfold all the proteins, reduce and alkylate the cysteine residues, before digestion into peptides by a proteolytic enzyme. The lysate undergoes chromatographic separation, typically by low-pH reversed-phase Colorectal cancer is the third-most prevalent, and second-most deadly, cancer worldwide (Global Cancer Observatory, 2018). In the United States, it is estimated there will be 147,950 new cases of colorectal cancer and will claim over 50,000 lives in 2020 alone (American Cancer Society, 2020). Early diagnosis and various medical treatments can reduce the fatality rate, but it requires further understanding the intricacies of cancer formation, survival, and spread at the molecular level. Edited by: Fangjun Wang, Chinese Academy of Sciences, China Keywords: colorectal cancer, proteomics, mass spectrometry, organoids, cell culture, tumors, biomarkers Proteomics of Colorectal Cancer: Tumors, Organoids, and Cell Cultures—A Minireview Philip H. Lindhorst1 and Amanda B. Hummon1,2* Philip H. Lindhorst1 and Amanda B. Hummon1,2* 1 Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, United States, 2 The Comprehensive Cancer Center, The Ohio State University, Columbus, OH, United States 1 Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, United States, 2 The Comprehensive Cancer Center, The Ohio State University, Columbus, OH, United States 1 Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, United States, 2 The Comprehensive Cancer Center, The Ohio State University, Columbus, OH, United States Proteomics, the study of the complete protein composition of a sample, is an important ﬁeld for cancer research. Changes in the proteome can serve as a biomarker of cancer or lead to the development of a targeted therapy. This minireview will focus on mass spectrometry-based proteomics studies applied speciﬁcally to colorectal cancer, particularly the variety of cancer model systems used, including tumor samples, two-dimensional (2D) and three-dimensional (3D) cell cultures such as spheroids and organoids. A thorough discussion of the application of these systems will accompany the review of the literature, as each provides distinct advantages and disadvantages for colorectal cancer research. Finally, we provide conclusions and future perspectives for the application of these model systems to cancer research as a whole. Reviewed by: As seen in a 2010 review, the ﬁeld of omics has tremendous potential for new molecular discoveries that change the way we treat colorectal cancer (Nambiar et al., 2010). Omics research analyzes cancer at the molecular level. For example, a genomics study of colorectal cancer patients may discover a recurring mutation that could be a factor in tumor formation, while a metabolomics study has the potential to discover a small molecule secreted into the bloodstream that could be used as a diagnostic biomarker for the presence of colorectal cancer. Shanghai Jiao Tong University, China Correspondence: Amanda B. Hummon hummon.1@osu.edu Correspondence: Amanda B. Hummon hummon.1@osu.edu Specialty section: This article was submitted to Metabolomics, a section of the journal Frontiers in Molecular Biosciences Received: 10 September 2020 Accepted: 20 November 2020 Published: 10 December 2020 Citation: Lindhorst PH and Hummon AB (2020) Proteomics of Colorectal Cancer: Tumors, Organoids, and Cell Cultures—A Minireview. Front. Mol. Biosci. 7:604492. doi: 10.3389/fmolb.2020.604492 Specialty section: This article was submitted to Metabolomics, a section of the journal Frontiers in Molecular Biosciences Received: 10 September 2020 Accepted: 20 November 2020 Published: 10 December 2020 Citation: Lindhorst PH and Hummon AB (2020) Proteomics of Colorectal Cancer: Tumors, Organoids, and Cell Cultures—A Minireview. Front. Mol. Biosci. 7:604492. doi: 10.3389/fmolb.2020.604492 Specialty section: This article was submitted to Metabolomics, a section of the journal Frontiers in Molecular Biosciences Received: 10 September 2020 Accepted: 20 November 2020 Published: 10 December 2020 Proteomics is the study of proteins and can encompass the identiﬁcation, quantiﬁcation, localization, turnover, and regulation in a biological system. Liquid chromatography-mass spectrometry (LC-MS) has seen substantial use for proteomics in recent decades in which it has been utilized to obtain a global proteomic proﬁle of a biological sample (Cravatt et al., 2007). In a mass spectrometry-based proteomics experiment, one method to obtain the identiﬁcation and quantiﬁcation of proteins is through bottom-up proteomics. As seen in Figure 1, proteins are extracted from a biological sample; the sample undergoes several steps to unfold all the proteins, reduce and alkylate the cysteine residues, before digestion into peptides by a proteolytic enzyme. The lysate undergoes chromatographic separation, typically by low-pH reversed-phase MINI REVIEW MINI REVIEW published: 10 December 2020 doi: 10.3389/fmolb.2020.604492 Frontiers in Molecular Biosciences \| www.frontiersin.org Citation: December 2020 \| Volume 7 \| Article 604492 1 Frontiers in Molecular Biosciences \| www.frontiersin.org Proteomics of Colorectal Cancer Lindhorst and Hummon FIGURE 1 \| Proteomics workﬂows for different colorectal cancer samples (Tirumalai et al., 2003; Ang et al., 2010; Quesada-Calvo et al., 2017). Figure created with BioRender.com. FIGURE 1 \| Proteomics workﬂows for different colorectal cancer samples (Tirumalai et al., 2003; Ang et al., 2010; Quesada-Calvo et al., 2017). Figure created with BioRender.com. chromatography before being analyzed by mass spectrometry. As peptides elute out of the column, the MS fragments the peptides to generate sequence-speciﬁc peaks. The methodology is outside the scope of this review; however, for a detailed explanation, Zhang et al. (2013) published an excellent overview of this approach. in cancer cells. In addition to obtaining the basal proteome, mass spectrometry-based proteomics experiments can also be used to further evaluate the eﬀect of a perturbation (adding a drug, knocking out a gene, etc.) by observing any proteomic alterations between control and treated groups. However, tumor heterogeneity is a major obstacle that has to be factored into any proteomics experiment due to the composition of cancer cells. Imperial et al. (2018) previously showed that the proteome throughout an individual tumor, as well between tumors at diﬀerent locations, may be diﬀerent. For a more detailed discussion of tumor heterogeneity, Lim and Lim (2018) provided Proteomics can be used in many facets of cancer research, beginning by being able to analyze the protein expressions in healthy and cancerous cells to identify diﬀerences in the protein abundance between the two populations. This information can be important to understand potential therapeutic targets December 2020 \| Volume 7 \| Article 604492 Frontiers in Molecular Biosciences \| www.frontiersin.org 2 Proteomics of Colorectal Cancer Lindhorst and Hummon biomarkers. Hao et al. (2017) used a novel algorithm to identify changes in abundance of entire protein pathways. They found elevated expression of proteins associated with chromatin modiﬁcation and gene expression, but decreased expression of proteins responsible for core matrix architecture. These results would support the high mutation rate observed in cancer. Saleem et al. (2019) compared the proteomes of healthy tissue, non-adenomatous colon polyps, non-metastatic tumors, and metastatic tumors to identify proteins with elevated expression in the cancerous samples, such as matrix metalloproteinase-9, and decreased expression, such as caveolin-1. Similarly, Knol et al. Citation: (2014) compared benign and malignant tumor proteomes by enriching the protein fractions for chromatin-binding proteins, which had shown a diﬀerence previously (Albrethsen et al., 2010). Wisniewski et al. (2012) found that the proteome of colon adenocarcinoma tissue was signiﬁcantly remodeled in comparison to normal tissue, which led them to compare the proteomes of colorectal mucosa, adenoma, and cancer (Wi´sniewski et al., 2015). They noted signiﬁcant changes in fatty acid metabolism and plasma membrane transporter proteins. New workﬂows using iTRAQ labeling (Jankova et al., 2011) or a centrifugal proteomic reactor (Liu et al., 2016) improve the data acquired from proteomic experiments. For further information on protein biomarkers, we suggest the following reviews: Tjalsma (2010); Lee et al. (2018), and Martins et al. (2019), which provide more information on recent advances and discoveries. an excellent review for solutions to overcoming this common problem. Additionally, it can be diﬃcult to procure primary tumor samples and patient approval has to be obtained for their use. Due to these obstacles, it is often more favorable to use models that simulate the colorectal cancer proteome. With this in mind, researchers have developed several in vitro models, with varying degrees of complexity to study colorectal cancer. This minireview highlights the use of mass spectrometry- based proteomics for primary colorectal tumor tissues studies and various models of colorectal cancer that are currently available. Since colorectal cancer proteomics is a rapidly evolving area of research, we will also provide our perspective on the future of this ﬁeld in the new decade. Primary Tumors For colorectal cancer research, no sample is more valuable than tissue directly from patients, as these samples provide the most accurate representation of the cancer’s proteome. Previously, the major focus of colorectal cancer proteomics research is the search for protein biomarkers for colorectal cancer, elucidated by comparing the proteomes of cancerous and non-cancerous tissue. IN VIVO COLORECTAL CANCER PROTEOMICS In vivo colorectal cancer samples, samples obtained from either a patient or mouse model, are the most desirable source of proteomic information and have been used for colorectal cancer research for a number of years. Patient-derived in vivo samples can include primary tumor tissue, formalin-ﬁxed paraﬃn- embedded (FFPE) tissue, liquid biopsies (plasma, serum, etc.), and stool samples. Much of the literature from the last 20 years focuses on these samples and has been previously reviewed (Tjalsma, 2010; Lee et al., 2018; Martins et al., 2019). In this section, we will brieﬂy focus on previous studies using speciﬁcally primary tumor tissue samples, then more thoroughly discuss why in vitro model systems are useful and seeing increased popularity in the last decade. In contrast with the previously mentioned comparative studies, other cancer proteomics studies have utilized diﬀerent approaches, for example, comparing the proteome with the genome or transcriptome. The relationship between the genome and proteome provides a more holistic view of colorectal cancer (Kang et al., 2012; Zhang et al., 2014; Vasaikar et al., 2019; Li et al., 2020). Frontiers in Molecular Biosciences \| www.frontiersin.org IN VITRO COLORECTAL CANCER PROTEOMICS Primary human samples are the most valuable and accurate representations of human cancer, but they are a limited resource. A solution to this problem is the use of cultured models, which oﬀer greater reproducibility and simplicity, and still have proteomes that are representative of primary colorectal cancer (Wang et al., 2017). The most common models for cancer research are cell lines. Cell lines have seen consistent use (Dumont et al., 2016) since the development of the HeLa cell line from cervical cancer in 1951 (Gey et al., 1952). While two-dimensional (2D) cell culture, where the cells are grown as a monolayer adhered to the bottom of the incubation ﬂask, has been the most common use of cell lines, three-dimensional (3D) cell culture of multicellular spheroids is also possible. These cell cultures are called spheroids due to their shape, and for information on methodology, we would recommend a recent review from Chatzinikolaidou (2016). Even more recently, a new in vitro model called an organoid has become popular in cancer research. Several reviews on organoids exist (Clevers, 2016; Drost and Clevers, 2018; Xu et al., 2018), but their potential for proteomics is still relatively unexplored. In Protein biomarkers are proteins that are notably up- or down-regulated in the cancer proteome as compared to the normal proteome. Ideally, these proteins can serve as diagnostic markers of the presence of cancer, provide us with more information on cancer formation or survival, or possibly lead us to treatments that target these proteins. Using comparative proteomics with healthy and cancerous tissues, recent approaches show recognizable diﬀerences between these populations. The diﬀerences in protein abundance result in the identiﬁcation of protein biomarkers, which are detailed in Table 1. Examples of protein biomarkers include the protein OLFM4, found by two separate labs as a biomarker for colorectal cancer using bottom-up proteomics (Besson et al., 2011; Quesada- Calvo et al., 2017). Zhang et al. (2010) identiﬁed transgelin-2 as a biomarker of colorectal cancer, while Yamamoto et al. (2016) identiﬁed aldolase A. Mori et al. (2017) discovered shock protein 47 as a biomarker for the metastasis of colorectal cancer to lymph nodes. Mikula et al. (2011) used a combination of proteomics and transcriptomics to search for new protein December 2020 \| Volume 7 \| Article 604492 3 Proteomics of Colorectal Cancer Lindhorst and Hummon TABLE 1 \| Protein biomarkers of colorectal cancer using mass spectrometry. IN VITRO COLORECTAL CANCER PROTEOMICS Although these two cell lines form poor spheroids (Stadler et al., 2018), they can provide valuable insight into proteins that may determine whether a cancer will metastasize. this section, we will review recent colorectal cancer proteomics research for all three in vitro models and discuss the inverse relationship between simplicity and proteomic accuracy as seen between them. IN VITRO COLORECTAL CANCER PROTEOMICS Yamamoto et al., 2016 Increased number of HSP47-positive spindle cells in CRC stroma linked to lymph node metastasis Mori et al., 2017 Increased expression along malignant progression from normal colon tissue to adenocarcinoma Mikula et al., 2011 Decreased expression along malignant progression from normal colon tissue to adenocarcinoma Mikula et al., 2011 Highly increased expression in E1 cell line, the metastatic derivative of HCT-116 CRC cell line, and metastatic primary tumor tissues Tan et al., 2012 Signiﬁcantly increased expression in highly invasive CRC cell lines and lymph node metastatic tumor microarrays Hu et al., 2018 Increased expression in the HCT-116 CRC cell line as compared to normal derived colon cell line Ludvigsen et al., 2020 Increased expression in CRC cells correlated with increased metastasis. Modulating its expression changed the expression of other cancer-related proteins Ala et al., 2020 Decreased expression associated with CRC progression and increased metastasis Torres et al., 2018 Increased expression in CRC organoids. Inhibition using a drug resulted in organoid disaggregation and death Bozzi et al., 2017 Increased expression in organoids where the removal of the APC gene activates an oncogenic Wnt response Michels et al., 2019 Decreased expression in organoids where the removal of the APC gene activates an oncogenic Wnt response Michels et al., 2019 Decreased expression in organoids where the removal of the APC gene activates an oncogenic Wnt response Michels et al., 2019 focused on utilizing a comparison discovery-based approach to identifying protein biomarkers. Tan et al. (2012) used the colon cancer cell line, HCT-116, to identify Stathmin-1 as a protein biomarker of cancer cell migration. Ala et al. (2020) identiﬁed the pro-oncogenic role of ERSP1 in the colorectal cell lines HCA24 and COLO320DM. Hu et al. (2018) used HCT-116 and RKO, selectively cultured to be highly invasive, to identify the Cdc42-Cdc42BPA signaling pathway as a marker for colon cancer invasion. Ludvigsen et al. (2020) compared a normal derived colon mucosa cell line (NCM460) and a colon cancer cell line (HCT116) to identify protein biomarkers with increased expression in colorectal cancer, including retinol-binding protein 1. 2D cell culture has also been used to compare non-metastatic (SW480) and metastatic colon cancer cell lines (SW620) (Zhao et al., 2007; Schunter et al., 2017; Torres et al., 2018). These two cell lines derive from the same patient (Stage II for SW480 and Stage III for SW620). IN VITRO COLORECTAL CANCER PROTEOMICS Inhibition using a drug resulted in organoid disaggregation and death Bozzi et al., 2017 Organoid HMGCS2, CEMIP, LRP1, DPP4 Up-regulated Increased expression in organoids where the removal of the APC gene activates an oncogenic Wnt response Michels et al., 2019 Organoid EPHA2, BCAM Down-regulated Decreased expression in organoids where the removal of the APC gene activates an oncogenic Wnt response Michels et al., 2019 TABLE 1 \| Protein biomarkers of colorectal cancer using mass spectrometry. Model type Protein Up/down-regulated Biomarker Primary tumor Transgelin-2 Up-regulated Correlation Primary tumor Matrix metalloproteinase-9 Up-regulated Increased e tumor tissue Primary tumor Caveolin-1 Down-regulated Decreased tumor tissue Primary tumor (FFPE) OLFM4, KNG1. Sec24c Up-regulated Increased e expression Primary tumor ALDH1A1, OLFM4, HSPE1, SORD Up-regulated Signiﬁcant e expression Primary tumor (FFPE) Cyclophilin A, Annexin A2, Aldolase A Up-regulated Increased e secretion of Primary tumor HSP47 Up-regulated Increased n stroma linke Primary tumor COL12A1, CALU, BGN Up-regulated Increased e colon tissue Primary tumor MAOA, ENTPD5, MOSC2 Down-regulated Decreased colon tissue 2D cell culture/primary tumor STMN1 Up-regulated Highly incre derivative of tumor tissue 2D cell culture/primary tumor Cdc42BPA Up-regulated Signiﬁcantly lines and lym 2D cell culture Retinol-binding protein 1 Up-regulated Increased e compared t 2D cell culture ERSP1 Up-regulated Increased e metastasis. of other can 2D cell culture SRSF3 Down-regulated Decreased increased m Organoid MIF/CD74 Up-regulated Increased e resulted in o Organoid HMGCS2, CEMIP, LRP1, DPP4 Up-regulated Increased e APC gene a Organoid EPHA2, BCAM Down-regulated Decreased APC gene a TABLE 1 \| Protein biomarkers of colorectal cancer using mass spectrometry. Biomarker expression References Correlation with lymph node metastasis Zhang et al., 2010 Increased expression in both non-metastatic and metastatic tumor tissue Saleem et al., 2019 Decreased expression in both non-metastatic and metastatic tumor tissue Saleem et al., 2019 Increased expression in early CRC stages, with decreasing expression in late stages (non-metastatic) Quesada-Calvo et al., 2017 Signiﬁcant expression in early CRC stages, but no change in expression in late stages (non-metastatic) Besson et al., 2011 Increased expression in colorectal cancer cells. Decreased secretion of aldolase A in human serum. IN VITRO COLORECTAL CANCER PROTEOMICS Model type Protein Up/down-regulated Biomarker expression References Primary tumor Transgelin-2 Up-regulated Correlation with lymph node metastasis Zhang et al., 2010 Primary tumor Matrix metalloproteinase-9 Up-regulated Increased expression in both non-metastatic and metastatic tumor tissue Saleem et al., 2019 Primary tumor Caveolin-1 Down-regulated Decreased expression in both non-metastatic and metastatic tumor tissue Saleem et al., 2019 Primary tumor (FFPE) OLFM4, KNG1. Sec24c Up-regulated Increased expression in early CRC stages, with decreasing expression in late stages (non-metastatic) Quesada-Calvo et al., 2017 Primary tumor ALDH1A1, OLFM4, HSPE1, SORD Up-regulated Signiﬁcant expression in early CRC stages, but no change in expression in late stages (non-metastatic) Besson et al., 2011 Primary tumor (FFPE) Cyclophilin A, Annexin A2, Aldolase A Up-regulated Increased expression in colorectal cancer cells. Decreased secretion of aldolase A in human serum. Yamamoto et al., 2016 Primary tumor HSP47 Up-regulated Increased number of HSP47-positive spindle cells in CRC stroma linked to lymph node metastasis Mori et al., 2017 Primary tumor COL12A1, CALU, BGN Up-regulated Increased expression along malignant progression from normal colon tissue to adenocarcinoma Mikula et al., 2011 Primary tumor MAOA, ENTPD5, MOSC2 Down-regulated Decreased expression along malignant progression from normal colon tissue to adenocarcinoma Mikula et al., 2011 2D cell culture/primary tumor STMN1 Up-regulated Highly increased expression in E1 cell line, the metastatic derivative of HCT-116 CRC cell line, and metastatic primary tumor tissues Tan et al., 2012 2D cell culture/primary tumor Cdc42BPA Up-regulated Signiﬁcantly increased expression in highly invasive CRC cell lines and lymph node metastatic tumor microarrays Hu et al., 2018 2D cell culture Retinol-binding protein 1 Up-regulated Increased expression in the HCT-116 CRC cell line as compared to normal derived colon cell line Ludvigsen et al., 2020 2D cell culture ERSP1 Up-regulated Increased expression in CRC cells correlated with increased metastasis. Modulating its expression changed the expression of other cancer-related proteins Ala et al., 2020 2D cell culture SRSF3 Down-regulated Decreased expression associated with CRC progression and increased metastasis Torres et al., 2018 Organoid MIF/CD74 Up-regulated Increased expression in CRC organoids. 2D CELL CULTURE Over the years, 2D cell culture has provided a simple, cost- eﬀective model for cancer research and many cell lines have been developed. Importantly, 2D cell culture can be used to answer fundamental questions about cancer cells. Nusinow et al. (2020) recently used quantitative proteomics to screen the cell lines in the Cancer Cell Line Encyclopedia (CCLE). The CCLE is a large-scale database of over 1,000 cancer cell lines and includes information regarding gene expression, genome sequencing, metabolite proﬁling, drug sensitivity screens, and targeted protein quantiﬁcation. The CCLE did not have deep proteome proﬁling until this study. In addition, similar to primary tumor samples, colorectal proteomics using 2D cell culture has recently December 2020 \| Volume 7 \| Article 604492 Frontiers in Molecular Biosciences \| www.frontiersin.org Frontiers in Molecular Biosciences \| www.frontiersin.org 4 Proteomics of Colorectal Cancer Lindhorst and Hummon monolayer cultures. Certain proteins and pathways that showed a change in the spheroids were associated with slower growth and decreased cell and DNA replication, mimicking in vivo conditions. In a subsequent study, Kim et al. (2018) compared the proteomes of 3D and 2D SW480 cell cultures after the addition of an enzyme inhibitor. Another colon cell line frequently used for spheroids is HCT-116, which Feist et al. (2015) used to detect over 1350 proteins with very little variation between the replicate spheroids. McMahon et al. (2012) characterized the diﬀerences in the proteome between the three cellular populations in spheroids, the proliferating outer layer, the senescent middle layer, and the necrotic core, for the HT29 cell line. These cellular populations are also present in in vivo conditions, making this study particularly valuable for comparisons between in vivo and in vitro models. In addition to spheroids grown using immortalized cell lines, Rajcevic et al. (2014) used patient tumor material to produce spheroids with more in vivo tissue characteristics than cell line-based spheroids. One feature of cell lines not available for primary tumor samples is in vitro manipulation. These manipulations include dosing with anticancer drugs (Bauer et al., 2012; Valdes et al., 2017; Raimondo et al., 2018; Schroll et al., 2018), inducing autophagy using hypoxia (Lai et al., 2016), and binding growth inhibitors (Michalak et al., 2016), followed by downstream proteomics, an analysis that looks for diﬀerences in the proteome after the manipulation. Fanayan et al. (2013) took a diﬀerent approach and performed a bottom-up proteomics experiment using chromosome-based data analysis. 2D CELL CULTURE The proteomic data were organized into clusters based on the MS-based data and the chromosomal location of genes. One cluster identiﬁed with this method included several tumor suppressor proteins, including caveolin-1 and caveolin-2, associated with a region on chromosome 7 often deleted in the pathogenesis of colorectal cancer. As seen above, there are many aspects of 2D cell culture that make them desirable for colorectal cancer proteomics. However, due to a lack of 3D structure, vasculature, and other in vivo tumor properties, information obtained from 2D cell culture is incomplete. With the introduction of 3D spheroids and organoids, their ability to provide a more biologically accurate environment (reviewed by Yamada and Cukierman, 2007) helps to ﬁll this gap in the knowledgebase. Furthermore, most of the in vitro manipulations performed above can also be used for these models as well. Although 2D cell culture will never become obsolete due to its ease and simplicity, 3D cell cultures have increasingly become more common as models for colorectal cancer. Recent studies in our laboratory have used spheroids to test chemotherapy drugs and the eﬀects of glucose. LaBonia et al. (2018) tested the proteomic changes that occur with treatment of FOLFIRI (folinic acid, 5-ﬂuorouracil, and irinotecan), a combination chemotherapy, on HCT-116 spheroids and observed that folinic acid penetrated into the core of the spheroids and several cancer-associated protein pathways were enriched. Schroll et al. (2016) showed that restricting glucose or serum from the growing spheroids resulted in similar proteomic changes. The eﬀect of combining glucose restriction with chemotherapies has also been examined using spheroids (Schroll et al., 2017, 2018). Feist et al. (2017) used spheroids to evaluate diﬀerences in histone post-translational modiﬁcations and the eﬀect of an epigenetic drug that targets these modiﬁcations. The use of spheroids in the proteomics studies above provides important information about how a treatment might aﬀect an in vivo tumor, without testing the conditions on actual patients. These data are the ﬁrst step toward verifying possible treatments for cancer that could be explored in clinical studies. 3D CELL CULTURE Since 2D cell cultures do not have the same structural features as tumors, there is an inherent loss of spatial information and the proteome will not be truly representative of a tumor. With the introduction of spheroids, the cellular environments of tumors are more accurately replicated in vitro, while still providing many of the same advantages of 2D cell culture, for instance, ease of use, versatility, and lower experimental cost. Alternatively, a shared disadvantage between 2D cell culture and spheroids is the presence of only cancer cells, so information on the interaction between healthy and cancerous cells that would be present in vivo is lost. Another in vitro model, organoids, can be grown to contain both populations, solving this issue and providing a more accurate mimetic (Johnson et al., 2020). This section will review recent proteomic literature for both spheroids and organoids. Organoids d Organoids are a relatively recent development in comparison to the other models discussed in this minireview. Xu et al. (2018) deﬁne organoids as “3D constructs” that can be developed from “embryonic stem cells, induced pluripotent stem cells, somatic cells, and cancer cells.” The use of stem cells is especially important for this technology, in that these cells can be used to make a variety of organoids, including intestinal organoids. Colorectal tumor organoids have very high potential for screening and thus the possibility for the development of organoid libraries has been explored (van de Wetering et al., 2015; Fujii et al., 2016). Another beneﬁt to organoids is that non-cancerous organoids can be grown, so comparisons between healthy and cancer proteomes can be made, something not possible for 2D cell culture and spheroids. Frontiers in Molecular Biosciences \| www.frontiersin.org Spheroids The ﬁrst proteomics investigation of spheroids compared the spheroids’ proteome with their 2D counterparts. Yue et al. (2016) compared both the proteome and phosphoproteome between spheroids and 2D cell cultures of HT29, a colon cell line. Observation of reproducible changes in abundance for several proteins between the populations suggested that spheroids may provide more accurate proteomic information compared to 2D One major disadvantage for organoid research is the use of a substance called Matrigel, an extracellular matrix necessary for preparing and embedding organoids. Unfortunately, Matrigel contains a variety of growth factor proteins that cause severe ion December 2020 \| Volume 7 \| Article 604492 Frontiers in Molecular Biosciences \| www.frontiersin.org 5 Proteomics of Colorectal Cancer Lindhorst and Hummon suppression (Abe et al., 2018). In their research, Abe and his colleagues performed phosphoproteomics analysis on colorectal cancer spheroids and organoids embedded in Matrigel. To avoid the ion suppression eﬀects, they incorporated an acetone precipitation of the digested peptides in their workﬂow, which increased the percentage of the MS/MS spectra associated with peptides from 8.8 to 26.9%. although primary tumor samples will continue to be valuable research tools, the application of proteomics to spheroids and organoids will continue to see increased representation as cell culture technology progresses and proteomics methodologies improve. These in vitro models demonstrate greater statistical reproducibility, due to the lack of tumor heterogeneity, and still maintain a relatively representative proteome. In addition, as these models are grown, they are more readily available than tumor samples that need to be collected from patients. While there are still challenges that need to be overcome, in vitro models provide a viable alternative to primary tumor samples, not only for colorectal cancer, but many other cancers as well. Gonneaud et al. (2017) wrote an excellent review of mass spectrometry-based proteomics using colon organoids. Bozzi et al. (2017) identiﬁed the MIF/CD74 axis as a target for therapeutics in colorectal cancer, in that its inhibition caused cancer cells to be vulnerable to oxidative stress-induced death. More recently, Lindeboom et al. (2018) used a multi-omics (metabolomics, proteomics, transcriptomics, epigenomics, and genomics) analysis to obtain a complete intramolecular view of intestinal organoids. Michels et al. (2019) used proteomic and transcriptomic proﬁling of colon organoids to observe physiologic and oncogenic responses to Wnt signaling, such as the up-regulated proteins HMGCS2 and CEMIP. REFERENCES Colorectal Cancer Establishes OLFM4 as a New Nonmetastatic Tumor Marker. Mol. Cell. 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Cell 177, 1035–1049. doi: 10.1016/j.cell.2019.03.030 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Wang, J., Mouradov, D., Wang, X., Jorissen, R., Chambers, M., Zimmerman, L., et al. (2017). Colorectal cancer cell line proteomes are representative of primary tumors and predict drug sensitivity. Gastroenterology 153, 1082–1095. doi: 10.1053/j.gastro.2017.06.008 Copyright © 2020 Lindhorst and Hummon. 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. Wi´sniewski, J., Du´s-Szachniewicz, K., Ostasiewicz, P., Ziółkowski, P., Rakus, D., and Mann, M. (2015). Absolute Proteome Analysis of Colorectal Mucosa, Adenoma, and Cancer Reveals Drastic Changes in Fatty Acid Metabolism and Plasma Membrane Transporters. J. Proteome. Res. 14, 4005–4018. doi: 10.1021/ acs.jproteome.5b00523 December 2020 \| Volume 7 \| Article 604492 Frontiers in Molecular Biosciences \| www.frontiersin.org 8
https://openalex.org/W2037464964	https://zenodo.org/record/1580882/files/article.pdf	English	null	Farm yard and dairy hygiene	Public health	1,919	public-domain	2,723	Presidential Address delivered at opening meeting of N.'vV. Branch in Manchester, Friday, October 10th, 1919. Fu1~gi.--These live only on food derived from animals or plants, they cannot live on soil alone, e.g., mushrooms, tree fungi, and they consist of threads of mycelium; these threads grow on surfaces or into the substance upon which the fungus is growing (feeding) and in trees is often the cause of the wood beginning to rot, due to its having made an inroad for exposure to weather. This manure heap must be viewed by the Medical Officer of Health with a rather cold eye. It ought to be 20 yards at least from the cowshed and dairy, walled round, the liquid manure run into a tank, a pump provided, to pump it into a liquid manure cart and taken on to the land ; this must be done often, as it rapidly undergoes ammonical changes and is a loss. The solid manure ought to be trodden down and made as solid as possible, it ripens better, it does notblow all over the yard and it looks tidier, and the more solidit is, the less can flies breed in it, after treading down, sprinkling with petroleum or Borax twice a week helps to prevent the breeding of flies. 2. Yeasts.--These cause fermentation--and we have (a) alcholic fermentation; (b) butyric, producing butyric acid in butter; (c) lactic, which sours milk and ripens cream ; (d) acetic, forming vinegar ; (e) proteolytic or peptonising fermentation as in the ripening of cheese ; (f) nitrifying, which converts ammonia into nitrites and nitrates. 2. Yeasts.--These cause fermentation--and we have (a) alcholic fermentation; (b) butyric, producing butyric acid in butter; (c) lactic, which sours milk and ripens cream ; (d) acetic, forming vinegar ; (e) proteolytic or peptonising fermentation as in the ripening of cheese ; (f) nitrifying, which converts ammonia into nitrites and nitrates. 3. Bacteria.---We all know about the divisions into streptococci or chains, micrococci, sarcinae, bacilli, etc., and milk contains many kinds of bacteria; by boiling or heating to about 200F. most are killed but the spores are not. Bacteria thrive on Proteids, e.g., meat, albumen and gluten. Presidential Address delivered at opening meeting of N.'vV. Branch in Manchester, Friday, October 10th, 1919. Presidential Address delivered at opening meeting of N.'vV. Branch in Manchester, Friday, October 10th, 1919. This he does from plants--and so long as the plants find a proper nidus the farmer does his job, with the help of CO~ and water from the heavens. As Medical Officer of Health for a large rural area consisting of about 200 square miles--and a population of about 33,000--with a birth rate of 15.9 an'd a death rate of 15.2 and an infant mortality rate of 73.6 per 1,000, and a phthisis death rate of .52, I think it may be of interest to most of you how this agricultural district presents itself to its Medical Officer of Health. p The nidus or upper tayers of soil are rich in bacteria, yeasts and fungi, and each has an impor~ tant share in the activities, causing the growth of the crop. This humus is not found in sand or barren soil, only in fertile soil, and is the remains of former dead leaves, roots, etc.--animal excrement. When one goes on a farm to inspect it under the various Acts and Orders--the manure heap comes in for some attention, and often a farmer's wealth is judged by the size of his midden--for manure is money--and contains vegetable tissures, digested and otherwise, hay and straw, fats from oil cakes, sugars and starches all in a state of decomposition, with nitrogen in the form of urea in the liquid manure perhaps running away, most valuable, con.. raining three-quarters of the whole of the nitrogen of the midden. Intestinal bacteria, then putrefactive, denitrifying and nitryfying bacteria, each hold their sway, causing chemical changes, and modifying the manure---this is advantageous, making it available for crop production. As a first consideration I think the most important is micro-organic life, fungi, yeasts and bacteria which enter into tile processes of cropping, either in the soil or water supply, and are, although probably the farmer does not know it, absolutely necessary for the soil to yield its crops. 1. FARM YARD AND DAIRY HYGIENE. BY waste and manures, contamination of soil by sewage which clears itself as do the polluted streams,nearIy all due to microorganisms, especially bacteria and yeasts. \¥. E. BURTON, J.P., M.R.C.S., L.R.C.P., D.P.H., M.O.H. SIW. Cheshire Combined Districts. The farmer's job is to make his farm which consists of soil, water and what he takes from the air, into food for man and beast. PUBLIC HEALTH. PUBLIC HEALTH. i9i 9. 41 FARM YARD AND DAIRY HYGIENE. BY \¥. E. BURTON, J.P., M.R.C.S., L.R.C.P., D.P.H., M.O.H. SIW. Cheshire Combined Districts. Presidential Address delivered at opening meeting of N.'vV. Branch in Manchester, Friday, October 10th, 1919. On two occasions I remember farmers losing several cattle every year from tuberculosis, although their cow- sheds were not perfect, but very good. I persuaded them to fence off the ponds and not allow tile water to be used for their cattle, this in each case ended their deaths from tuberculosis, the water evidently being the source of T.B. infection. Now I pass on to milk control. Cleanliness is the pass word. First the cow well bred, from a good milking strain, free from tuberculosis both on sires side and on dams side, one often asks was the bull alright ? Hardly a butt owner knows whether he was or he was not, this would not do for the Argentine! Still as no calf has ever been born with tuberculosis that may be considered not to matter, but it does, it produces a good nidus for infection. According to experiments at Charity Farm, "Woburn, calves from tuberculous parents were removed immediately after birth to a building about a mile away that had never housed cattle and put under the care of a man who only looked after them. The milk given these calves, obtained from a neighbouring farm, was raised to a temp. of 190F., by being imrnersed in vessels in a boiling copper, to make sure no bacilli were introduced. When old enough to be weaned the calves were taken to fields exclusively for their use. No evidence of T.B. was found in any of these animals after slaughtering, showing that by. means of isolation it is possible to rear healthy stock from tuberculous parents. Dairies ought to be well b, uilt with a northern aspect if possible and good impervious flooring, no drain inside, well lighted and ventilated, good supply of good water for milk cooling and boiler for scalding cans--cans often are not rinsed in cold water before being scalded, the former is 'tt~e only quick way of doing the job properly. Presidential Address delivered at opening meeting of N.'vV. Branch in Manchester, Friday, October 10th, 1919. The yard must be noted ; it is important to have a good yard surface, the first thing a cow does on being turned out of tile shiffon is to defaecate, I do not know why but it does, so the yard ought to be cleaned and swept at the same time that the shippon is. Yeasts thrive on starches and sugars. Fu,~gi thrive on both of above but perhaps prefer woody tissues. So we get decay in fruits, fermentation of juices, vegetable decay, putrefaction of animal bodies, souring of milk, ripening of cheese, decaying of PUBLIC HEALTH. DECEMBER, 42 Shippons ought to be built in a longitudinal direction about 9 to 10 feet high, each cow having about 500 cubic feet air space if turned out, as they are in Rural Districts, for tile greater portion of the day, ventilation through and through, as welt as cross. Light so that every portion of the shippon can be seen, even the corners. Feedi~lg troughs of earthenware; good groups 4 in. deep and 2 feet wide, with 6 feet behind the cows, all well paved and concreted between the blue brick or flags of concrete and falling to a self-cleansing yard gulley outside the cowshed, not near a window or door, the same to apply to stable and piggeries. The calf house requires attention, on ahnost every farm it is the worst building on the homestead, the darkest and most ill ventilated, sour smelling, with often several feet deep of manure in it, and ,, et the farmer says he is trying to stamp out tuberculosis ; he would not put his children in the worst rooms in his house. excrement soaking in from tile top. In these cases I advise the well being pumped dry, cleaned out, render the sides in coficrete to the impervious stratum, raise the well a foot and render in concrete and place a manhole with an airtight lid on, as a cover for the w, ell; this means that the person has to go up a couple of steps to do the pumping, but it prevents pollution from soakage ; it is necessary to fix the pump with iron stays and not wood, which soon become rotten. The drinking pond is often a source of danger, especially near the yard, and ought to be made so that no excreta can possibly get into it. Presidential Address delivered at opening meeting of N.'vV. Branch in Manchester, Friday, October 10th, 1919. In cheese making, unless pigs are kept, the whey soon becomes a nuisance and causes bad brook and river pollution, which can be traced for miles if allowed to run to a watercourse, fish flee from it and so do passers by, the smell from decomposing whey being very offensive; prevention, i.e., for- bidding wiley to be run to a watercourse is the only cure, whey ought to be run into a tank by means of an open earthenware channel (washable) and pumped out when required to feed pigs. Tile water supply to a farm is often defective, the wells being shallow wells, with no doubt plenty of bacteria in the water from the bad surroundings; the water supply that I advise, if a pipe supply is not available, is the artesian bore with a wind-mill fixed to pump the water to a tank, near the milk cooler, • tile waste water from the cooler to be run into a large watering trough in the yard for the cattle to drink out of, the overflow from this to a trapped gutley and on to the other drainage. In one case the artesian bore and windmill supplies two very large dairy farms, the supply to the second farm gravitating from the first ; even if the well % a deep well the water is often contaminated, The cow ought to be well groomed and welt fed and the udder wiped over with a wet cloth just before milking. The milkers' hands ought to be clean, and they should wear washable overalls and on no account to hanctle milk if he or any of his family is suffering from or recovering from infectious diseases. PUBLIC HEALTH. z9T9. 43 s the curd. The curd contains the nutritious food irnaterial in mill<, and the whey contains the milk sugar. In many parts of Cheshire milking machines are being used, of the suction type. These can milk six or more cattle at a time into covered milk pails, and if intelligently used, produce a clean milk. The secret is washing the machinery in cold water after use, before sterilizing. Cheeses are of 3 kinds: 1, hard ; 2, blue vein; 3, soft ; according to how much water or whey is contained in them. lgxamples of: (1) Hard pressed, are Cheshire, Cheddar, American, Swiss and Dutch. Presidential Address delivered at opening meeting of N.'vV. Branch in Manchester, Friday, October 10th, 1919. Cooling and sieving tile milk is the next thing to do and the sooner the cooling the better. It~ought to be taken straight from the cow and immediately cooled down to a temp. of 40 F. This makes the milk resist bacterial changes for a longer period than if not cooled, and the milk carries better if it has to be sent a distance away. (2) Blue vein, Stilton, VC'ensleydale, Gor- gonzola. (3) Soft cheese, Coiumbia, Camembert and crealgl. (3) Soft cheese, Coiumbia, Camembert and crealgl. Latterly lactic starters, i.e., pure B. Iactis cultures have been used to procure a more complete and uniform souring of the curd. The growth of acid organisms checks the growth of bart. that would be likely to spoil the cheese. This assists ripening and is a necessary step in the chemical changes that take place to bring about proper ripening. 7With regard to sieving this only removes large particles of dirt, hair, &c. There are several kinds of milk that one ought to recognise, e.g.: ..... (a) Ropey miik or slimy milk from cattle suffer- ing from Garget udder and is ropey when drawn. This is not fit for use--or it may become ropey after some hours, and is due to the action of B. lactis viscosus ; it may come from one cow and infect the wholeherd's milk, and the cause is difficult to find out at once; or bad water for can washing may cause it or when drinking ponds are low. Mould cultures are added to some cheeses to bring about proper ripening, e.g., Camembert ; and we all know the rnoulds of a good Stilton or Gorgonzola, probably produced artificially. Cheese can, like butter, be made from sterilized milk and the curd cut into small dice or large dice for a keepi~g cheese or a cheese to be used quickly. Be, fore closing I should like to mention Farm Pests. VVood-pigeons and rats are perhaps tile most destructive, and the best way to get rid of them is by having organised shooting days for a given week in the year for the whole country, to attack the pigeons, and a similar week of laying poison or rat virus for rats, as unless everybody acts at the same time the pigeons and rats are only driven from one place to another. (b) Blue milk. Presidential Address delivered at opening meeting of N.'vV. Branch in Manchester, Friday, October 10th, 1919. Generally from poverty, and may be skimmed, or due to a rare infection from the B2 cyonogenes. (c) Red milk due to a crushed quarter. (d) Bad tastes in milk due to turnip, rancid taste, putrid taste, or drug tastes from a beast undergoing a course of physic. t pass over milk borne diseases, tuberculosis, typhoid, scarlet fever, and diphtheria and diarrhceal diseases as superfluous in this paper, so go on to butter making. Usually cream is skimmed or separated from the milk, kept for 12 to 24 hours to ripen before it is churned. The process of ripening makes it churn easily and increases the butter yield, especially if the cream is separated, it also produces the butter flavour and aroma of goodbutter. During the ripening of the cream bacilli grow very rapidly, especially the B. acidi Iactiei, but there are other bacilli, as butter made from pasteurised cream and ripened by cultures of B. lactis has nothing like so good a flavour. Cleanliness and plenty of dry lime dusted into the nests of fowls keep down fleas in the hen runs. Lice on horses and cattle may be prevented in the winter by good grooming and sponging down with a solution of Kerol or Jeyes' Fluid. Cleanliness and plenty of dry lime dusted into the nests of fowls keep down fleas in the hen runs. Lice on horses and cattle may be prevented in the winter by good grooming and sponging down with a solution of Kerol or Jeyes' Fluid. LONDON UNIVERSITY EXANIINERSHIPS.--PrO- fessor Matthew Hay, M.O.H., for Aberdeen, and Professor of Public Health and Forensic Medicine in the University of Aberdeen, and Dr. Robert A. Lyster, County Medical Officer for Hampshire and Lecturer hi Public Health and Forensic Medicine, St. Bartholomew's Hospital, London, E.C., have been appointed Examiners in Hygiene and Forensic Medicine in the University of London. Cheese making. Cheese equals casein and milk fat ripened. Rennet is added to milk, this coagu- lates the casein which seperates from the whey and
https://openalex.org/W2809549308	https://elar.urfu.ru/bitstream/10995/73593/1/10.3390-su10062052.pdf	English	null	Sustainability of the Urban Transport System under Changes in Weather and Road Conditions Affecting Vehicle Operation	Sustainability	2,018	cc-by	12,312	Received: 28 April 2018; Accepted: 14 June 2018; Published: 16 June 2018 Abstract: The paper suggests a methodological approach for assessing the sustainability of the urban transport system. Parameters were selected for assessing the sustainability of the transport system and signiﬁcant factors affecting sustainability were determined. Parameters of the sustainability of the system when changes in the weather and road conditions affect vehicle operation were estimated on the basis of the simulation modeling. An integral indicator of sustainability was introduced to evaluate the sustainability of the transport ﬂow management subsystem and the methodological approach to its calculation was substantiated. The results from changing the parameters of the trafﬁc ﬂow were demonstrated in the case of a signiﬁcant amount of precipitation and the constraints put on the movement of vehicles on the road infrastructure unit due to snow-removal operations and road trafﬁc accidents. Also, the parameters of road trafﬁc under the reconstruction of the main street of regulated trafﬁc into a street of uninterrupted trafﬁc were presented. Keywords: transport system; sustainability; road trafﬁc; transport model; operating conditions; snow-removal operations; vehicle Sustainability of the Urban Transport System under Changes in Weather and Road Conditions Affecting Vehicle Operation Dmitriy Zakharov 1, Elena Magaril 2,* ID and Elena Cristina Rada 3 ID Dmitriy Zakharov , Elena Magaril and Elena Cristina Rada 1 Department of Road Transport Operation, Industrial University of Tyumen, Volodarskogo Street, 625000 Tyumen, Russia; zaharovda@tyuiu.ru 2 Department of Environmental Economics, Ural Federal University, Mira Street, 19, 620002 Ekaterinburg, Russia 1 Department of Road Transport Operation, Industrial University of Tyumen, Volodarskogo Street, 625000 Tyumen, Russia; zaharovda@tyuiu.ru 2 Department of Environmental Economics, Ural Federal University, Mira Street, 19, 620002 Ekaterinburg, Russia y y 2 Department of Environmental Economics, Ural Federal University, Mira Street, 1 620002 Ekaterinburg, Russia g 3 Department of Theoretical and Applied Sciences, Insubria University of Varese, via G.B. Vico, 46, I-21100 Varese, Italy; elena.rada@unitn.it 3 Department of Theoretical and Applied Sciences, Insubria University of Varese, via G.B. Vico, 46, I-21100 Varese, Italy; elena.rada@unitn.it * C d l67@ l T l 7 912 63 98 544 * Correspondence: magaril67@mail.ru; Tel.: +7-912-63-98-544 * Correspondence: magaril67@mail.ru; Tel.: +7-912-63-98-544 Sustainability 2018, 10, 2052; doi:10.3390/su10062052 sustainability sustainability sustainability sustainability www.mdpi.com/journal/sustainability 1. Introduction Transport services for the urban population is an important area of activity for municipal authorities. To this end, a transport system is formed which generally represents a coherent assembly of workers, vehicles and equipment, and elements of transport and transportation infrastructure, including a management system aimed at the efﬁcient transportation of goods and passengers. A crucial function of the transport system is to meet the demands of the city residents and allow their unrestricted mobility. The transport system is part of a more complex life-support system of the city. At the same time, the transport system is a complex system in itself and includes several subsystems: urban passenger public transport, the route network, the street and road network, and the management of trafﬁc lights These systems include elements such as intelligent transport systems, the trafﬁc ﬂow of light and heavy vehicles, and vehicles of individual owners, organizations, and taxi companies. An important factor affecting transport system operation is road safety. To achieve a high safety indicator, various measures are applied, including those related to the Internet of Things (IoT) [1–3], adaptive lighting control [4–11], etc. Providing efﬁcient transport services to the population with minimal budgetary costs is a highly relevant task. No less signiﬁcant a task is meeting the demand for the movement of the city Sustainability 2018, 10, 2052; doi:10.3390/su10062052 www.mdpi.com/journal/sustainability Sustainability 2018, 10, 2052 2 of 17 residents under conditions of temporary constraints or the cessation of both vehicular and pedestrian trafﬁc [12–15]. Temporary limitations may be associated with adverse weather conditions, the construction and maintenance of roads, facilities associated with engineering infrastructure and capital construction, mass rallies and public events, and increased trafﬁc during holidays. g y A prediction algorithm is also used to estimate the effect of weather on many other types of human activity [16–19]. Reducing the impact of the negative consequences of temporary trafﬁc constraints or reduced public transport services can be achieved in two ways: by creating a reserve for the transit and carrying capacity of the road network and by the prompt reorganization of trafﬁc in the event of external disruptive factors. Developing road infrastructure and increasing the capacity of the street-road network (through the reconstruction of streets of regulated trafﬁc to the main streets of uninterrupted trafﬁc) is one of the possible ways to improve the sustainability of the transport system. 1. Introduction Both of these approaches include a system of measures aimed at solving the problem. In order to determine the optimal solution and then form an appropriate system of measures, it is necessary to adequately assess changes in the state of the transport system, i.e., its sustainability, which is one of the most signiﬁcant properties [20–27]. Sustainability is considered in relation to the system of urban public transportation, individual transportation providers, and municipal routes, as well as uneven spatial distribution of the severity of road accidents [28]. Sustainability as a characteristic of the transport system and its individual elements in relation to weather conditions was studied in References [29–32]. The application of “big data” and digital technologies in the urban transport system ensures a more accurate assessment of its performance [33]. Road trafﬁc accidents and incidents on the roads have a signiﬁcant impact on the short-term sustainability indicators of the urban transportation system [34–37]. Studies on the environmental assessment and justiﬁcation of the methods for reducing emissions of CO2 and toxic substances within the exhaust gases of vehicles are also relevant [37–47]. The sustainability of the transport system can be enhanced by improving the adaptation properties of vehicles, as well as the choice of the rational application area of vehicles and vehicle brands and models most suitable to the speciﬁc operating conditions [48,49]. Implementation of the “Mobility as a Service (MaaS)” concept in cities is one method to improve the sustainability and efﬁciency of urban transport systems. It is crucial to take into account the interaction of different modes of transportation and types of transport in the formation of the transport supply [50–52]. The development of autonomous and self-driving vehicles will have a signiﬁcant impact on the ainability of the urban transport system in the future. It was suggested in Reference [53] that sustainability should be considered instead of efﬁciency when choosing a road design, the cost of which would be justiﬁed by the efﬁcient handling of accidents and other disruptions. Selecting appropriate indicators for sustainability assessment is a complex process [33], which determines the signiﬁcance of research in this direction. The aim of this work is to develop an approach for assessing the sustainability of the urban transport system under varying weather, climate, and road conditions for vehicle operation. • amount and intensity of precipitation; 2. Materials and Methods The research was carried out in Tyumen—a transport hub as well as an important business and industrial center in Siberia, Russian Federation. Tyumen has a humid continental climate with warm, somewhat humid summers and long, cold winters. The sustainability of the transport system was assessed with respect to the inﬂuence of the following factors: 3 of 17 Sustainability 2018, 10, 2052 • duration of road cleaning; • number of road sections with constraints or the cessation of vehicle trafﬁc due to road trafﬁc accidents. These factors have an impact on variations in the operating conditions, which leads to changes in the trafﬁc parameters. The system theory and simulation modeling were applied in this work to study the transport system of the city. Simulation modeling was carried out using the PTV Vissim program. Vissim’s trafﬁc ﬂow model is a stochastic, timestep-based, microscopic model that treats driver-vehicle units as basic entities. The trafﬁc ﬂow model contains a psycho-physical car following a model for longitudinal vehicle movement and a rule-based algorithm for lateral vehicle movement. The models deployed are based on Wiedemann’s extensive research work [54]. The simulation model of the trafﬁc was made for the main street of regulated trafﬁc and had the following characteristics: the length of the highway in the main direction was 3.5 km, the length of all segments (road sections) in the model was 14 km, the number of trafﬁc lanes was 6, the number of crossroads was 7, the number of trafﬁc lights was 8, and the total number of vehicles in the system was 26,392 units. The total mileage of vehicles within the frame of modeling was 16,280 km. The simulation period was taken to be equal to 1 h during the morning under the maximum load of the street-road network. For the vehicles that did not have enough time to enter the simulation area or ﬁnish their movement within it, the simulation time period was increased. This allowed the movement of all vehicles to be completed and meant that the trafﬁc parameters could be obtained for different modeling scenarios with the same transport operation. A linear object (part of the street) was simulated in the study with the detailing of each vehicle and pedestrian (micromodel). This type of modeling was chosen having taking into account the recommendations of certiﬁed experts on transport simulation in PTV Vision programs (developer PTV GROUP, Karlsruhe, Germany). 2. Materials and Methods It is critical that the micromodel be able to accept variable trafﬁc parameters that correspond to different road and weather conditions. In macro models (for example, in the PTV Visum program) the standard parameters which correspond to normal weather conditions are usually applied, but these would not allow the research goal to be achieved. The composition of the ﬂeet of vehicles was determined by video recording and counting the number of vehicles of different classes (cars and trucks, buses) at each crossroad. When conducting the study during a period of snowfall, the minimum lateral separation distance for all types of vehicles (distance at rest), maximum visibility distances and limit of rear visibility, instantaneous speed on the selected road sections, and distance traveled by the vehicle at a constant speed mode, under acceleration and deceleration measurements, were performed on the approach to the crossroad, on the descent and entry onto the overpass, and on the road section with uninterrupted trafﬁc. Based on the results of the measurements taken, the acceleration and deceleration of vehicles were calculated during start-up and braking. The speed value was read from the engine electronic control unit using special equipment. The estimation of trafﬁc parameters under adverse weather conditions was carried out after changing the values of the initial model parameters, which are given in Table 1. The list of factors, important in assessing the sustainability of the transport system under varying weather, climate, and road conditions, as well as their weight, were determined on the basis of questionnaires and expert assessments. The expert team included researchers from Tyumen Industrial University, trafﬁc police specialists in the Tyumen region, representatives of the enterprises of the transport complex, and the administration of Tyumen city. The methodology proposed by the authors allows changes in the sustainability of the transport system to be assessed under different weather-climatic conditions. It is necessary to perform preliminary investigations, determine the values of the basic parameters on a small section of the road, and then apply the speciﬁed parameters to the simulation model. 4 of 17 Sustainability 2018, 10, 2052 Table 1. Parameters of the vehicle trafﬁc under normal and adverse weather conditions. 2. Materials and Methods Parameters of the Simulation Model Value under Normal Conditions Value under Adverse Weather Conditions Relative Change of the Parameter Value, % Maximum speed, km/h 60.0 40.0 −33.3 Maximum visibility distances, m 250.0 150.0 −40.0 Maximum limit of back visibility, m 150.0 50.0 −66.7 Inherent maximum deceleration, m/s2 −4.0 −3.2 −20.0 Inherent acceptable deceleration, m/s2 −1.0 −0.8 −20.0 Acceptable deceleration of vehicle behind, m/s2 −1.0 −0.8 −20.0 Minimum collision distance before/behind, m 0.5 1.5 200.0 Maximum deceleration for full braking, m/s2 −3.0 −2.0 −33.3 Minimum side distance for all types of vehicles (distance at rest), m 0.2 0.3 50.0 Table 1. Parameters of the vehicle trafﬁc under normal and adverse weather conditions. 3. Results Investigations of the trafﬁc management system as a subsystem of the urban transport system were carried out. The system has a set of properties: organization, integrity, emergence, functionality, structural properties, sustainability, reliability, survivability, adaptability. The property of adaptability of the transport system in relation to the controlled crossroads was considered in Reference [55]. The state of the system is inﬂuenced by a large number of external and internal factors. With regard to the conditions of vehicle operation, external factors are generally divided into weather-climatic, road, and transport factors. These groups of factors have a signiﬁcant effect on the efﬁciency of the transport system and its individual elements [56,57]. The degree of the factor impact on the transport system depends on the property being studied. The main signiﬁcant characteristics of the factors affecting the sustainability of the transport system include: • the duration of the disturbing factor’s inﬂuence on the system; • the strength of the factor; • the object of the negative effect of the factor (on the element of the system or relationship between the elements). The system is composed of interconnected elements. Disturbance of the equilibrium state of the system is caused by a factor’s inﬂuence on the elements or the breaking-down of the connection between them. This study considers the impact of a negative factor (snowfall) on road conditions (an element of the transport system). By estimating the degree of the trafﬁc parameters’ variation, one can speak of a change in the sustainability of the transport system. For example, snowfall affects the state of the carriage-way of the road. If there is snow cover on the road surface, the coefﬁcient of tire adhesion to the supporting surface decreases from 0.7–0.8 to 0.2–0.3. The value of the coefﬁcient of tire adhesion to the road surface depends on the design and state of the tire tread; thus, this indicator cannot be taken into account when modeling. The inﬂuence of weather, climate, and road conditions for operation on the sustainability of the transport system reveals itself to the greatest extent in the autumn period with the ﬁrst snowfall accompanied by a large amount of precipitation. During this period, many car owners are still using summer tires instead of winter tires. Under adverse road conditions the coefﬁcient of adhesion of the summer tires to the road surface signiﬁcantly reduces, reaching the value of 0.1 [58]. hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System 3.1. Methodological Approach to Assessing the Sustainability of the Urban Transport System 3.1. Methodological Approach to Assessing the Sustainability of the Urban Transport System To assess changes in the sustainability of the transport system in relation to traffic management, the authors propose the introduction of a traffic management coefficient KT, which characterizes the degree of increase in the total travel time under adverse climatic, road, or transport conditions in comparison with the standard conditions, given by Equation (1): ′ To assess changes in the sustainability of the transport system in relation to trafﬁc management, the authors propose the introduction of a trafﬁc management coefﬁcient KT, which characterizes the degree of increase in the total travel time under adverse climatic, road, or transport conditions in comparison with the standard conditions, given by Equation (1): 𝐾𝑇= 𝑡, (1) ute under adverse conditions (in minutes), while t is the d d diti (i i t ) KT = t′ t , (1) (1) mean route travel time on route under standard conditions (in minutes). It is advisable in assessing changes in sustainability to take into account the impact of environmental degradation on the street-road network, especially in places with a large number of where t′ is the mean route travel time on route under adverse conditions (in minutes), while t is the mean route travel time on route under standard conditions (in minutes). mean route travel time on route under standard conditions (in minutes). It is advisable in assessing changes in sustainability to take into account the impact of environmental degradation on the street-road network, especially in places with a large number of where t′ is the mean route travel time on route under adverse conditions (in minutes), while t is the mean route travel time on route under standard conditions (in minutes). g , p y p g pedestrians (pedestrian crossings, stopping points). To take into account the contribution of individual toxic substances to the total negative impact, their relative environmental hazard must be considered. This is taken into account when calculating It is advisable in assessing changes in sustainability to take into account the impact of environmental degradation on the street-road network, especially in places with a large number of pedestrians (pedestrian crossings, stopping points). hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System damage De to the atmospheric air caused by toxic substances, emitted with the exhaust gases of vehicles (for example, according to the methodology proposed for Russia [59]), given by Equation (2): 𝐷𝑒= 𝑑∙𝐾𝑒∙𝐽𝑑∙∑ 𝐺𝑖 𝑁 𝑖=1 ∙𝐾𝑖 (2) where d is an indicator of specific damage to the atmospheric air, caused by a mass unit of a toxic To take into account the contribution of individual toxic substances to the total negative impact, their relative environmental hazard must be considered. This is taken into account when calculating damage De to the atmospheric air caused by toxic substances, emitted with the exhaust gases of vehicles (for example, according to the methodology proposed for Russia [59]), given by Equation (2): e ton); Ke is the coefficient of environmental situation and Jd is the index-deflator for the recalculation of specific damage es; Gi is the actual mass of the i-th toxic substance emitted to the d i d f ti ( i t ) d K i i di t f th l ti De = d·Ke·Jd· N ∑ i=1 Gi·Ki (2) (2) atmospheric air during the considered period of time ( intons); and Ki is an indicator of the relative environmental hazard of the i-th toxic substance, calculated as a quantity inversely proportional to the maximum allowable concentration of the i-th toxic substance (MACi) (in relative ton/ton). To assess changes in the environmental sustainability of the transport system, the authors propose the introduction of a coefficient of environmental compatibility KE, which is determined by a relative change in the damage caused by toxic emissions, given by Equation (3): KE = 𝐷𝑒′ 𝐷𝑒= ∑ 𝐺´𝑖∙𝐾𝑖 𝑖=1 𝑁 ∑ 𝐺𝑖∙𝐾𝑖 𝑖=1 𝑁 = 𝐺´ 𝐺 (3) where d is an indicator of speciﬁc damage to the atmospheric air, caused by a mass unit of a toxic substance (in monetary unit/relative ton); Ke is the coefﬁcient of environmental situation and ecological signiﬁcance of the region; Jd is the index-deﬂator for the recalculation of speciﬁc damage from current prices to comparable ones; Gi is the actual mass of the i-th toxic substance emitted to the atmospheric air during the considered period of time ( intons); and Ki is an indicator of the relative environmental hazard of the i-th toxic substance, calculated as a quantity inversely proportional to the maximum allowable concentration of the i-th toxic substance (MACi) (in relative ton/ton). 3. Results Under adverse road conditions, drivers usually act in two ways: Under adverse road conditions, drivers usually act in two ways: • they decrease the maximum speed of the vehicle to reduce the length of the braking dista • they decrease the maximum speed of the vehicle to reduce the length of the braking distance, or • they increase the distance between the moving car ahead to reduce the probability of collision of vehicles. t ey dec ease t e a u speed o t e e c e to educe t e e gt o t e b ak g d sta ce, o • they increase the distance between the moving car ahead to reduce the probability of collision of vehicles. The inﬂuence of the maximum speed on the total travel time and average speed for an urban main street of regulated trafﬁc is shown in Figure 1. 5 of 17 Sustainability 2018, 10, 2052 (a) (b) Figure 1. Influence of maximum speed of vehicles on the average speed (a) and total travel time (b). Figure 1. Inﬂuence of maximum speed of vehicles on the average speed (a) and total travel time (b). (b) (a) i I fl f d f h l (a) (b) (a) Figure 1. Influence of maximum speed of vehicles on the average speed (a) and total travel time (b). Figure 1. Inﬂuence of maximum speed of vehicles on the average speed (a) and total travel time (b). According to the experts conclusions made on the basis of the questionnaire, sustainability as a characteristic of the urban transport system is subject to changes in weather, climate, and road conditions for vehicle operation, including the impact of these changes on the route timetable, environmental safety of vehicle operation, accidents, and operating costs of the transport process. The system approach allows the consolidation of these heterogeneous factors for an integrated assessment. According to the experts’ conclusions made on the basis of the questionnaire, sustainability as a characteristic of the urban transport system is subject to changes in weather, climate, and road conditions for vehicle operation, including the impact of these changes on the route timetable, environmental safety of vehicle operation, accidents, and operating costs of the transport process. The system approach allows the consolidation of these heterogeneous factors for an integrated assessment. hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System To assess changes in the environmental sustainability of the transport system, the authors propose the introduction of a coefﬁcient of environmental compatibility KE, which is determined by a relative change in the damage caused by toxic emissions, given by Equation (3): 6 of 17 Sustainability 2018, 10, 2052 6 of 17 KE = D′ e De = ∑i=1 N Gi·Ki ∑i=1 N Gi·Ki = G G (3) (3) where De, De is the environmental damage to the atmospheric air, Gi, Gi is the actual mass of the i-th toxic substance entering the atmospheric air during the considered period of time, G′, G is the equivalent mass of toxic emissions, taking into account the relative toxicity (in relative ton) when moving under adverse and standard conditions, respectively. To account for the operating costs in assessing changes in the sustainability of the transport system, it is proposed to take into account the fuel consumption and its change under adverse conditions. For this purpose, the authors propose the introduction of a fuel efﬁciency coefﬁcient, KQ, given by Equation (4): KQ = ∑Q′ ∑Q , (4) (4) where ∑Q′ is the total fuel consumption of vehicles on the route or road segment considered when driving under adverse conditions (in liters), and ΣQ is the total fuel consumption of vehicles on the route or road segment considered when driving under standard conditions (in liters). where ∑Q′ is the total fuel consumption of vehicles on the route or road segment considered when driving under adverse conditions (in liters), and ΣQ is the total fuel consumption of vehicles on the route or road segment considered when driving under standard conditions (in liters). The fuel consumption in transport vehicles is directly proportional to the emissions of carbon dioxide in the process of operation, which makes it possible to assess changes in the sustainability of the transport system in terms of fuel efﬁciency, calculating the ratio of carbon dioxide emissions under adverse and standard weather conditions, given by Equation (5): KQ = ∑CO′ 2 ∑CO2 , (5) (5) where ∑CO2′ is the total CO2 emissions emitted by all vehicles under adverse conditions (in tons), and ∑CO2 is the total CO2 emissions emitted by all vehicles under standard conditions (in tons). hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System 7 of 17 Sustainability 2018, 10, 2052 According to the results of the expert evaluation, the trafﬁc management coefﬁcient KT has the greatest weight under the conditions considered. This is determined by the signiﬁcant problems in the organization of transport services for the population, insufﬁcient road capacity, and the poor quality of public transport operation. After relevant improvements to the transport system have been implemented, the further efforts of specialists can be concentrated on reducing the number of road accidents, fuel consumption, and emissions of harmful substances with exhaust gases of vehicle internal combustion engines (ICEs). The value of the weight coefﬁcient λT might decrease in the future and the values of the λE and λQ coefﬁcients might increase. When assessing the changes in the sustainability of the transport system, the trafﬁc safety factor KMVC was not taken into account because, according to the experts’ opinion, its contribution to the integral indicator of sustainability in Tyumen is negligible, when considering the experience of driving in adverse weather conditions for most local drivers. Under adverse weather conditions, as a rule, drivers operate their vehicles more carefully. This is conﬁrmed by statistics on the number of road accidents per month of the year, presented in Table 2. As can be seen from the data presented, in three of the four winter months the number of road accidents is less than the average annual values. An increase in values observed in December is due to a signiﬁcant increase in trafﬁc intensity (the number of trips) during winter holidays. Table 2. Statistics on the number of trafﬁc accidents by month in winter. Period Number of Accidents, Units during the Period 2015 2016 November 118 83 December 128 126 January 122 99 February 108 77 In a year 1481 1395 Average number of accidents per month during the year 123.4 116.3 Table 2. Statistics on the number of trafﬁc accidents by month in winter. The criterion of fuel efﬁciency KQ was calculated from the change in fuel consumption. The integral indicator of sustainability, taking into account the weight of the factors, can be calculated by Equation (8): K = 0.85·KT + 0.05·KE + 0.1·KQ (8 (8) 3.2.1. hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System where ∑CO2′ is the total CO2 emissions emitted by all vehicles under adverse conditions (in tons), and ∑CO2 is the total CO2 emissions emitted by all vehicles under standard conditions (in tons). Thus, Equations (4) and (5) can be used to calculate KQ depending on the type of source rmation available (fuel consumption, CO2 emissions). To take into account socioeconomic factors, it is proposed to apply a trafﬁc safety factor KMVC. The coefﬁcient shows how many times the number of accidents on a given section of the road network increases under adverse road conditions in comparison with the standard conditions, given by Equation (6): KMVC = N′ MVC NMVC , (6) (6) where N′ MVC is the number of motor vehicle collisions (MVCs), or road incidents, on the road network section under adverse conditions (in units), and NMVC is the number of MVCs, or road incidents, on the road network section under standard conditions (in units). where N′ MVC is the number of motor vehicle collisions (MVCs), or road incidents, on the road network section under adverse conditions (in units), and NMVC is the number of MVCs, or road incidents, on the road network section under standard conditions (in units). The authors suggest that a change in transport system sustainability can be estimated using an integral indicator of sustainability, K, taking into account the weight of each factor λi (0 ≤λi ≤1; ∑N i=1 λi = 1), which is evaluated by the coefﬁcient Ki, as given by Equation (7): K = n ∑ i=1 Ki·λi = λT·KT + λE·KE + λQ·KQ + λMVC·KMVC, (7) (7) where n is the number of criteria involved in the evaluation. λi may vary for different municipalities and can be determined by the method of expert assessments considering the research purpose. 3.2. Assessment of Changes in the Sustainability of the Transport System of Tyumen City under Changes in Weather, Climate, and Road Conditions for Vehicle Operation 3.2. Assessment of Changes in the Sustainability of the Transport System of Tyumen City under Changes in Weather, Climate, and Road Conditions for Vehicle Operation The proposed approach was tested in assessing changes in the sustainability of the transport system of the main street of regulated trafﬁc of Tyumen city (Russia). The proposed approach was tested in assessing changes in the sustainability of the transport system of the main street of regulated trafﬁc of Tyumen city (Russia). hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System Parameters of the Trafﬁc Flow Value under Normal Conditions Value under Worsening Weather Conditions Relative Change of the Parameter Value, % Mean delay time, s 201 247 23 Average number of stops 4.1 4.5 9 Average speed, km/h 9 7 −17 Mean delay time in trafﬁc congestion, s 165 211 28 Total travel time, h 1811.0 2196.2 21 Total delay time, h 1471.7 1811.5 23 Total number of stops 109,029 119,244 9 Delay time of vehicles waiting for entry, h 686.4 1115.4 63 rs of the Trafﬁc Flow Value under Normal Conditions Value under Worsening Weather Conditions Relative Change of the Parameter Value, % The results presented show that when the highway capacity decreases due to worsening road conditions caused by snowfall, the trafﬁc parameters become worse. Under such conditions the average delay time increased by 23%, the average speed decreased by 17%, and the value of the trafﬁc management coefﬁcient KT was equal to 1.21. g q The delay time of vehicles waiting for entry increased by 63%, which demonstrates deterioration in the trafﬁc quality. Under worsening road conditions, the fuel consumption of vehicles and the amount of emissions of harmful substances with exhaust gases of vehicle ICEs increase. This is due to an increase in the number of stops and trafﬁc non-uniformity. The time when vehicles move in an unsteady mode of operation also increases. In the simulations, the emissions of carbon monoxide (CO), nitrogen oxides (NOx), and volatile organic compounds (VOC) were taken into account. The indicators of relative hazard Ki (in relative ton/ton) of the speciﬁed pollutants were assumed to be equal to 0.4 for CO, 16.5 for NOx, and 0.7 for VOC, respectively [59]. The relevant role of NOx has also been conﬁrmed in the latest literature. The inﬂuence of different regimes on the emissions of individual pollutants was ignored, which reduced the reliability of the estimation, but at the same time increased the availability of the method since there was no requirement for additional voluminous information for calculations. By taking into account this simpliﬁcation, the change in emissions of all substances and the reduced mass of emissions with regard to their relative hazard will directly correlate with fuel consumption. The values of fuel consumption and mass of emissions under different road conditions are given in Table 4. Table 4. hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System Change in the Road Trafﬁc Parameters in the Case of Precipitation When a Large Amount Falls within a Short Period of Time In the case of precipitation, when a large amount falls within a short period of time, the road conditions for vehicle operation become signiﬁcantly worse. In winter, in the cold-temperate climatic zone (the Tyumen conditions), precipitation is characterized by several indicators: • the average decadal height of the snow cover is 38 cm; • the average decadal height of the snow cover is 38 cm; • the maximum decadal height of the snow cover is 63 cm; • the maximum decadal height of the snow cover is 63 cm; • the number of days in a year with snow cover is 161 days; • the number of days in the winter with solid precipitation is 67 days, including 10.6 days in November, 15.3 days in December, and 13.0 days in January [60]. • the number of days in the winter with solid precipitation is 67 days, including 10.6 days in November, 15.3 days in December, and 13.0 days in January [60]. The amount of precipitation for the period November–March in Tyumen is 107 mm. If an amount of precipitation in the range of 7 to 19 mm falls within 12 h (from 7 to 19 cm of snow), the weather conditions are considered to be difﬁcult, with a precipitation amount of more than 20 mm indicating extremely difﬁcult conditions. y The results of the trafﬁc modeling under conditions of heavy snowfall and the surface of the road having freshly fallen, unconsolidated snow with a thickness of more than 7 cm are given in Table 3. Sustainability 2018, 10, 2052 8 of 17 Table 3. Change in the parameters of the trafﬁc ﬂow under adverse conditions. Table 3. Change in the parameters of the trafﬁc ﬂow under adverse conditions. hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System Inﬂuence of adverse weather conditions on fuel consumption and the environmental compatibility of the transport system. Table 4. Inﬂuence of adverse weather conditions on fuel consumption and the environmental compatibility of the transport system. Indicators Value under Normal Conditions Value under Adverse Weather Conditions CO emission, kg 40.54 46.15 NOX emission, kg 7.89 8.98 VOC emission, kg 9.40 10.70 Equivalent mass of emissions, relative kg 152.98 174.12 Fuel consumption, L 580 660 If the road conditions worsen, these values increase by 13.8%. The values of the coefﬁcients KQ and KE are equal to 1.14. The value of the integral indicator of sustainability K is equal to 1.2. A minor difference between the value of the integral indicator and the value of the partial coefﬁcient of sustainability by the trafﬁc management criterion is due to the highest weight value of this indicator being equal to 0.85. g q Decreasing the speed value under worsening road conditions leads to an increase in the travel time. Consequently, the value of the sustainability coefﬁcient of the system increases, which indicates deterioration in the quality of transport services for the city’s population. Sustainability 2018, 10, 2052 9 of 17 3.2.2. Change in the Road Trafﬁc Parameters during Snow-Removal Operations 3.2.2. Change in the Road Trafﬁc Parameters during Snow-Removal Operations The primary snow removal operations include treatment using anti-icing materials, shoveling and sweeping snow, and forming a snow bank for subsequent removal. The secondary operations consist of removal of snow, chipping ice, and the removal of snow-ice formations. In the case of a large amount of precipitation, there can be a situation where a considerable period of time elapses between the operations of the ﬁrst and second stages. This is typical for cities where there is not enough equipment for snow removal (dump trucks, loading and unloading machinery) and its disposal (snow melting units). After carrying out technological operations of the ﬁrst stage, the snow bank remains at the edge of the roadway until the moment of snow removal. The snow bank can reach 20–60 cm in height and 100–150 cm in base width. For urban roads with a lane width of 3–3.25 m, this can result in the actual cessation of trafﬁc on the rightmost lane. This leads to a decrease in the capacity of the road sections with the snow bank formed. hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System When the transport demand exceeds the capacity, transport congestion occurs and the trafﬁc parameters worsen. When creating a simulation model of the road trafﬁc during the snow-removal period, the following assumptions were made: • the rightmost lane of the road in the main direction is occupied by a snow bank and special snow-removing equipment for its loading and removal, • the gap in the snow bank begins 50 m before the crossroad and ends 50 m after the crossroad. If the gap in the snow bank before the crossroad turns out to be smaller, then the parameters of the trafﬁc ﬂow become signiﬁcantly worse. If the gap in the snow bank before the crossroad turns out to be smaller, then the parameters of the trafﬁc ﬂow become signiﬁcantly worse. The change in the parameters of the trafﬁc ﬂow in the presence of a snow bank on the main street is given in Table 5. The change in the parameters of the trafﬁc ﬂow in the presence of a snow bank on the main street is given in Table 5. Table 5. Change in the trafﬁc ﬂow parameters during snow-removal operations. g p g p Parameters of the Trafﬁc Flow Value under Normal Conditions Value in the Presence of a Snow Bank Relative Change of the Parameter Value, % Mean delay time, s 201 311 55 Average number of stops 4.1 4.9 19 Average speed, km/h 9 6 −31 Mean delay time in trafﬁc congestion, s 165 268 63 Total travel time, h 1811.0 2616.9 44 Total delay time, h 1471.7 2277.7 55 Total number of stops 109,029 130,287 19 Delay time of vehicles waiting for entry, h 686.4 2570.4 274 With a decrease in the road capacity due to the snow bank formed on the rightmost lane, the trafﬁc parameters became considerably worse. Under such conditions, the mean delay time increased by 55%, the average speed reduced by 31%, and the delay time of vehicles waiting for entry increased by 274%. This characterizes a signiﬁcant deterioration in the quality of trafﬁc. 274%. This characterizes a signiﬁcant deterioration in the quality of trafﬁc. The calculated value of the trafﬁc management coefﬁcient KT was equal to 1.44. The values of the indicators presented in Tables 3 and 5 characterize the simulated system as a whole. On some sections of the street-road network, changes in the indicators can be even more signiﬁcant. hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System For instance, in the model of the street of regulated trafﬁc with the presence of a snow bank on the carriageway, the decrease in the average speed in the main direction was 49%. With the road conditions worsening and the presence of the snow bank on the road, fuel consumption and the amount of emissions of harmful substances from the exhaust gases of motor vehicles increased by 26% (Table 6). The value of the coefﬁcients KQ and KE was 1.26. The value of the integrated coefﬁcient of the transport system sustainability K was 1.41. 10 of 17 Sustainability 2018, 10, 2052 Table 6. Change in fuel consumption and the environmental compatibility of the transport system under adverse weather conditions during snow-removal operations. Indicators Value under Normal Conditions Value in the Presence of the Snow Bank along the Street-Road Network CO emission, kg 40.54 51.10 NOX emission, kg 7.89 9.94 VOC emission, kg 9.40 11.84 Equivalent mass of emissions, relative kg 152.98 192.73 Fuel consumption, L 580 731 Table 6. Change in fuel consumption and the environmental compatibility of the transport system under adverse weather conditions during snow-removal operations. In the subsequent assessment, the simulation object considered was transformed into a main street of uninterrupted trafﬁc. The separation of transport and pedestrian ﬂows in space was achieved through the creation of two road junctions, a tunnel, and four aboveground as well as one underground pedestrian crosswalks. The trafﬁc ﬂow parameters for this model and their comparison are given in Table 7. Table 7. Change in the trafﬁc ﬂow parameters when creating the main street of uninterrupted trafﬁc. Table 7. Change in the trafﬁc ﬂow parameters when creating the main street of uninterrupted trafﬁc. hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System Parameters of the Trafﬁc Flow Value for the Main Street of Regulated Trafﬁc Value for the Main Street of Uninterrupted Trafﬁc Relative Change of the Parameter Value, % Mean delay time, s 201 74 −63 Average number of stops 4.1 1.1 −73 Average speed, km/h 9.0 24.4 171 Mean delay time in trafﬁc congestion, s 165 61 −63 Total travel time, h 1811 1029 −43 Total delay time, h 1472 541 −63 Total number of stops 109,029 28,962 −73 Total delay time in trafﬁc congestion, h 1208 446 −63 Parameters of the trafﬁc ﬂow in the model of the street of uninterrupted trafﬁc were substantially improved: the average speed increased by 171% and the total travel time was reduced by 43%. This allows the basic values of the indicators in Equations (1)–(6) to be reduced. Changes in the trafﬁc ﬂow parameters for adverse weather and road conditions along the main street of uninterrupted trafﬁc are given in Table 8. Table 8. Parameters of the trafﬁc ﬂow for the main street of uninterrupted trafﬁc under adverse weather conditions during snow-removal operations. Table 8. Parameters of the trafﬁc ﬂow for the main street of uninterrupted trafﬁc under adverse weather conditions during snow-removal operations. Parameters of the Trafﬁc Flow Value under Normal Conditions Value under Adverse Weather Conditions Value in the Presence of a Snow Bank along the Street-Road Network Mean delay time, s 74 80 131 Average number of stops 1.1 1.4 2.1 Average speed, km/h 24.4 23.4 17.3 Mean delay time in trafﬁc congestion, s 61 62 110 Total travel time, h 1029 1072 1450 Total delay time, h 541 585 962 Total number of stops 28,962 36,423 54,707 Delay time of vehicles waiting for entry, s 15 179 29 The parameters of trafﬁc ﬂow, fuel economy, and environmental compatibility under adverse weather and road conditions for the model of the main street of uninterrupted trafﬁc are much better than those in the model of the street of regulated trafﬁc, even under normal conditions. Figure 2 presents the examples of fuel efﬁciency and the environmental compatibility for the street of uninterrupted trafﬁc under various operating conditions in comparison with the street of regulated trafﬁc. hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System by reducing the number of trips made by residents of the city in private vehicles and using the route transport. At the same time, the sustainability of the public transport system also significantly changes. Because it is not possible to increase the carrying capacity of public transport during such periods, the quality of passenger transportation and travel comfort significantly decrease. The development of road infrastructure helps to ensure the sustainability of the transport system in the event of road accidents and incidents. In this study, the traffic flow was simulated after Increasing the sustainability of the transport system in adverse weather conditions is possible by reducing the number of trips made by residents of the city in private vehicles and using the route transport. At the same time, the sustainability of the public transport system also signiﬁcantly changes. Because it is not possible to increase the carrying capacity of public transport during such periods, the quality of passenger transportation and travel comfort signiﬁcantly decrease. y y, an accident (a collision of two cars on the approach to the crossroad at a distance of 10 m from the stop line in the center of the simulation model) within one lane using two variants: the main street of regulated and uninterrupted traffic under normal weather conditions. After the accident, the parameters of the traffic flow on the main street of uninterrupted traffic will be better than when driving on the main street of regulated traffic without an accident (Table 9). Fi 3 h h i h i l i di f i bili i d diff The development of road infrastructure helps to ensure the sustainability of the transport system in the event of road accidents and incidents. In this study, the trafﬁc ﬂow was simulated after an accident (a collision of two cars on the approach to the crossroad at a distance of 10 m from the stop line in the center of the simulation model) within one lane using two variants: the main street of regulated and uninterrupted trafﬁc under normal weather conditions. Figure 3 shows how many times the integral indicator of sustainability varies under different weather-climatic and road conditions in comparison with the initial value of the indicator. The indicator value for the main street of regulated traffic under normal weather conditions corresponds to 1. hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System 11 of 17 ted 11 of 17 ted Sustainability 2018, 10, 2052 uninterrupted traffic u ffi (a) Fi 2 P t f f l d th i (b) l ibili f h ffi fl d (a) (b) Figure 2. Parameters of fuel economy and the environmental compatibility of the traffic flow under different operating conditions for the main streets of uninterrupted and regulated traffic: (a) total fuel consumption (in liters); (b) equivalent mass of emissions of harmful substances (in relative kg). Figure 2. Parameters of fuel economy and the environmental compatibility of the trafﬁc ﬂow under different operating conditions for the main streets of uninterrupted and regulated trafﬁc: (a) total fuel consumption (in liters); (b) equivalent mass of emissions of harmful substances (in relative kg). (b) (a) Figure 2. Parameters of fuel economy and the environmental compatibility of the traffic flow under different operating conditions for the main streets of uninterrupted and regulated traffic: (a) total fuel consumption (in liters); (b) equivalent mass of emissions of harmful substances (in relative kg). Figure 2. Parameters of fuel economy and the environmental compatibility of the trafﬁc ﬂow under different operating conditions for the main streets of uninterrupted and regulated trafﬁc: (a) total fuel consumption (in liters); (b) equivalent mass of emissions of harmful substances (in relative kg). , , p p of the traffic flow for the main street of uninterrupted traffic will be less than that for the main street of regulated traffic. The indicator of sustainability of the transport system KT under worsening road conditions is 1.04 (for the regulated main street it is 1.22), while in the presence of a snow bank it is 1.41 (for the regulated main street it is 1.44). Increasing the sustainability of the transport system in adverse weather conditions is possible by reducing the number of trips made by residents of the city in private vehicles and using the route The inﬂuence of adverse weather, climate, and road conditions of operation on the parameters of the trafﬁc ﬂow for the main street of uninterrupted trafﬁc will be less than that for the main street of regulated trafﬁc. The indicator of sustainability of the transport system KT under worsening road conditions is 1.04 (for the regulated main street it is 1.22), while in the presence of a snow bank it is 1.41 (for the regulated main street it is 1.44). hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System After the accident, the parameters of the trafﬁc ﬂow on the main street of uninterrupted trafﬁc will be better than when driving on the main street of regulated trafﬁc without an accident (Table 9). Figure 3 shows how many times the integral indicator of sustainability varies under different weather-climatic and road conditions in comparison with the initial value of the indicator. The indicator value for the main street of regulated trafﬁc under normal weather conditions corresponds to 1. Table 9. Parameters of the trafﬁc ﬂow for the main streets of regulated and uninterrupted trafﬁc in the case of a road accident. Table 9. Parameters of the trafﬁc ﬂow for the main streets of regulated and uninterrupted trafﬁc in the case of a road accident. Parameters of the Trafﬁc Flow Main Street of Regulated Trafﬁc Main Street of Uninterrupted Trafﬁc Without Road Accident With Road Accident Without Road Accident With Road Accident Mean delay time, s 201 331 74 185 Average number of stops 4.1 7.6 1.1 4.1 Average speed, km/h 9.0 5.9 24.4 13.6 Mean delay time in trafﬁc congestion, s 165 273 61 146 Total travel time, h 1811 2766 1029 1,841 Total delay time, h 1471 2427 541 1355 Total number of stops 109,029 200,937 28,962 107,425 Trafﬁc management coefﬁcient 1.22 2.42 0.27 0.80 Main Street of Regulated Trafﬁc Main Street of Uninterrupted Trafﬁc Main Street of Regulated Trafﬁc Main Street of Uninterrupted Trafﬁc 12 of 17 55 425 80 Sustainability 2018, 10, 2052 y Total number of s T ffi Figure 3. Integral indicator of sustainability of the transport system for the main streets of uninterrupted and regulated traffic under changing weather conditions. Figure 3. Integral indicator of sustainability of the transport system for the main streets of uninterrupted and regulated trafﬁc under changing weather conditions. Figure 3. Integral indicator of sustainability of the transport system for the main streets of uninterrupted and regulated traffic under changing weather conditions. Figure 3. Integral indicator of sustainability of the transport system for the main streets of uninterrupted and regulated trafﬁc under changing weather conditions. Thus, the development of road infrastructure allows the sustainability of the transport system to be improved. hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System For a combination of these measures, the share of each measure in the plan is expedient to • the introduction of additional trips of scheduled passenger transport, or the adjustment of public transport timetables. public transport timetables. For a combination of these measures, the share of each measure in the plan is expedient to d i ki i h di d l f h i bili f Ch i • the introduction of additional trips of scheduled passenger transport, or the adjustment of public transport timetables. determine taking into account the predicted value of the system sustainability factor. Choosing a complex of effective measures will reduce the negative impact of adverse external factors on the urban transport system. For a combination of these measures, the share of each measure in the plan is expedient to determine taking into account the predicted value of the system sustainability factor. Choosing a complex of effective measures will reduce the negative impact of adverse external factors on the urban transport system. hodological Approach to Assessing the Sustainability of the Urban Transport System dological Approach to Assessing the Sustainability of the Urban Transport System Increasing the sustainability of the transport system, including creating a main street of uninterrupted traffic and increasing the capacity of the road, allows the introduction of measures to improve road safety (reducing the speed of traffic, preventing vehicles from moving into the lane of oncoming traffic, preventing the intersection of the trajectory of vehicles and pedestrians). The properties of sustainability and safety are interrelated and their joint development generates a synergistic effect. The measures of rapid response of municipal authorities and traffic police that degrade the Thus, the development of road infrastructure allows the sustainability of the transport system to be improved. Increasing the sustainability of the transport system, including creating a main street of uninterrupted trafﬁc and increasing the capacity of the road, allows the introduction of measures to improve road safety (reducing the speed of trafﬁc, preventing vehicles from moving into the lane of oncoming trafﬁc, preventing the intersection of the trajectory of vehicles and pedestrians). The properties of sustainability and safety are interrelated and their joint development generates a synergistic effect. The measures of rapid response of municipal authorities and traffic police that degrade the sustainability of the urban transport system include:  the identification of places and the installation of traffic signs with variable information; The measures of rapid response of municipal authorities and trafﬁc police that degrade the sustainability of the urban transport system include:  the identification of places and the installation of traffic signs with variable information;  the prompt change of the road traffic organization scheme by the traffic police; • the identiﬁcation of places and the installation of trafﬁc signs with variable information; the identification of places and the installation of traffic signs with variable information;  the prompt change of the road traffic organization scheme by the traffic police; • the identiﬁcation of places and the installation of trafﬁc signs with variable information;  the prompt change of operating modes of traffic signal installations on the sect • the prompt change of the road trafﬁc organization scheme by the trafﬁc police; street-road network with the greatest load; the restriction of entry to certain areas; the prompt change of operating modes of trafﬁc signal installations on the sections of the street-road network with the greatest load;  the introduction of additional trips of public transport timetables • the restriction of entry to certain areas; public transport timetables. 4. Discussion As reported in a number of studies [61–65], in order to increase the sustainability of the transport system, the recommended measures include: reducing the number of trips taken by private transport, promoting the more extensive use of bicycles and public transport, and promoting taxis, car-sharing, and pedestrian movements. This concept has found wide application in most cities of the world and its implementation ensures the availability of transport, its ongoing safety, and a reduction in negative impacts on the environment. The results obtained by the authors supplement these conclusions with reference to the operation of the transport system under adverse weather conditions, as illustrated in Figure 2. g The inﬂuence of the transport ﬂow on the environment was considered in Reference [61]; however, the authors did not take into account the travel time. An advantage of the presented study is the analysis of linkages between emissions into the atmosphere due to an increase in the amount of fuel Sustainability 2018, 10, 2052 13 of 17 consumed and the travel time. Unusual weather conditions further increase the amount of emissions of harmful substances, since, at the same distance, the travel time increases due to a decrease in the average speed. g p Reference [62] considered the actual change in transport demand on the road with a road load factor of less than one (1). Our investigations were carried out at the maximum intensity of movement during the day under normal and adverse weather conditions. In both cases, the value of transport demand was assumed to remain constant. However, due to a decrease in the capacity of the road in the case of snowfall, the value of the road load factor increased and became more than one. Compared to Reference [61], in which the assessment of the degree of change in the transport demand was also carried out, in the presented study additional important information was obtained: the transport supply was evaluated relative to the transport demand under adverse weather conditions. In contrast to Reference [63], the present study did not take into account the transport mode distribution depending on the travel length, because the object of study is one of the subsystems of the city’s transport system—transport ﬂow management. Studies on changes in the distribution of the number of trips made by different ways and modes of transportation, taking into account weather conditions, are very topical. 4. Discussion For instance, in Siberia in winter, the choice of transportation mode depends on the air temperature. Residents of cities choose transport with comfortable conditions and abstain from walking or cycling. In Reference [64], for the selection of measures for the development of the urban transport system, three categories of indicators of a sustainable transport system were offered, which were characterized by the possibility of use: A—in almost all situations, B—if necessary/possible; C—in the case when speciﬁc social needs arise. This study demonstrated the changes in sustainability of the system under the inﬂuence of adverse weather conditions, which are typical for both Western Siberia and other northern regions. The term sustainability has several deﬁnitions [64–66], since it is applied in different ﬁelds of science and technology for various technical and social systems. In addition, the deﬁnition of the term sustainability depends on the stage of the lifecycle of the system. When using the term sustainability with regard to the impact of weather conditions on the urban transport system, the authors based their discussion on the following considerations: • The main emphasis in the study is given to the change in the parameters of trafﬁc in adverse weather and climate conditions. These conditions are supposed to be disruption factors. The ability of the system to resist changes under the inﬂuence of disruption factors determines its sustainability. • If the weather conditions worsen, the system’s sustainability and effectiveness are degraded. • Dealing with adverse weather conditions allows an improvement of sustainability. • The development of the street and road network is only one of the possible measures to increase sustainability. Other activities can include improving the route network and increasing the number of public transport vehicles, introducing parking fees and increasing existing fees, adopting the use of remotely controlled vehicles, introducing smart transport systems, and many others [55]. Further studies could be directed at obtaining correcting coefﬁcients for mathematical models of trafﬁc parameters under adverse weather and climate conditions. Models of parameter determination are widely used in software complexes for transport planning and simulation. However, in some programs weather conditions are not considered and in some programs they are underestimated. The inclusion of correcting factors that take into account weather conditions in such models would improve the simulation accuracy for the operational management of transport ﬂows. 5. Conclusions Theoretical and experimental studies carried out by the authors on the inﬂuence of weather, climate, and road conditions for vehicle operation on the sustainability of the transport system made it 14 of 17 Sustainability 2018, 10, 2052 possible to formulate recommendations for its improvement. With worsening weather conditions and during snow-removal operations, the sustainability of the urban transport system was found to have deteriorated substantially. Developing road infrastructure and increasing the capacity of highways can improve the sustainability of the transport system and reduce the negative consequences of adverse weather and road conditions. The simulation model of trafﬁc developed makes it possible to determine the parameters of the trafﬁc ﬂow under adverse weather conditions and inform the city residents about the predicted time for a trip to the city center. The simulation allows one to determine the maximum number of vehicles in a transport system, which, if exceeded, would lead to a trafﬁc collapse in the urban system. These results are necessary to inform drivers about the trafﬁc situation and adjust an intelligent transport system, in particular when imposing restrictive measures on the trafﬁc to the central part of the city and choosing measures to improve the sustainability of the urban passenger transport system. Author Contributions: Investigation, D.Z.; methodology, E.M.; validation, E.C.R. D.Z., E.M., and E.C.R. developed the hypothesis and theoretical part of the study, analyzed the results of the study, and wrote the text of the paper. D.Z. developed a simulation model of the trafﬁc ﬂow and conducted experimental studies. Funding: This research received no external funding. Acknowledgments: The work was supported by the Act 211 of the Russian Federation Government, contract No. 02.A03.21.0006. 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A Comprehensive Analysis of the Association of Highway Trafﬁc with Winter Weather Conditions. Procedia Soc. Behav. Sci. 2013, 104, 497–506. [CrossRef] 63. Jin, Y.; Denman, S.; Deng, D.; Rong, X.; Ma, M.; Wan, L.; Mao, Q.; Zhao, L.; Long, L. Environmental impacts of transformative land use and transport developments in the Greater Beijing Region: Insights from a new dynamic spatial equilibrium model. Transp. Res. Part D 2017, 52, 548–561. [CrossRef] 64. Litman, T. Sustainable Transportation Indicators. Transp. Res. Board Ann. Meet. 2009, 9, 1–13. 65. Hidalgo, D.; Huizenga, C. Implementation of sustainable urban transport in Latin America. Res. Transp. Econ. 2013, 40, 66–77. [CrossRef] 66. Martos, A.; Pacheco-Torres, R.; Ordóñez, J.; Jadraque-Gago, E. Towards successful environmental performance of sustainable cities: Intervening sectors. A review. Renew. Sustain. Energy Rev. 2016, 57, 479–495. [CrossRef] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. 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https://openalex.org/W37099971	https://thescipub.com/pdf/ajassp.2012.227.230.pdf	English	null	Investigation of the Critical Clearing Time of Power System with Synchronous Machine Model Including Saliency	American journal of applied sciences	2,012	cc-by	2,091	American Journal of Applied Sciences 9 (2): 227-230, 2012 ISSN 1546-9239 © 2012 Science Publications American Journal of Applied Sciences 9 (2): 227-230, 2012 ISSN 1546-9239 © 2012 Science Publications INTRODUCTION excluding saliency. Thus it is very interesting to investigate the effect of salient pole of generator whether it affects on the CCT or not. This paper investigated the critical clearing time of the system equipped with different types of synchronous generator. The mathematical model of non-salient pole and salient pole synchronous machine model in power system are symmetrically derived. The critical clearing time of both models are tested and compared on various cases. excluding saliency. Thus it is very interesting to investigate the effect of salient pole of generator whether it affects on the CCT or not. From the energy utilization point of view, electricity is still being regarded as mighty means of energy carrier. Nowadays, the demand of electricity has dramatically increased and a modern power system becomes a complex network of transmission lines interconnecting the generating stations to the major loads points in the overall power system in order to support the high demand of consumers. The complicated network causes the stability problem. Currently, power engineers are much more concerned about stability problem due to blackout in Northeast United States, Scandinavia, England and Italy (Kumkratug, 2010; Osuwa and Igwiro, 2010; Santos et al., 2010). They have proposed many methods to improve stability of power system such as load shedding, Flexible AC Transmission System (FACTS), (Al-Husban, 2009; Darabi et al., 2008; Kumkratug, 2011; Magaji and Mustafa, 2009; Taher et al., 2008; Zarate-Minano et al., 2010). This paper investigated the critical clearing time of the system equipped with different types of synchronous generator. The mathematical model of non-salient pole and salient pole synchronous machine model in power system are symmetrically derived. The critical clearing time of both models are tested and compared on various cases. Investigation of the Critical Clearing Time of Power System with Synchronous Machine Model Including Saliency Prechanon Kumkratug Division of Electrical Engineering, Faculty of Engineering at Si Racha, Kasetsart University, 199 M.6, Tungsukhla, Si Racha, Chonburi, 20230, Thailand Abstract: Problem statement: Most of synchronous generator applied in power system is the salient pole type. Most of previous researches use non-salient model to evaluate the critical clearing time. Thus the results of critical clearing time of power system may be error. Approach: Thus this paper investigate the critical clearing time of power system with synchronous machine. The mathematical model of non-salient pole and salient pole synchronous machine model in power system are symmetrically derived. The critical clearing time of both models are tested and compared on various cases. Results: The critical clearing time of the power system with synchronous including saliency is slightly higher that of non saliency model. Conclusion: It is found from simulation results that even swing curve of both models is obviously different. Their critical clearing times are slightly different. Key words: Power system stability, transient stability, critical clearing time, FACTS devices, salient pole, non-salient pole And: (c) dI Isin = θ (6) dI Isin = θ (6) The synchronous voltage in quadrature axis is given by Eq. 7: (c) (c) q b d d E V cos X I ′ = δ +∑ (7) (7) Fig. 1: Single machine infinite bus system (a) schematic diagram (b) equivalent circuit (c) net series reactance diagram Fig. 1: Single machine infinite bus system (a) schematic diagram (b) equivalent circuit (c) net series reactance diagram The output power of generator is given by Eq. 8: e b P V Icos = θ (8) e b P V Icos = θ (8) From the (5), (6) and (8), the output power of generator is given by Eq. 9 and 10: e b q d P V (I cos I sin ) = δ + δ (9) From Fig. 2: b q q V sin X I δ = ∑ (10) e b q d P V (I cos I sin ) = δ + δ (9) (9) From Fig. 2: From Fig. 2: (10) b q q V sin X I δ = ∑ (10) b q q V sin X I δ = ∑ Here ∑Xq is the net equivalent reactance of the system in quadratur axis and given by Eq. 11: Fig. 2: Phasor diagram m e 1 [P P ] M ω = − ɺ (2) q q t 1 2 X X X X X ∑ = + + + (11) m e 1 [P P ] M ω = − ɺ (2) (11) From (7) and (10), the Id is given by Eq. 12: From (7) and (10), the Id is given by Eq. 12: The δ, ω and Pm in (1)-(2) are the rotor angle, speed, mechanical input power and moment of inertia, respectively of synchronous machine. q b d d E V cos I X ′ − δ = ∑ (12) (12) The output electrical power of synchronous machine (Pe) excluding saliency is given by Eq. 3: The output power of synchronous generator including saliency is given by Eq. MATERIALS AND METHODS Mathematical model: Figure 1a shows the single line diagram of power system. First consider the non-salient pole model of generator equipped in power system as can be shown in Fig. 1b. Here XL is the equivalent reactance of each transmission line. The generator is represented by a synchronous voltage in quadrature axis ( q E′ ) behind direct transient reactance ( d X′ ). The Vb is The evaluation of Critical Clearing Time (CCT) of power system is one of the most important research areas for power engineers because it indicates the robustness of the faulted power system. The dynamic behavior of synchronous generator plays very important role to determine the CCT of power system. There are two type of synchronous machine; salient pole and non-salient pole. Many previous researches use synchronous generator the voltage at infinite bus. Fig. 1c is the equivalent reactance circuit of Fig. 1b. Here X1 is equivalent circuit of transmission line L1 and line L2 whereas X2 is equivalent circuit of transmission line L3 and line L4. The dynamic equation for evaluating critical clearing tine is given by Eq. 1 and 2: (1) δ = ω ɺ 227 Am. J. Applied Sci., 9 (2): 227-230, 2012 d d t 1 2 X X X X X ′ ∑ = + + + (4) (a) (b) (c) (4) (a) Here let consider the synchronous machine in salient pole type. Figure 2 shows the phasor diagram of power system with synchronous machine including saliency. (b) Here I is the generator current at terminal and it is consist two component; direct axis and quature axis as given by Eq. 5 and 6: qI Icos = θ (5) And: dI Isin = θ (6) qI Icos = θ (5) qI Icos = θ qI Icos = θ (5) (b) And: 13: q b e d E V P sin X ′ = δ ∑ (3) q b e d E V P sin X ′ = δ ∑ (3) d q 2 b e b d d q X X E V P sin V sin 2 X X X ′ − ′ = δ + δ ∑ ∑ ∑ (13) Here ∑Xd is the net equivalent reactance of the system in direct axis and given by Eq. 4: (13) 228 9 (2): 227-230, 2012 Fig. 4: Swing curve of the system for tcl = 170 msec Fig. 5: Swing curve of the system for tcl = 175 msec Am. J. Applied Sci., 9 (2): 227-230, 2012 Fig. 3: Power curve of synchronous machine It is well known that the power curve (Pe-δ) provides very important information of analyzing the critical clearing time of power system. Figure 3 shows the swing curve of the sample system as shown in Fig. 1a. The system parameters are: Fig. 4: Swing curve of the system for tcl = 170 msec Fig. 5: Swing curve of the system for tcl = 175 msec Fig. 3: Power curve of synchronous machine Fig. 3: Power curve of synchronous machine Fig. 4: Swing curve of the system for tcl = 170 msec Fig. 3: Power curve of synchronous machine It is well known that the power curve (Pe-δ) provides very important information of analyzing the critical clearing time of power system. Figure 3 shows the swing curve of the sample system as shown in Fig. 1a. The system parameters are: The system parameters are: Fig. 5: Swing curve of the system for tcl = 175 msec H=5, Xt = 0.1 pu, d X′ =0.25 pu, Xq=0.6 puXL1=0.5 pu, XL2=0.5 pu, XL3=0.5 pu, XL4=0.5 pu, Pm=0.8 pu, q E′ =1.22 pu, Vb=1 pu H=5, Xt = 0.1 pu, d X′ =0.25 pu, Xq=0.6 puXL1=0.5 pu, XL2=0.5 pu, XL3=0.5 pu, XL4=0.5 pu, Pm=0.8 pu, q E′ =1.22 pu, Vb=1 pu DISCUSSION There are three interesting simulation results on this investigation. First, the swing curve of salient pole and non-salient pole are obviously different. With tcl = 170 m sec, the maximum generator rotor angle (δmax) of non-salient pole type is around 121.19 whereas the salient pole type is around 127.71. Second, the time of maximum rotor angle (tmax) of salient pole is less than that of non-salient pole as can be seen in Fig. 3. Third, the critical clearing time of different types of synchronous generator model are slightly different. It can be seen from the figure that the at output electrical power 0.8 pu the rotor angle of non-salient pole is 31.64 degree whereas for the salient pole is 45.35 degree. The maximum value of the output power for salient pole is higher than that of non-salient pole. The derived equation will be further investigate and compared the critical clearing time by implementing in MATLAB/SIMULINK. RESULTS This paper investigated the critical clearing time of the system equipped with different types of synchronous generator. The mathematical models are symmetrically derived. There are three interesting simulation results on this paper. First, the maximum generator rotor angle of salient pole and non-salient pole are significantly different. Second, the time of maximum rotor angle (tmax) of salient pole is less than that of non-salient pole. Third, the critical clearing time of different types of rotor pole are insignificantly different. It can be confirmed that with critical clearing time evaluation, the mathematical model of Consider the diagram of sample system is shown in Fig. 1a. It is considered that three phase fault appears at line 2 near bus i and the fault is cleared by opening both circuit breakers. Figure 4 shows the rotor angle of the system with clearing time (tcl) for 170 m sec. Based on the simulation, the critical clearing time of the system with synchronous generator of non-salient pole is around 174-175 m sec where that of the salient pole is 175-176 m sec. Figure 5 shows the swing curve of the system with tcl = 175 m sec. 229 Am. J. Applied Sci., 9 (2): 227-230, 2012 Magaji, N. and M.W. Mustafa, 2009. Optimal thyristor control series capacitor neuro-controller for damping oscillations. J. Comput. Sci., 5: 980-987. DOI: 10.3844/jcssp.2009.980.987 synchronous generator in non-salient is not only simpler but also able to be applied for the system with salient pole. REFERENCES j p Osuwa, J.C. and E.C. Igwiro, 2010. Uninterruptible power supply using solar rechargeable battery. Physics. Int., 1: 77-82. DOI: 10.3844/pisp.2010.77.82 Al-Husban, A.N., 2009. An eigenstructure assignment for a static synchronous compensator. Am. J. Eng. Applied Sci., 2: 812-816. DOI: 10.3844/ajeassp.2009.812.816 Santos, S.P.Y., E. Delbone, E.F. Carvalho and L.N. Martins, 2010. Synchronous generator disturbance provoked by induction motor starting. Am. J. Applied Sci., 7: 962-968. DOI: 10.3844/ajassp.2010.962.968 Darabi, A., S.A. Soleamani and A. Hassannia, 2008. Fuzzy based digital automatic voltage regulator of a synchronous generator with unbalanced loads. Am. J. Eng. Applied Sci., 1: 280-286. DOI: 10.3844/ajeassp.2008.280.286 Taher, S.A., R. Hematti, A. Abdolalipour and M. Nemati, 2008. Decentralized controller design for static synchronous compensator using robust quantitative feedback theory method. Am. J. Eng. Applied Sci., 1: 66-75. DOI: 10.3844/ajeassp.2008.66.75 Kumkratug, P., 2010. Application of interline power flow controller to increase transient stability of power system. J. Comput. Sci., 6: 1490-1493. DOI: 10.3844/jcssp.2010.1490.1493 Zarate-Minano, R., T.V. Custsem, F., Milano and A.J. Conejo, 2010. Securing transient stability using time-domain simulations within an optimal power flow. IEEE Trans. Power Syst., 25: 243-253. DOI: 10.1109/TPWRS.2009.2030369 Kumkratug, P., 2011. optimal control design of static synchronous series compensator for damping power system oscillation. J. Comput. Sci., 7: 844- 848. 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https://openalex.org/W4306924572	https://researchonline.lshtm.ac.uk/id/eprint/4668089/1/Nanyeenya_etal_2022_Low-level-viraemia-an-emerging.pdf	English	null	Low-level viraemia: An emerging concern among people living with HIV in Uganda and across sub-Saharan Africa	African journal of laboratory medicine	2,022	cc-by	4,937	African Journal of Laboratory Medicine ISSN: (Online) 2225-2010, (Print) 2225-2002 Page 1 of 5 Review Article Affiliations: 1Department of Epidemiology and Biostatistics, School of Public Health, Makerere University College of Health Sciences, Kampala, Uganda 2Ministry of Health Central Public Health Laboratories, Kampala, Uganda 2Ministry of Health Central Public Health Laboratories, Kampala, Uganda Keywords: HIV/AIDS; low-level viraemia; viral load testing; non-suppression; virologic failure. Low-level viraemia: An emerging concern among people living with HIV in Uganda and across sub-Saharan Africa Authors: Nicholus Nanyeenya1,2 Noah Kiwanuka1 Damalie Nakanjako3 Gertrude Nakigozi4 Simon P.S. Kibira5 Susan Nabadda2 Charles Kiyaga2 Isaac Sewanyana2 Esther Nasuuna6 Fredrick Makumbi1 Attaining viral load (VL) suppression for over 95% of the people living with HIV on antiretroviral therapy is a fundamental step in enabling Uganda and other sub-Saharan African countries to achieve global Sustainable Development Goal targets to end the HIV/AIDS epidemic by 2030. In line with the 2013 World Health Organization recommendations, several sub-Saharan African countries, including Uganda, use a threshold of 1000 HIV viral RNA copies/mL to determine HIV viral non-suppression. The United States Centers for Disease Control and Prevention and the International Association of Providers of AIDS Care deem this threshold very high, and hence recommend using 200 copies/mL to determine viral non- suppression. Using 1000 copies/mL as a threshold ignores people living with HIV who have low-level viraemia (LLV; HIV VL of at least 50 copies/mL but less than 1000 copies/mL). Despite the 2021 World Health Organization recommendations of using intensive adherence counselling for people living with HIV with LLV, several sub-Saharan African countries have no interventions to address LLV. However, recent studies have associated LLV with increased risks of HIV drug resistance, virologic failure and transmission. The purpose of this narrative review is to provide insights on the emerging concern of LLV among people living with HIV receiving antiretroviral therapy in sub-Saharan Africa. The review also provides guidance for Uganda and other sub-Saharan African countries to implement immediate appropriate interventions like intensive adherence counselling, reducing VL thresholds for non- suppression and conducting more research to manage LLV which threatens progress towards ending HIV by 2030. Authors: Nicholus Nanyeenya1,2 Noah Kiwanuka1 Damalie Nakanjako3 Gertrude Nakigozi4 Simon P.S. Kibira5 Susan Nabadda2 Charles Kiyaga2 Isaac Sewanyana2 Esther Nasuuna6 Fredrick Makumbi1 Introduction By the end of 2020, an estimated 37.7 million people were living with HIV/AIDS; two-thirds (67%) were living in sub-Saharan Africa, and 27.5 million were accessing antiretroviral therapy (ART).1 The increased access to ART has necessited the monitoring of its efficacy among people living with HIV, hence the increased scale-up of HIV viral load (VL) monitoring. As the world strives to control the HIV epidemic, attaining VL suppression for over 95% of people living with HIV on ART is a fundamental step in enabling Uganda, and other sub-Saharan African countries, to achieve the global targets of ending the HIV/AIDS epidemic by 2030, as stipulated in target 3.3 of the Sustainable Development Goal 3.1,2,3 HIV viral non-suppression is defined as HIV RNA viral copies equal to or greater than 1000 copies/mL as recommended by the World Health Organization (WHO).4 This is also an indication of HIV virologic treatment failure in Uganda, and several other sub-Saharan African countries like Kenya, Zambia and Sierra Leone, among others.5 Low-level viraemia (LLV) defined as having an HIV VL of at least 50 copies/mL but less than 1000 copies/mL (≥ 50 copies/mL to < 1000 copies/mL) is considered as being virally suppressed. Despite the recent 2021 WHO recommendation to offer intensive adherence counselling (IAC) to people living with HIV on ART with LLV,6 no special intervention is given to people living with HIV with LLV in Uganda and several other sub-Saharan African countries; yet, LLV has been associated with various clinically poor outcomes like HIV drug resistance and virologic failure.7,8 This narrative review was completed in January 2022, and the reviewed articles were published from March 2000 to June 2021. Several databases including Google Scholar, PubMed, and Science Direct were searched 6Infectious Diseases Institute, Makerere University College of Health Sciences, Kampala, Uganda How to cite this article: Nanyeenya N, Kiwanuka N, Nakanjako D, et al. Low level viraemia: An emerging concern among peop with HIV in Uganda and across sub-Saharan Africa. Afr J Lab Med. 2022;11(1), a1899. https://doi.org/10.4102/ajlm.v11i1.1899 Copyright: © 2022. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. g ; ( ),t p // g/ / j Copyright: © 2022. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. How to cite this article: Nanyeenya N, Kiwanuka N, Nakanjako D, et al. Low-level viraemia: An emerging concern among people living with HIV in Uganda and across sub-Saharan Africa. Afr J Lab Med. 2022;11(1), a1899. https://doi.org/10.4102/ajlm.v11i1.1899iti Definition and risk factors The WHO defines LLV as a low but detectable VL that is, viraemia (≥ 50 copies/mL to < 1000 copies/mL).26 People living with HIV/AIDS on ART with viraemia ≥ 50 copies/mL to < 1000 copies/mL are virally suppressed, but have LLV.5, 27 Previous studies have associated LLV with baseline CD4 + T cell counts below 200 cells/mm3, ART duration longer than 60 months, ART regimens comprising of zidovudine, lamivudine and nevirapine (aOR: 2.26, p < 0.001) or didanosine-based regimen, and existing subtype B′ infection.27, 28 A number of sub-Saharan African countries initiated the scale-up of VL testing from 2014 to 2015, following the WHO 2013 recommendation.15, 16 Uganda began scaling up HIV VL testing for all eligible people living with HIV on ART in 2014.16 Upon initiation of ART, the first VL test is done for people living with HIV at six months, and then at 12 months, after which it is repeated annually for suppressed adults, and every six months for suppressed children and adolescents. People living with HIV with a VL result below 1000 copies/mL have a suppressed VL and are routinely given adherence counselling in which they are encouraged to continue with ART, and no other intervention is given.5 A decreased VL is associated with better clinical outcomes and slowed disease progression,17 and is also associated with reduced HIV incidence at community level.17, 18 However, there is limited data about the cut-off of viral copies at which the slowed disease progression and reduced HIV incidence at community level happens. Dates: Read online: Scan this QR code with your smart phone or mobile device to read online. Read online: Scan this QR code with your smart phone or mobile device to read online. http://www.ajlmonline.org Open Access Page 2 of 5 Page 2 of 5 Review Article sessions for three months. After the IAC sessions, a VL test is repeated in the fourth month to determine whether they have achieved VL suppression.5, 6, 12 If the repeat VL result after IAC is still non-suppressed, this is considered as virologic failure provided non-adherence to ART is ruled out. A switch committee is then convened to discuss switching the patient to another ART line.5 Several predisposing factors like poor ART adherence, unawareness about the ART benefits and other existing chronic illness, among others, have been associated with VL non-suppression.19, 20, 21 People living with HIV on ART with non-suppressed VL are at increased risk of faster progression to AIDS, which is also associated with poor clinical outcomes.21, 22 using search terms like ‘low-level viraemia’, ‘viral load testing’, ‘non-suppression threshold’ and ‘virologic failure’. This review aims at guiding Uganda and other sub-Saharan African countries to take immediate appropriate interventions to manage LLV. Antiretroviral therapy monitoring Highly active ART has been the springboard in the management and control of HIV/AIDS over the years; the goal of highly active ART is to lead to HIV viral suppression with an increase in the body immunity function.4 Globally, access to ART has increased, thereby improving the HIV treatment outcomes,1 and this has been a great milestone in achieving epidemic control. In Uganda, an estimated 1.2 million (out of 1.4 million) people living with HIV were on ART by 2019.9 As the efforts to scale-up people living with HIV on ART continue, there is an inevitable need to monitor the efficacy of ART among people living with HIV10 and several methods of ART monitoring are recommended by WHO. These include patient monitoring involving clinical events and adherence monitoring, immunological monitoring involving CD4 cell count, and virologic monitoring comprising HIV VL testing.11, 12 Tremendous progress has been achieved by several sub- Saharan African countries in establishing comprehensive VL testing programmes23; for instance, in Uganda, the number of annual VL tests has steadily increased annually from 16 411 VL tests (2% of people living with HIV on ART) in 2014 to 1 332 335 VL tests (95% of people living with HIV on ART) in 2020.24 Challenges affecting the scale-up of VL testing, like suboptimal sample transportation and results delivery, limited human resource in health facilities, unawareness about VL testing among HIV healthcare providers and people living with HIV, protracted procurement processes, and poor adherence to national and WHO VL testing guidelines, have limited full coverage of VL testing in the country.23, 25 The other key important issue to note with the introduction of VL testing is LLV. Virologic monitoring Virologic monitoring which comprises HIV VL testing is the preferred way of monitoring treatment outcomes in people living with HIV on ART, and WHO recommends use of a threshold of 1000 copies/mL to determine VL non- suppression, indicative of either poor drug adherence or virologic failure.6 However, the United States Centers for Disease Control and Prevention and International Association of Providers of AIDS Care recommend a threshold of 200 copies/mL for VL non-suppression.13, 14 Review Article transmission was greatly associated with VL at delivery, with noted risks of 0.25%, 2.0% and 8.5% among women with VL < 50 copies/mL, 50 copies/mL – 1000 copies/mL and > 1000 copies/mL at delivery, respectively (p < 0.001). This implies that women with LLV at delivery had eight times the risk of HIV vertical transmission, as compared to women with a non-detectable VL < 50.46 the performance of dried blood spot samples for VL testing is lower, when compared to the gold standard sample type plasma, with a low sensitivity and specificity to detect treatment failure, and favours using a threshold of 1000 copies/mL.29, 32 transmission was greatly associated with VL at delivery, with noted risks of 0.25%, 2.0% and 8.5% among women with VL < 50 copies/mL, 50 copies/mL – 1000 copies/mL and > 1000 copies/mL at delivery, respectively (p < 0.001). This implies that women with LLV at delivery had eight times the risk of HIV vertical transmission, as compared to women with a non-detectable VL < 50.46 Slowed disease progression has been demonstrated in people living with HIV on ART with a VL of less than 1000 copies/ mL, 17, 33 though the exact threshold at which LLV predicts disease progression varies, and remains debatable.34 Furthermore, a reduced risk of HIV transmission for people living with HIV on ART with VL results less than; 1000 copies/mL,35 1500 copies/mL,36 and less than 1700 copies/ mL,37 has been shown. No HIV transmission risks have been demonstrated below 400 copies/mL,38 but the range of viraemia between 400 copies/mL and 1000 copies/mL at which HIV transmission occurs, still remains unclear.39 In Malawi, a study was conducted that comprised 1274 mothers and described VL suppression among HIV- positive mothers at 4–26 weeks postpartum, the factors associated with VL suppression, and the impact of VL suppression levels on mother-to-child transmission. This study indicated that 8.7% of these HIV-positive mothers had LLV and were more likely to be adolescents, who had been on ART for less than six months, with suboptimal adherence. LLV was associated with 7.0% risk of mother-to-child transmission, as compared to 0.9% risk for mothers with a non-detectable VL.47 Proposed interventions Reduced drug adherence has been associated with residual LLV,52 and this implies that interventions to enhance treatment adherence could be offered to people living with HIV on ART with LLV to achieve a non-detectable VL, which is the recommended target of ART in various international guidelines.53, 54 In the recent 2021 WHO consolidated guidelines, IAC has been recommended to be offered to people living with HIV with LLV,6 though most sub-Sahara African countries have not yet started offering this intervention. Furthermore, there is no data in Uganda and other sub-Sahara African countries to determine whether IAC can be effective in achieving a non-detectable VL among people living with HIV on ART with LLV. Few sub-Sahara African countries like South Africa7, 46 and Malawi47 have undertaken research to understand LLV and its implications, which has created a knowledge gap about the subject, thereby affecting key policy decisions in the region. Furthermore, in South Africa, LLV occurred in 23% of people living with HIV on ART with an incidence of 11.5 per 100 person-years of follow-up (95% confidence interval: 11.4–11.7), during first-line ART. In this study, LLV was associated with increased hazards ratios of virological failure and the subsequent switch to second-line ART, as compared with a non-dectectable VL of less than 50 copies/mL; and the risk of virological failure was increased more with increased ranges of LLV.7 This study did not look at HIV drug resistance testing results in LLV, because they were not available. Prevalence and effects Sustained VL suppression has been shown to reduce the occurrence of HIV drug resistance among people living with HIV on ART and also leads to improved treatment outcomes.40, 41, 42 However new emerging resistance mutations have been demonstrated in people living with HIV on ART with LLV,7,8 where persistent LLV has been associated with increased risk of virologic failure43; which is also associated with increased risk of adverse treatment outcomes.44 Elsewhere, HIV drug resistance was detected in about 30.0% of people living with HIV with a VL test having the first episode of LLV in British Columbia; and these patients had an increased risk of developing virologic failure, compared to those without HIV drug resistance.48 Another study indicated that there were 44.0% accumulated resistance mutations in 47 people living with HIV on ART with two or more episodes of LLV, and surprisingly the median viraemia was 267 copies/mL; and virologic failure followed in 16.0% of these people living with HIV.49 In the United Kingdom, 30.0% of people living with HIV with LLV acquired at least one drug resistant mutation50; and major resistant mutations were detected in 12.7% of people living with HIV with LLV.51 For the case of the sub-Sahara African region, 19.3% of the participants had LLV while 7.8% of the participants had persistent LLV in a study, which aimed to evaluate virologic failure and its predictors in four African countries including Uganda, Kenya, Tanzania and Nigeria.45 Furthermore, 57.5% of participants with persistent LLV (plasma HIV RNA > 50 copies/mL at two consecutive visits) in this study would later have confirmed virologic failure. However this study did not examine HIV drug resistance, which could be a key driver of virologic failure (failure of people living with HIV with a non-suppressed VL to suppress after three sessions of IAC, and poor drug adherence has been ruled out), and also assessment of drug adherence was mainly through self reports which could have been affected by social desirability and recall bias.45 Justification for using a threshold of 1000 copies/ mL for viral load non-suppression The use of 1000 copies/mL as a threshold for determining VL non-suppression in sub-Saharan African countries including Uganda, has generated enormous debate over the years, since it can lead to the accumulation of people living with HIV on ART categorised as suppressed VL, but having LLV.29 The recommendation by WHO to use this threshold of 1000 copies/mL30 was based on a public health perspective where dried blood spot samples were used instead of plasma as a specimen type for HIV-1 VL testing, facilitate the decentralisation of specimen collection and can increase access to VL testing in resource-limited settings like Uganda due to their cost-effectiveness, as compared to plasma.31 Unfortunately, People living with HIV/AIDS who have been on ART for at least six months with a VL of 1000 copies/mL or more are non-suppressed. These people are offered monthly IAC http://www.ajlmonline.org Open Access Page 3 of 5 Page 3 of 5 Review Article Conclusion Antiretroviral drug resistance in HIV-1-infected patients experiencing persistent low-level viremia during first-line therapy. J Infect Dis. 2011;204(4):515–520. https://doi.org/10.1093/infdis/jir353 9. UNAIDS. Uganda. Country fact sheet. [homepage on the Internet]. [cited 2021 Jun 21]. Available from: https://www.unaids.org/en/regionscountries/countries/uganda 10. World Health Organization. Antiretroviral therapy for HIV infection in infants and children: Towards universal access: Recommendations for a public health approach. Geneva: World Health Organization; 2010. Conclusion In another South African study looking at HIV viraemia and mother-to-child transmission risk after ART initiation in pregnancy in Cape Town, the risk of early HIV vertical There is an increasing frequency of LLV among people living with HIV on ART in Uganda and across other sub-Sahara African countries. This LLV is associated with increased risks of http://www.ajlmonline.org Page 4 of 5 Page 4 of 5 Review Article HIV drug resistance and virologic failure which can affect the efficacy of ART, and lead to accelerated HIV disease progression. With the advancing global targets to end the HIV epidemic, reduce transmission rates, and improve the clinical outcomes of the people living with HIV on ART, there is an inevitable need to re-assess the use of the threshold of 1000 copies/mL to determine VL non-suppression, and to establish strategies to address the rising proportions of people living with HIV on ART with unmanaged LLV in the region. More research about the association between LLV and drug resistance and virologic failure in sub-Sahara Africa also needs to be done. 5. Consolidated guidelines for prevention and treatment of HIV in Uganda.pdf [homepage on the Internet]. [cited 2021 Jun 20]. Available from: http://library. health.go.ug/publications/hivaids/consolidated-guidelines-prevention-and- treatment-hiv-uganda 5. Consolidated guidelines for prevention and treatment of HIV in Uganda.pdf [homepage on the Internet]. [cited 2021 Jun 20]. Available from: http://library. health.go.ug/publications/hivaids/consolidated-guidelines-prevention-and- treatment-hiv-uganda 5. Consolidated guidelines for prevention and treatment of HIV in Uganda.pdf [homepage on the Internet]. [cited 2021 Jun 20]. Available from: http://library. health.go.ug/publications/hivaids/consolidated-guidelines-prevention-and- treatment-hiv-uganda 6. World health Organization. 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https://openalex.org/W2046263802	https://www.scielo.br/j/ni/a/czrKJQgZRr8MpkCFSNqQnTq/?lang=en&format=pdf	English	null	Metabolic substrates are not mobilized from the osmoregulatory organs (gills and kidney) of the estuarine pufferfishes Sphoeroides greeleyi and S. testudineus upon short-term salinity reduction	Neotropical ichthyology/Neotropical Ichthyology	2,008	cc-by	7,486	Viviane Prodocimo, Carine F. Souza, Cristina Pessini, Luiz Claudio Fernandes and Carolina A. Freire Viviane Prodocimo, Carine F. Souza, Cristina Pessini, Luiz Claudio Fernandes and Carolina A. Freire The marine-estuarine species of pufferfishes Sphoeroides testudineus and S. greeleyi are very efficient osmoregulators. However, they differ with respect to their tolerance of salinity reduction. During low tide S. testudineus remains in diluted estuarine waters, whereas S. greeleyi returns to seawater (SW). The hypothesis tested here was that the short-term mobilization of metabolic substrates stored in their main osmoregulatory organs would correlate with this differential tolerance. Fishes exposed to 5‰ (for 6 h) were compared to those kept in 35‰. Branchial and renal contents of triglycerides, protein and glycogen were evalu ated, and total ATPase activity accounted for the tissues’ metabolism. Plasma osmolality, chloride and glucose, hematocrit, and muscle water content were also measured. Total triacylglycerol content was higher in S. greeleyi than in S. testudineus in both salinities and in both organs. Kidney glycogen contents were higher in S. greeleyi than in S. testudineus in 5 and 35‰. Total ATPase activity was reduced in 5‰ when compared to 35‰ in the kidney of S. greeleyi, and was higher in the gills of S. greeleyi than in those of S. testudineus, in both salinities. Upon exposure to dilute SW, both species displayed a similar osmoregulatory pattern: plasma osmolality and chloride were reduced. Again in both species, stability in muscle water content indicated cellular water content control. Although the metabolic substrates stored in the osmoregulatory organs of both species were not mobilized during these short-term sea water dilution events, some differences could be revealed between the two species. S. greeleyi showed more metabolic reserves (essentially triacylglycerols) in these organs, and its gills showed higher total ATPase activity than those S. testudineus. Os baiacus marinhos Sphoeroides testudineus e S. greeleyi são freqüentadores de estuários e eficientes osmorreguladores. No entanto, eles diferem quanto à sua capacidade de tolerar a redução de salinidade. Durante a descida de maré S. testudineus permanece no estuário com salinidade reduzida e S. greeleyi retorna para a água do mar. A hipótese testada neste estudo foi a de que a mobilização em curto prazo dos substatos metabólicos armazenados nos órgãos osmorregulatórios poderia explicar esta tolerância diferencial a redução de salinidade. Peixes expostos a 5‰ (por 6 h) foram comparados com os mantidos em 35‰ . Neotropical Ichthyology, 6(4):613-620, 2008 Copyright © 2008 Sociedade Brasileira de Ictiologia Neotropical Ichthyology, 6(4):613-620, 2008 Copyright © 2008 Sociedade Brasileira de Ictiologia Departamento de Fisiologia, Setor de Ciências Biológicas, Universidade Federal do Paraná, Centro Politécnico, 81531-990 Curitiba, Paraná, Brazil. vprodocimo@ufpr.br Key words: Fish gill, Fish kidney, Glycogen, Metabolism, Osmoregulation, Tetraodontidae. tor de Ciências Biológicas, Universidade Federal do Paraná, Centro Politécnico, 81531-990 Curitiba, Paraná, Introduction In the freshwater euryhaline tilapia (Oreochromis mossam bicus) liver glycogen content increased after 6 hours in 23‰ (Nakano et al., 1998), but rapid mobilization of the local reserves of branchial glycogen has been observed to fuel ion transport mechanisms in ionocytes of the gills of tilapia upon short-term (3 hours) increase in salinity (25‰) (Tseng et al., 2007). In the marine teleosts red grouper (Epinephelus akaara) and black sea bream (Mylio macrocephalus), short- term (6 hours) exposure to 7‰ salinity (from 30‰) did not induce consumption of liver glycogen content (Woo & Wu, 1982). We are not aware of any study using marine/estuarine teleosts in a short-term protocol of salinity reduction with further evaluation of branchial and renal metabolic substrate store, in a protocol somewhat related to their natural habitat. i The pufferfish Sphoeroides testudineus (Linnaeus, 1758) is an abundant teleost in bays and estuaries along the Brazil ian coast, in places with salinities ranging from 34‰ to 0‰ (Figueiredo & Menezes, 2000). Sphoeroides greeleyi Gilbert, 1900 is also common along the Brazilian coast, but is found in areas of higher salinity (Figueiredo & Menezes, 2000; Vendel et al., 2002), and has been observed to be more sensi tive to salinity reduction either in the laboratory (Prodocimo & Freire, 2001) or in the field (Prodocimo & Freire, 2004), when compared to S. testudineus. The hypothesis tested here was that the two species of pufferfishes, with distinct toler ances of seawater dilution, might differ in their short-term use of metabolic substrates stored in their main osmoregulatory organs, when challenged with salinity alteration. The distur bance in the osmotic homeostasis of these fish would impose a metabolic demand of the energetic substrates stored in their osmoregulatory organs, thus potentially more readily available (Tseng et al., 2007). This hypothesis is supported by previous reports that rapid alterations in transport mechanisms (includ ing changes in Na+, K+-ATPase activity) ensue when estuarine fishes endure salinity changes in their environment (Mancera & McCormick, 2000; Sakamoto et al., 2000; Marshall, 2003; Wood & Laurent, 2003; Prodocimo et al., 2007). ) The energy requirement of the gills and kidney is partially maintained by using the metabolic substrates stored in these organs (Tseng et al., 2007). Introduction macrocephalus) (Woo & Wu, 1982) presented no changes in their hepatic glycogen content and protein after long periods (10-14 days) of exposure to dilute seawater (~ 5‰). Marine teleosts are hypo-osmotic to seawater. Their gills act to maintain plasma homeostasis by secreting the excess of salt taken up as they drink seawater (Jobling, 1995; Evans et al., 1999, 2005). The kidney is not as relevant as are the gills for osmotic and ionic regulation because renal tubules essentially secrete multivalent ions (Mg+2 and SO4 -2) (Bey enbach & Baustian, 1989; Beyenbach et al., 1993; Hentschel & Zierold, 1994; Jobling, 1995). Estuarine teleosts, while facing fast salinity changes, maintain the homeostasis of their extracellular fluid activating energy-consuming osmoregula tory mechanisms (mainly in the gills) in order to switch from salt secretion to salt absorption mechanisms or vice-versa (Mancera & McCormick, 2000; Prodocimo & Freire, 2001; 2004; Scott et al., 2004; Evans et al., 2005; Sangiao-Alvarellos et al., 2005; Prodocimo et al., 2007). Vectorial salt transport against electrochemical gradients is ultimately ATP-driven (e.g., Stahl & Baskin, 1990; Evans et al., 2005). In fact, the major ATPase involved with the energization of salt transport in gills of euryhaline fishes is the Na,K-ATPase (reviewed in Evans et al., 1999, 2005). In the osmoregulatory organs, the Na,K-ATPase is the major contributor to total ATPase activity (Venturini et al., 1992). Thus, in those organs, ATPase activity changes can indeed at least partially represent changes in the organs’ energy budget, and can be related to the Na,K-ATPase activity, in fishes submitted to osmotic stress (Perry & Walsh, 1989; Venturini et al., 1992; Díaz et al., 1998). Both hypo- regulation in high salinity and hyper-regulation in low salin ity demand energy consumption, for gradient maintenance. Although fish metabolism measured by oxygen consumption as a function of salinity displays a variable response, it is often reported as an "U-shaped" curve: higher metabolism in very low (frequently fresh water) or high salinities (often hypersaline), versus lower metabolism in intermediary, more isosmotic salinities (e.g., Jensen et al., 1998; Kelly et al., 1999; Imsland et al., 2003; Laiz-Carrión et al., 2005). The effects of short-term (hours) salinity changes on metabolic reserves of fish have been poorly investigated (Woo & Wu, 1982; Nakano et al., 1998; Tseng et al., 2007). Introduction However, there is scant infor mation about the mobilization of their metabolic substrates according to the salinity of acclimation in fish (Hansen et al., 1999; Laiz-Carrión et al., 2002, 2005; Sangiao-Alvarellos et al., 2003, 2005). Still, metabolic changes in branchial and renal tissues have been essentially associated to long peri ods (days) of exposure to different salinities (Hansen et al., 1999; Laiz-Carrión et al., 2005; Sangiao-Alvarellos et al., 2003, 2005). The liver has been the major focus of studies on metabolism versus salinity (Woo & Fung, 1981; Woo & Wu, 1982; Nakano et al., 1998). It is proposed that the liver is the main site of glycogen storage and it provides plasma with glucose, which is then taken up for energy generation in the gills (e.g., Vijayan et al., 1996; Sangiao-Alvarellos et al., 2005). However, marine teleosts such as the sea bream Chrysophrys major (Woo & Fung, 1981), the red grouper (Epinephelus akaara), and also the black sea bream (Mylio Viviane Prodocimo, Carine F. Souza, Cristina Pessini, Luiz Claudio Fernandes and Carolina A. Freire O conteúdo branquial e renal de triacilglicerol, proteína e glicogênio foram avaliados, e a atividade ATPásica total foi quantificada para representar o metabolismo do tecido. Osmolalidade, cloreto e glicose plasmáticos, o hematócrito e o conteúdo de água no músculo também foram mensurados. O conteúdo total de triacilglicerol foi maior em S. greeleyi do que em S. testudineus em ambas as salinidades e em ambos os órgãos. O conteúdo renal de glicogênio foi maior em S. greeleyi quando comparado a S. testudineus na duas salinidades. A atividade ATPásica total foi reduzida em 5‰ quando comparada a 35‰ no rim de S. greeleyi, e foi maior nas brânquias de S. greeleyi quando comparada aos valores para S. testudineus em ambas as salinidades. Após a exposição a água do mar diluída, ambas as espécies apresentaram padrão osmorregulatório similar: osmolalidade e cloreto plasmáticos foram reduzidos. Em ambas as espécies a estabilidade do conteúdo de água no músculo indicou capacidade de regular o conteúdo de água intracelular. Os substratos metabólicos estocados nos órgãos osmorregulatórios de ambas as espécies não foram mobilizados durante a exposição de curto-prazo a diluição da água do mar, porém algumas diferenças foram reveladas entre as duas espécies. S. greeleyi apresentou maior reserva metabólica (essencialmente triacilglicerol) em seus órgãos, e suas brânquias apresentaram maior atividade ATPásica total quando comparado a S. testudineus. Key words: Fish gill, Fish kidney, Glycogen, Metabolism, Osmoregulation, Tetraodontida 613 Metabolic substrates from the osmoregulatory organs of estuarine pufferfishes Metabolic substrates from the osmoregulatory organs of estuarine pufferfishes 614 Material and Methods Adults, male or female of S. testudineus (~ 15 cm body length, ~ 40 g body mass, deposited at the Museum of Natural History Capão da Imbuia, Curitiba, MHNCI 11700) and S. greeleyi (~ 10 cm, ~ 20 g, MHNCI 11701) were obtained from Baguaçu river tidal creek (25°33’06.33’’S, 48°23’41.63’’W), at the south margin of Paranaguá Bay, State of Paraná, Brazil, in May and August, 2003, May and June 2004, June 2006, and finally in February, 2007. Fish were obtained during flow tide using a fyke type net installed across the creek channel. The net has remained in place for 6 hours, until the high tide peak, V. Prodocimo, C. F. Souza, C. Pessini, L. C. Fernandes & C. A. Freire 615 Brazil). Proteins were read according to the Biuret method, and triacylglycerols using an enzymatic method: lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase, and peroxidase. Tissue glycogen content was determined as rou tinely in the literature, according to Keppler & Decker (1984), using approximately 0.07 g of gills or kidneys. Total protein concentration of these tissue samples was measured according to Bradford (1976). when the net was retrieved and the animals were transferred to plastic gallons with aerated water from the collection site. Water salinity was verified (29-30‰) immediately after fish ing the animals. Fishes were then transferred to the laboratory, where they were acclimated for ~7 days in a 160 L tank with 30‰, temperature of 21±1°C, constant aeration and biological filtration, under natural photoperiod. Fish were daily fed ad libitum with fresh prawns and earthworms. Sphoeroides testudineus and S. greeleyi were exposed for 6 hours to full-strength seawater 35‰ or to diluted seawater 5‰. Experiments were performed in 30-liter aquaria with constant aeration and temperature of 21±1°C, and a maximum number of 3 fish per experimental aquarium. Seawater 35‰ was prepared appropriately by mixing 30‰ seawater with the first thaw of frozen seawater. Experimental fishes submitted to 35‰ were directly transferred from the stock tank of 30‰ to the experimental aquarium 35‰. Seawater was diluted with filtered dechlorinated tap water from ~30‰ down to ~10-12‰ along 4 hours (flux of 0.7 ml/seg) to avoid osmotic shock to fishes exposed to 5‰. The fish then remained 10-12 hours in salinity 10-12‰ overnight, and were then transferred to 5‰, where they remained for 6 hours. Results Plasma osmolality and chloride levels in fishes of both species were lower in 5‰ than in 35‰. On the contrary, the hematocrit of both species was higher in 5‰ than in fishes exposed to 35‰. Further, the hematocrit of S. greeleyi was higher than that of S. testudineus in both salinities. Muscle wa ter content and plasma glucose of S. testudineus and S. greeleyi were both unaffected by salinity reduction (5‰) (Table 1). Table 1. Plasma osmolality (mOsm.kgH2O-1), chloride (mM), plasma glucose (mg.dL-1), hematocrit (%), and muscle water content (%) of Sphoeroides testudineus and S. greeleyi after short-term exposure to seawater of 5‰ or 35‰ salinity, mean ± standard error. * = salinity effect (5‰ ≠ 35‰), p<0.05; & = species effect (S. testudineus ≠ S. greeleyi), p<0.05. Table 1. Plasma osmolality (mOsm.kgH2O-1), chloride (mM), plasma glucose (mg.dL-1), hematocrit (%), and muscle water content (%) of Sphoeroides testudineus and S. greeleyi after short-term exposure to seawater of 5‰ or 35‰ salinity, mean ± standard error. * = salinity effect (5‰ ≠ 35‰), p<0.05; & = species effect (S. testudineus ≠ S. greeleyi), p<0.05. Fishes were then anaesthesized with benzocaine (in ethanol, 80 mg/l of aquarium water), were opened by ventral incision, and a blood sample was withdrawn through cardiac puncture using an heparinized insulin syringe. Blood samples were centrifuged for 10 min (2100×g) and plasma was im mediately frozen at −20°C. Blood was also collected into glass capillaries for immediate hematocrit reading. All gill arches and the whole kidney were removed for the assays of total ATPase activity, triacylglycerol and glycogen contents, and total protein concentration. A dorsal axial muscle sample (~0.40 g) was removed for tissue water content determination. These procedures have been approved by the Committee on Ethics in Animal Experimentation (CEEA) of the Institute of Biological Sciences of the Universidade Federal do Paraná (UFPR), certificate number 072, issued on May 3rd, 2004. Salinity S. testudineus S. Material and Methods Those fish submitted to salinity reduction had this protocol of transfer because of the large difference in salinity. Two-way ANOVA was performed to identify the effect of salinity (5‰ and 35‰) or species (S. testudineus and S. greeleyi), on all parameters, followed by Tukey post hoc test. Statistical significance was indicate by p <0.05. Results greeleyi (Prodocimo & Freire, 2006), its hematocrit increased, but not as much as in S. greeleyi. The opposite salinity challenge can lead to the same response. There are several reports of increased hematocrit upon exposure/acclimation of fishes to salinity increase. In this case, loss of extracellular water to the external medium would be the primary cause, although partially compensated by red cell shrinkage. However, eventu ally, regulatory volume increase of the red cells, coupled to an increase in red cell counts (long-term change) in order to cope with increasing demand for oxygen delivery following the increased metabolism would at the end lead to increased hematocrit (Plaut, 1998; Claireaux & Audet, 2000; Brown et al., 2001; Martinez-Álvarez et al., 2002). In fact, in either direction, the response is variable, depending on the time of exposure, magnitude of the salinity challenge imposed, degree of change in plasma osmolality, and volume regulatory capac ity of the red blood cells. Despite of the changes in hematocrit seen in the pufferfishes, their muscle cells apparently have a greater or faster capacity for tissue water regulation (as also in Prodocimo & Freire, 2006), as in both species no differences in muscle tissue water content were noted, compatible with a previous report on the red grouper Epinephelus akaara and the black sea bream Mylio macrocephalus also submitted to dilute seawater (Woo & Wu, 1982). The values of muscle water content (~ 80%) for both pufferfishes were similar to previ ous values reported for other euryhaline species (Feldmeth & Waggoner, 1972; Plaut, 1998; Kelly & Woo, 1999; Claireaux & Audet, 2000; Brown et al., 2001; Martinez-Álvarez et al., 2002), as well as for the same species (Prodocimo & Freire, 2006). of 35‰ was higher in the kidney of S. greeleyi than in that of S. testudineus (p<0.05). Glycogen content of gills and kidney of both species was not modified after exposure to 5‰ (Table 2). Kidney glycogen content in the S. greeleyi was higher when compared to S. testudineus, in both salinities (p<0.05). S. greeleyi presented higher levels (~twofold) of triacylglyc erol (Table 2) both in branchial and renal tissue, and in both salinities, than its congener S. testudineus (p<0.05). Finally, total protein concentration of both tissues and in both species was not affected by salinity reduction (Table 2). Table 2. Results greeleyi Plasma Osmolality 5‰ 308.5±2.4 (n=11)* 304.6±7.1 (n=11)* 35‰ 343.0±9.4 (n=11) 341.7±4.0 (n=11) Plasma Chloride 5‰ 144.3±2.5 (n=11)* 148.9±5.1 (n=11)* 35‰ 155.2±2.9 (n=11) 161.6±2.4 (n=11) Hematocrit 5‰ 27.9±1.1 (n=9)* 30.5±1.2 (n=11)& 35‰ 25.7±1.1 (n=11) 27.4±1.2 (n=11)& Muscle Water Content 5‰ 80.7±2.0 (n=6) 81.0±0.3 (n=11) 35‰ 82.7±0.9 (n=6) 81.3±0.8 (n=11) Plasma Glucose 5‰ 22.9±2.8 (n=11) 19.0±1.7 (n=6) 35‰ 24.8±2.9 (n=13) 23.7±2.9 (n=5) Plasma osmolality (vapor pressure micro-osmometer Vapro 5520, Wescor, USA), chloride (commercial kit, Labtest, Brazil), and glucose (commercial glucose oxidase kit, Labor clin, Brazil) were assayed in thawed samples. Hematocrit was determined after 12,000 rpm centrifugation (10 min, Presvac centrifuge, Argentina). For muscle water content determina tion, muscle samples were weighed on an analytical balance, dried at 100°C for 24h, then weighed again, and total water content was expressed as a percentage of the wet mass. Gills and kidneys were homogenized (Potter-S, in ice) in imidazole buffer 10 mM pH 7.5, and total ATPase activity was quantified in the supernatant (1000xg, 10 min) as the ap pearance of inorganic phosphate (Fiske & Subbarow, 1925), using Tris-ATP 3 mM as substrate, at 20 °C, as described in detail previously (Freire et al., 1995; Romão et al., 2001). These same samples had their total protein and triacylglycerol content assayed, using commercially available kits (Labtest, Branchial total ATPase activity (Table 2) in both species did not change upon salinity reduction (p>0.05). Renal total ATPase activity (Table 2) in S. greeleyi decreased upon expo sure to dilute seawater (p<0.05) and the activity in seawater Metabolic substrates from the osmoregulatory organs of estuarine pufferfishes 616 motic challenge, and water influx is expected. Hematocrit and muscle tissue water are simple and useful techniques frequent ly employed to access osmotic challenges to the intracellular compartment (Plaut, 1998; Marshall et al., 1999; Claireaux & Audet, 2000; Brown et al., 2001; Martinez-Álvarez et al., 2002; Prodocimo & Freire, 2006). Hematocrit results obtained for S. testudineus and S. greeleyi were similar to those re ported for other teleosts, of ~25-30% (Plaut, 1998; Claireaux & Audet, 2000; Brown et al., 2001; Martinez-Álvarez et al., 2002). Salinity reduction (5‰) lead to increased hematocrit in both pufferfishes, a result which may reflect red cell swelling, and the results in the S. greeleyi were higher than those of S. testudineus. As S. testudineus apparently has a higher capacity for tissue water content regulation regulation than S. Results Total ATPase activity (mmolPi.mgProt/h-1), triac ylglycerol (mg.Triac/mg.Prot) and glycogen content (mmol glycosyl units/g), and protein concentration (mg/mL) of bran chial and renal tissues of S. testudineus and S. greeleyi after short-term exposure to seawater of 5‰ or 35‰ salinity, mean ± standard error. = salinity effect (5‰ ≠ 35‰), p<0.05; & = species effect (S. greeleyi ≠ S. testudineus), p<0.05. Gill Kidney Salinity S. testudineus S. greeleyi S. testudineus S. greeleyi Total ATPase activity 5‰ 1.2±0.1 (n=8) 1.7±0.2 (n=8)& 1.7±0.2 (n=7) 1.4±0.2 (n=8)* 35‰ 1.3±0.04 (n=7) 1.7±0.1 (n=7)& 1.4±0.2 (n=9) 2.1±0.3 (n=8)& Glycogen content 5‰ 23.7±0.4 (n=8) 23.6±1.1 (n=6) 17.9±0.7 (n=8) 23.1±1.38 (n=4)& 35‰ 24.8±0.8 (n=8) 23.3±0.5 (n=5) 19.9±1.0 (n=8) 21.2±1.0 (n=3)& Triacylglycerol concentration 5‰ 0.082±0.004 (n=6) 0.152±0.018 (n=6)& 0.065±0.009 (n=6) 0.130±0.018 (n=6)& 35‰ 0.081±0.005 (n=6) 0.147±0.012 (n=6)& 0.068±0.006 (n=6) 0.137±0.027 (n=6)& Protein concentration 5‰ 5.7±0.3 (n=8) 6.1±0.7 (n=6) 8.3±0.2 (n=7) 8.1±0.7 (n=3) 35‰ 6.4±0.4 (n=8) 6.1±0.3 (n=5) 8.7±0.3 (n=7) 8.3±0.7 (n=4) Discussion Discussion This result is in agreement with that obtained in Sparus aurata, in which a direct relationship has been detected between renal Na+,K+-ATPase activity and salinity (Sangiao-Alvarellos et al., 2003). It should be pointed out that the marine euryhaline Dicentrarchus labrax displayed an opposite response, with increased renal ATPase activity when the fish were exposed to diluted seawater (3‰) and FW for 24 hours, due to the role of the renal tubules in salt absorption contributing to plasma hyper-regulation (Venturini et al., 1992). Still a different response may occur, in which the renal Na+,K+-ATPase is unaffected by salinity (McCormick et al., 1989). This issue is worth of additional investigation, also clarifying the quantitative role played by the Na+,K+-ATPase with respect to total ATPase activity and tissue metabolism in fish gills and kidneys. Except for the renal activity in low salinity, S. greeleyi in general displayed higher total ATPase activities than S. testudineus. This finding may indicate that S. greeleyi, which prefers higher salinities (Prodocimo & Freire, 2001, 2004, 2006), uses more ATP mainly in its gills either to hypo-regulate in full-strength seawater, or to hyper-regulate in dilute seawater. Besides higher ATPase activity levels, S. greeleyi also displayed higher levels of triacylglycerol in its gills and kidney than S. testudineus, confirming a species difference in metabo lism. The meaning of this difference cannot be explained by the present results; we cannot ascertain whether the rate of syn thesis and storage is higher or the rate of degradation is lower in S. greeleyi than in S. testudineus. We cannot in fact infer that triacylglycerol stored in the gills and kidney would be a source of energy for the osmoregulation of these pufferfishes, as S. greeleyi displayed higher branchial ATPase activity than S. testudineus in diluted seawater. Plasma triacylglycerol have been shown to increase during osmoregulatory challenges imposed on Sparus auratus submitted to salinities of 6‰ or 55‰ for 14 days, thus suggesting the use of these lipids as energy source for osmoregulation (Sangiao-Alvarellos et al., 2005). In accordance, the same species submitted to the isosmotic salinity of 12‰ also for 14 days displayed in no change in plasma triacylglycerol, in agreement with expected metabolism for osmoregulatory function (Sangiao-Alvarellos et al., 2003; Laiz-Carrión et al., 2005). Lipids as substrates to power ion transport in osmoregulatory organs should be the subject of future investigations. Discussion Plasma osmolality and chloride concentration found here in those two species of pufferfishes of the genus Sphoeroi des were in agreement with previous studies using the same species (respectively ~300 mOsm.kgH2O-1, and ~150 mM) (Prodocimo & Freire, 2001, 2004, 2006). The results also fell within the range reported for other marine/estuarine teleosts (e.g., Maren et al., 1992; Claiborne et al., 1994; Zadunaisky et al., 1995; Zadunaisky, 1996; Jensen et al., 1998; Marshall et al., 1999). When facing salinity changes, plasma osmolality and chloride concentrations directly reflect the fishes capacity of extracellular fluid homeostasis. Alterations in these con centrations indicate disturbances in this function. Although both species are euryhaline and efficient osmoregulators, as expected for estuarine fishes, a decrease in plasma concen trations was noted upon short-term exposure of the fish to diluted seawater, confirming previous studies (Prodocimo & Freire, 2004, 2006). l No mobilization of the metabolic substrates stored in the gills or kidney or in plasma glucose have been detected in S. testudineus and S. greeleyi upon this short-term exposure to salinity reduction. These results, coupled to the observation of no change in the total ATPase activity in both organs, means that branchial and renal metabolism remain stable upon this salinity challenge of short duration, a situation somewhat similar to what that these fishes could endure during ebb tide. No mobilization of the metabolic substrates stored in the gills or kidney or in plasma glucose have been detected in S. testudineus and S. greeleyi upon this short-term exposure to salinity reduction. These results, coupled to the observation of no change in the total ATPase activity in both organs, means that branchial and renal metabolism remain stable upon this salinity challenge of short duration, a situation somewhat similar to what that these fishes could endure during ebb tide. The lack of change in branchial total ATPase activity in both species when facing seawater dilution may either mean that this parameter does not adequately reflect the intensity of intermediary metabolism supplying ATP for this organ, or The lack of change in branchial total ATPase activity in both species when facing seawater dilution may either mean that this parameter does not adequately reflect the intensity of intermediary metabolism supplying ATP for this organ, or If there is extracellular fluid dilution, tissues endure os V. Prodocimo, C. F. Souza, C. Pessini, L. C. Fernandes & C. A. Discussion Freire 617 ingly, glycemia in the pufferfishes was very much lower (~20 mg/dL, or ~1mM) than values reported either for freshwater or marine teleosts. This is consistent with the observation that pufferfish gills store lots of glycogen, thus not requiring the mobilization of liver glycogen, and blood transport of glucose. The freshwater rainbow trout displayed values in the range ~36-144 mg/dL (Soengas et al., 1992; Figueroa et al., 2000), and the tilapia Oreochromis mossambicus ~50-120 mg/dL (Nakano et al., 1998) or 36-108 mg/dL (Vijayan et al., 1996). Other marine teleosts such as the sea bass Dicentrarchus labrax (~80 mg/dL, Roche et al., 1989), the sea breams Sparus auratus (80-90 mg/dL, Mancera et al., 1993; 72-90 mg/dL, Sangiao-Alvarellos et al., 2005), Sparus sarba (~60-75 mg/ dL, Kelly & Woo, 1999), and Chrysophrys major (~30-45 mg/ dL, Woo & Fung, 1981), the angelfish Pomacanthus imperator (~10-50 mg/dL, Woo & Chung, 1995), and the red grouper Epinephelus akaara (~30-50 mg/dL, Woo & Wu, 1982) had also values well above those measured for the pufferfishes. The marine teleosts Sparus sarba and S. aurata, submitted to salinities of 6-7‰ for 6-24 hours displayed increased plasma glucose levels (Mancera et al., 1993; Kelly & Woo, 1999; Sangiao-Alvarellos et al., 2005). It may be that, being estua rine and more euryhaline, reduced salinities for a short period are not as stressful and energy demanding for the pufferfishes as it apparently is for less euryhaline marine teleosts. that the challenge imposed really does not mean a significant change in the energy budget of the gills. Actually it is pos sible that either to hypo-regulate in full-strength seawater, or to hyper-regulate in dilute seawater both require increased levels of branchial ATP supply (“U-shaped” curves, e.g. Stuenkel & Hillyard, 1980; Jensen et al., 1998; Kelly et al., 1999; Imsland et al., 2003; Lin et al., 2004; Laiz-Carrión et al., 2005). Another possibility is that structural changes such as covering the apical pits of chloride cells turns off salt se cretion, as reported for estuarine fish facing reduced salinities (Sakamoto et al., 2000; Daborn et al., 2001; Marshall, 2003), thus preventing the need to increase ATPase consumption and the activation of energy-consuming processes. In the kidney of S. greeleyi, total ATPase activity was reduced in dilute seawater when compared to full-strength seawater. Discussion It may well be that divalent ion secretion, one of the main respon sibilities of the renal tubule when the fish is hypo-regulating in seawater, is turned off in dilute seawater (Jobling, 1995; Dantzler, 2003). This result is in agreement with that obtained in Sparus aurata, in which a direct relationship has been detected between renal Na+,K+-ATPase activity and salinity (Sangiao-Alvarellos et al., 2003). It should be pointed out that the marine euryhaline Dicentrarchus labrax displayed an opposite response, with increased renal ATPase activity when the fish were exposed to diluted seawater (3‰) and FW for 24 hours, due to the role of the renal tubules in salt absorption contributing to plasma hyper-regulation (Venturini et al., 1992). Still a different response may occur, in which the renal Na+,K+-ATPase is unaffected by salinity (McCormick et al., 1989). This issue is worth of additional investigation, also clarifying the quantitative role played by the Na+,K+-ATPase with respect to total ATPase activity and tissue metabolism in fish gills and kidneys. Except for the renal activity in low salinity, S. greeleyi in general displayed higher total ATPase activities than S. testudineus. This finding may indicate that S. greeleyi, which prefers higher salinities (Prodocimo & Freire, 2001, 2004, 2006), uses more ATP mainly in its gills either to hypo-regulate in full-strength seawater, or to hyper-regulate in dilute seawater. that the challenge imposed really does not mean a significant change in the energy budget of the gills. Actually it is pos sible that either to hypo-regulate in full-strength seawater, or to hyper-regulate in dilute seawater both require increased levels of branchial ATP supply (“U-shaped” curves, e.g. Stuenkel & Hillyard, 1980; Jensen et al., 1998; Kelly et al., 1999; Imsland et al., 2003; Lin et al., 2004; Laiz-Carrión et al., 2005). Another possibility is that structural changes such as covering the apical pits of chloride cells turns off salt se cretion, as reported for estuarine fish facing reduced salinities (Sakamoto et al., 2000; Daborn et al., 2001; Marshall, 2003), thus preventing the need to increase ATPase consumption and the activation of energy-consuming processes. In the kidney of S. greeleyi, total ATPase activity was reduced in dilute seawater when compared to full-strength seawater. It may well be that divalent ion secretion, one of the main respon sibilities of the renal tubule when the fish is hypo-regulating in seawater, is turned off in dilute seawater (Jobling, 1995; Dantzler, 2003). Discussion Glycogen stored in branchial and renal tissues of the puff erfishes was very stable and unchanged due to exposure to reduced salinity. This may indicate that indeed fuel (glucose) may come from the main glycogen storage organ (the liver), being taken up by the gills, as previously suggested (Soengas et al., 1992; Vijayan et al., 1996; Laiz-Carrión et al., 2002; Sangiao-Alvarellos et al., 2003, 2005). Alternatively, the gills of the pufferfishes may provide the glucose they need, since they have glycogen levels ~20-100-fold higher than glycogen contents of the gills of Sparus auratus (Sangiao-Alvarellos et al., 2003, 2005; Laíz-Carrión et al., 2002, 2005), measured using the same methodology (Keppler & Decker, 1974). Indeed, this mobilization of branchial glycogen has been recently demonstrated in the gills of the Mozambique tilapia (Tseng et al., 2007). The third metabolic substrate evaluated, protein, was also unchanged upon the salinity challenge imposed. This is an expected outcome, since the preferred metabolic substrates glycogen and lipids were also unchanged (Black & Love, 1986). Protein concentration was assayed in the plasma and was also reported as stable upon salinity reduction in Sparus auratus (Laiz-Carrión et al., 2005; Sangiao-Alvarellos et al., 2003, 2005). Plasma glucose was also maintained constant, and un changed upon short-term exposure to dilute seawater. Interest nclusion, besides the ecological difference in salin In conclusion, besides the ecological difference in salinity Metabolic substrates from the osmoregulatory organs of estuarine pufferfishes 618 Claireaux, G. & C. Audet. 2000. Seasonal changes in the hypo- osmoregulatory ability of brook charr: the role of environmental factors. Journal of Fish Biology, 56: 347-373. preference/tolerance, both species have confirmed previous results of an almost identical short-term osmoregulatory behaviour when facing salinity reduction, exception only for the higher hematocrit in S. greeleyi. In addition, they both did not display any clear sign of rapid metabolic substrate mobi lization from stores in the osmoregulatory organs (gills and kidney). Euryhaline estuarine fish, when facing fast salinity changes, have been reported to rapidly activate several mecha nisms to counteract changing gradients (Jacob & Taylor, 1983; Marshall, 2003). Among these actions, they rapidly activate the Na+,K+-ATPase (Mancera & McCormick, 2000), promote covering of the apical pits of the chloride cells (Sakamoto et al., 2000), and end up modulating unidirectional fluxes (Wood & Laurent, 2003; Prodocimo et al., 2007). Acknowledgements The authors wish to acknowledge the immense help from Dr. Henry L. Spach of the Marine Studies Center (CEM) of the Universidade Federal do Paraná (UFPR) in collecting the fishes. Thanks also to the biologist Marcos P. T. Veiga for help with fish maintenance and in setting up the experiments. This study has been financed by CAPES (doctorate fellowship to VP), DAAD-German Academic Exchange Service (donation of laboratory equipment to CAF). This work represents part of a Doctoral Dissertation by VP. Figueiredo, J. L. & N. A. Menezes. 2000. Manual de peixes marinhos do sudeste do Brasil. VI Teleostei (5). Museu de Zoologia, São Paulo: Universidade de São Paulo. Figueroa, R. I., R. Rodríguez-Sabarís, M. Aldegunde & J. L. Soengas. 2000. Effects of food deprivation on 24 h-changes in brain and liver carbohydrate and ketone body metabolism of rainbow trout. Journal Fish Biology, 57: 631-646. Fiske, C. H. & Y. Subbarow. 1925. The colorimetric determination of phophorus. Journal of Biological Chemistry, 66: 375-388. Freire, C. A., E. Kinne-Saffran, K. W. Beyenbach & R. K. H. Kinne. 1995. Na-D-glucose cotransport in renal brush-border membrane vesicles of an early teleost (Oncorhynchus mykiss). American Journal of Physiology, 269: R592-R602. Discussion This fast pattern is strikingly in contrast with the slow responses observed in euryhaline migrating fish such as anguilids and salmonids (Madsen et al., 1996; Wilson et al., 2004), and apparently do not demand fast and significant depletion of metabolic stores in osmoregulatory organs. Daborn, K., R. R. F. Cozzi & W. S. Marshall. 2001. Dynamics of pavement cell-chloride cell interactions during abrupt salinity change in Fundulus heteroclitus. Journal of Experimental Biol ogy, 204: 1889-1899. Dantzler, W. H. 2003. Regulation of renal proximal and distal tubule transport: sodium, chloride and organic anions. Comparative Biochemistry and Physiology A, 136: 453-478. Díaz, M., S. Cozzi, E. Almansa, M. Casariego, A. Bolaños, J. Cejas & A. Lorenzo. 1998. Characterization of intestinal Na+-K+- ATPase in the gilthead seabream (Sparus aurata L.). Evidence for a tissue-specific heterogeneity. Comparative Biochemistry and Physiolology B, 121: 65-76. Evans, D. H., P. M. Piermarini & W. T. W. Potts. 1999. Ionic transport in the fish gill epithelium. Journal of Experimental Zoology, 283: 641-652. Evans, D. H., P. M. Piermarini & K. P. Choe. 2005. The multifunc tional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physi ological Reviews, 85: 97-177. Feldmeth, C. R. & J. P. Waggoner. 1972. Field measurements of tolerance to extreme hypersalinity in the California killifish, Fundulus parvipinnis. Copeia, 1972: 592-594. Literature Cited F. Souza, C. Pessini, L. C. Fernandes & C. A. Freire 619 Prodocimo, V. & C. A. Freire. 2004. Estuarine pufferfishes (Spho eroides testudineus and S. greeleyi) submitted to seawater dilu tion during ebb tide: a field experiment. Marine and Freshwater Behaviour and Physiology, 37: 1-5. Kelly, S. P., I. N. K. Chow & N. Y. S. Woo. 1999. Haloplasticity of balck seabream (Mylio macrocephalus): hypersaline to freshwa ter acclimation. 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https://openalex.org/W4287124117	https://europepmc.org/articles/pmc8477482?pdf=render	English	null	The Nereid on the rise: Platynereis as a model system	Zenodo (CERN European Organization for Nuclear Research)	2,021	cc-by	19,613	Abstract The Nereid Platynereis dumerilii (Audouin and Milne Edwards (Annales des Sciences Naturelles 1:195–269, 1833) is a marine annelid that belongs to the Nereididae, a family of errant polychaete worms. The Nereid shows a pelago- benthic life cycle: as a general characteristic for the superphylum of Lophotrochozoa/Spiralia, it has spirally cleaving embryos developing into swimming trochophore larvae. The larvae then metamorphose into benthic worms living in self-spun tubes on macroalgae. Platynereis is used as a model for genetics, regeneration, reproduction biology, devel‑ opment, evolution, chronobiology, neurobiology, ecology, ecotoxicology, and most recently also for connectomics and single-cell genomics. Research on the Nereid started with studies on eye development and spiralian embryogen‑ esis in the nineteenth and early twentieth centuries. Transitioning into the molecular era, Platynereis research focused on posterior growth and regeneration, neuroendocrinology, circadian and lunar cycles, fertilization, and oocyte maturation. Other work covered segmentation, photoreceptors and other sensory cells, nephridia, and population dynamics. Most recently, the unique advantages of the Nereid young worm for whole-body volume electron micros‑ copy and single-cell sequencing became apparent, enabling the tracing of all neurons in its rope-ladder-like central nervous system, and the construction of multimodal cellular atlases. Here, we provide an overview of current topics and methodologies for P. dumerilii, with the aim of stimulating further interest into our unique model and expanding the active and vibrant Platynereis community. Keywords: Annelida, Spiralia, Marine model species, Evo-devo, Integrative biology The Nereid on the rise: Platynereis as a model system B. Duygu Özpolat1* , Nadine Randel2* , Elizabeth A. Williams3* , Luis Alberto Bezares‑Calderón4, Gabriele Andreatta5 , Guillaume Balavoine6 , Paola Y. Bertucci7, David E. K. Ferrier8 , Maria Cristina Gambi9 , Eve Gazave6 , Mette Handberg‑Thorsager10 , Jörg Hardege11 , Cameron Hird4 , Yu‑Wen Hsieh10 , Jerome Hui12, Kevin Nzumbi Mutemi7 , Stephan Q. Schneider13 , Oleg Simakov14 , Hernando M. Vergara15, Michel Vervoort6 , Gáspár Jékely4 , Kristin Tessmar‑Raible5* , Florian Raible5* and Detlev Arendt7,16* © 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://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Özpolat et al. EvoDevo (2021) 12:10 https://doi.org/10.1186/s13227-021-00180-3 Özpolat et al. EvoDevo (2021) 12:10 https://doi.org/10.1186/s13227-021-00180-3 EvoDevo Natural habitat and life cycle Habitath Correspondence: dozpolat@mbl.edu; nr479@cam.ac.uk; e.williams2@exeter. ac.uk; L.A.Bezares-Calderon@exeter.ac.uk; g.jekely@exeter.ac.uk; kristin. tessmar@mfpl.ac.at; florian.raible@univie.ac.at; arendt@embl.de 1 Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543, USA 2 Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK The Platynereis genus shows almost cosmopolitan dis- tribution and has been found in the Mediterranean, the North Sea, North Atlantic from North America to Bra- zil, Persian Gulf, Red Sea, Indian Ocean, Pacific, and along the coasts of Southwest Africa [1]. P. dumerilii as a species complex [2, 3] is widely distributed through- out the European Seas from Scandinavia and Great Britain to the French Atlantic coast, Iberian Peninsula and Canary Islands, all along the Mediterranean coasts and the Black Sea [4, 5]. 3 Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, UK 4 Living Systems Institute, University of Exeter, Stocker Road, Exeter, UK 5 Max Perutz Labs, University of Vienna, Dr. Bohr‑Gasse 9/4, 1030 Vienna, Austria 7 European Molecular Biology Laboratory, Developmental Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany Full list of author information is available at the end of the article © 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://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Özpolat et al. EvoDevo (2021) 12:10 Özpolat et al. EvoDevo (2021) 12:10 Page 2 of 22 Page 2 of 22 Platynereis dumerilii occurs preferentially in shal- low vegetated habitat, both on hard bottoms covered with macroalgae [6–9], as well as associated with sea- grass beds [10, 11]. During the pre-mature adult stage, P. dumerilii dwells in mucous tubes that it secretes and constructs by incorporating small pieces of algae from the surrounding environment [12]. This behavior is called “gardening”, since the algal species are often used as food by the worms. It is also thought to reduce the risk of predation [13]. Tubes are most commonly found attached to various macrophytes growing on shallow hard bottoms, including seagrass, brown, green and red algae [7, 8, 14–17]. have a head, a pygidium, and three cheatigerous segments bearing parapodia and chaetae (bristles). After around 2 weeks post-fertilization they begin to grow by adding new segments to their posterior end from the posterior growth zone in front of the pygidium. The juveniles then choose a spot to secrete their mucous tube, usually the stem or leaves of macrophytes or macroalgae. Upon addition of their 6th segment, juveniles undergo a cephalic metamorphosis in which the first cheatiger- ous segment fuses with the head and transforms into posterior tentacular cirri [22–24]. Juveniles feed and grow for at least 2 months. Gonial clusters start to pop- ulate most of the trunk segments when worms reach 35–40 segments in length [25]. Growth and maturation of gametes starts in the coelom when the worms are approximately 50 segments in size, and undergo matu- ration in worms ~ 70 segments in size [14, 23, 26–28]. During sexual metamorphosis, the gut degenerates, the eyes increase in size, and the parapodia and muscula- ture profoundly change into the swimming form [22, 29]. Spawning of P. dumerilii peaks around 1 week after the full moon phase [20]. Aspects of sexual metamorpho- sis in this heteronereid phase are regulated internally by levels of the sesquiterpenoid hormone, methylfarnesoate [30]. In the Mediterranean Sea, Scotland, and the Black Sea, P. dumerilii is reported to reproduce from late spring to late fall [18, 31, 32]. After metamorphosis, the sexually mature male and female P. dumerilii leave their tubes and swim to the sea surface, where they perform a nuptial dance, which culminates in gamete release and external fertilization. Following spawning, the adults die. Most recorded collections of P. dumerilii occur at depths down to 10 m, with sea surface salinity of 30–35 practical salinity units (PSU; equivalent to o/00) and at temperatures between 10 and 15 °C [1]. However, the species is also reported at greater depths, associated with rocky banks and gorgonians, although these records [6] seem associated with macroalgae drifting from shallower habitat and under decay. However, the question of the depth distribution of P. dumerilii is still open and requires further investigation, for example whether speciation is involved. P. dumerilii is also reported for tidal flats cov- ered by brown Laminaria beds [17], in algal decaying debris [18], and associated with the flowering spathes of Zostera marina [19]. Lab-based experiments suggest that the juveniles and pre-mature adults are most active at night, when they emerge from their tubes to hunt for food [20]. P. dumerilii juveniles and pre-mature adults feed primarily on benthic diatoms, foraminifera, cocco- lithophores, and macroalgae fragments collected close to their tube entrance, or from the epiphytic community of their macrophyte hosts [13, 14]. Lab culture and field collection Laboratory culture Platynereis dumerilii has been cultivated throughout its life cycle and studied in the laboratory for more than 70 years. Thus, one of the key advantages of this model system is controlled breeding, which enables the estab- lishment and maintenance of inbred, transgenic and mutant strains (Additional file 2: Table S2). Carl Hauen- schild established culture conditions and provided a first description of the life cycle [23]. He also established the distinction between the two naturally co-occurring sib- ling species P. dumerilii and P. massiliensis [36, 37]. Using P. dumerilii, Carl Hauenschild was the first to show that moon-controlled monthly cycles still persist in the labo- ratory, and that natural moonlight can be mimicked (at least to some extent) by a light bulb [38, 39]. Current laboratory cultures of P. dumerilii, which range in scale from a few hundred to tens of thousands of worms per culture, all trace back to the initial culture established by Carl Hauenschild in the 1950s, and originate from the Bay of Naples (Italy) with sporadic in-crossing from other Life cycleh The lifecycle of P. dumerilii features several significant tran- sitions (Fig. 1c). The morphology of developmental and life cycle stages, as well as the sequence of developmental transitions and metamorphoses have been described in great detail [14, 21–23]. Eggs and sperm are shed into the water, where fertilization and embryonic development occur. Embryos secrete a protective jelly coating, which gives them added buoyancy and prevents polyspermy [21]. After 24 h at 18 °C, free-swimming trochophore larvae hatch and spend about 2 days as plankton. These larvae do not feed, and live on maternally provided yolk platelets and lipid droplets. After 3 days in the plankton, the late tro- chophores start searching the sea floor in preparation for their settlement. They then undergo a habitat transition during which they settle to the benthic zone, and start feed- ing after 6 days only. These small, three-segmented juvenile errant worms (also called young worms, or nectochaetes) Page 3 of 22 Özpolat et al. EvoDevo (2021) 12:10 Özpolat et al. EvoDevo cations such as Arcachon [21 22] Laboratory culture colony under "summer" day and night light conditions Fig. 1 a Phylogenetic position of P. dumerilii in Annelida and in Metazoa [33, 34]. b Platynereis head morphology (from [35]). Ac: acicula; Buccal Segm: buccal segment; F.C. (I, II): tentacular cirri; Kief.: jaw; Palp: palpae; Parapod: parapodia; Tent: antennae. c Life cycle of P. dumerilii. Embryonic, arval, juvenile, and adult stages are shown, along with research areas that focus on the different life cycle stages (boxes). Immature adults: no visible maturing gametes; pre-mature adults: maturing gametes in the coelom; mature adults: fully metamorphosed and ready to spawn Fig. 1 a Phylogenetic position of P. dumerilii in Annelida and in Metazoa [33, 34]. b Platynereis head morphology (from [35]). Ac: acicula; Buccal Segm: buccal segment; F.C. (I, II): tentacular cirri; Kief.: jaw; Palp: palpae; Parapod: parapodia; Tent: antennae. c Life cycle of P. dumerilii. Embryonic, larval, juvenile, and adult stages are shown, along with research areas that focus on the different life cycle stages (boxes). Immature adults: no visib maturing gametes; pre-mature adults: maturing gametes in the coelom; mature adults: fully metamorphosed and ready to spawn locations such as Arcachon [21, 22]. Laboratory culture techniques were more recently improved [40, 41]. colony under "summer" day- and night-light conditions. Life cycleh Keeping the worms under different lunar light-cycle conditions also allows mature worms to be obtained throughout the month, therefore, with a large colony size, embryos are always available. As P. dumerilii repro- duces only once before death, it is important to keep a minimum colony size in order to obtain maturing males Egg-to-egg life cycle can be as short as 11 weeks depending on culturing conditions, but regularly takes 3 months or longer. Sexual maturation is easily synchro- nized by using artificial moon light, and mature worms can be obtained throughout the year by keeping the Page 4 of 22 Özpolat et al. EvoDevo (2021) 12:10 Özpolat et al. EvoDevo lab by shaking the thalli in seawater, or by inspection under a stereomicroscope. Generally, P. dumerilii has a large dispersal potential, having a pelagic larval stage, as they can survive weeks without food by relying on mater- nally provided resources [8]. History of the term Nereididae History of the term Nereididae In Greek mythology, Nereids were the 50 daughters of Nereus, the old man of the sea. They symbolized the beauty and kindness of the seas and were often referred to as goddesses of sea or sandy beaches. Some millennia later, another group of sisters would be named after Nereids: the family of Annelids known as the Nereididae. Among the ranks of Nereididae, Platynereis dumerilii (Audouin & Milne Edwards) [209] is the type species of the genus Platynereis (Kin- berg 1865) [210] (Fig. 1a). While Platynereis dumerilii and its sisters may not be widely appreciated for their beauty as the original Nereids (Fig. 1b), these impres- sive worms quickly become the beloved lab compan- ions of those who study them. Unlike the daughters of Nereus who lived in the Aegean Sea, today’s Nereids are all over the world, including many research labs on several continents. Collecting fully mature adults can be carried out using a strong light at night as the fully mature worms leave their benthic habitat and move close to the ocean surface to swarm and reproduce. As the swarming time is highly synchronized by the lunar cycle, timing of mature worm collection can be planned accordingly. Sexually mature adult worms can be attracted to the light, detected based on their characteristic circular movements, and collected using a fine net [40, 43]. While collecting P. 1. Platynereis embryology 1. Platynereis embryology Laboratory culture of earlier life stages of P. dumerilii (embryos and larvae) so far involved natural sea water transported from the sea. However, recent efforts to develop new culturing methods solely based on artificial seawater from commercially available sea salt mixes have proven successful (Hird et al., unpublished). In addition, progress has been made on standardizing and improving feeding, such as spirulina powder instead of spinach for juveniles [41] and using algae cocktails and live feeds for faster larval settlement and early development, respec- tively (Hird et al., unpublished). One of the critical future advances will be developing and improving cryopreser- vation methods for larvae or sperm for maintaining and sharing transgenic or mutant strains [42]. As a member of the spiralian clade, the Platynereis embryo shows highly stereotypical and synchronous development with a spiral cleavage pattern [44]. P. dumerilii embryos have unequal cleavage, and dur- ing the first two cell divisions, 4 distinct blastomeres called A, B, C, and D, are formed; the D blastomere (or the D-quadrant) is the largest of these (Fig. 2a). The spiral cleavage is then easily followed from 4- to 64-cell stages and comprises a series of invariant asymmetric cell divisions (Fig. 2a). Therefore, embry- onic cells can be individually identified by their posi- tion and size, and the cell lineages can be linked to particular cell fates similar to the nematode C. ele- gans and ascidian embryos [45–49]. Since the spiral- ian mode of embryogenesis is utilized by many phyla within Spiralia, it constitutes the starting point for many divergent adult body plans [44, 50–52]. Indeed, P. megalops embryos were among the first to be used for cell lineage and cell-fate mapping in the late nineteenth century, and these appear identical to P. dumerilii [53]. Several studies built upon these early works with microscopy, microinjection of embry- Major interests and research questions Below we present major research interests following the life cycle stages, starting with embryogenesis: Below we present major research interests following the life cycle stages, starting with embryogenesis: Life cycleh dumerilii is a great field experience, most labs do not need to do any collecting, and researchers who are interested in cultur- ing P. dumerilii can simply request larvae from estab- lished labs, which will happily share animals. This may indeed be the preferable option because these lab strains have been used to generate the genomic and transcrip- tomic resources, and therefore are better suited for molecular studies. and females at the same time to be able to propagate and maintain a particular strain. For wild-type strains, lab colony sizes can be as small as 500–1000 worms at a time (Hird et al., unpublished). Based on the number of mature animals needed, it is easy to scale up or down P. dumerilii cultures [41]. While practices vary across labs, the worms typically can be cultured in food-safe, non- reactive plastic containers with 500–1500 mL sea water. Major interests and research questions Below we present major research interests following the life cycle stages, starting with embryogenesis: Field collection The subsequent spiral cleavages with alternating division axes lead to a 64-cell stage embryo with A, B, C, and D quadrants (described in Hsieh 2020 [70]). b–b’’’ P. dumerilii cell lineage with 4d lineage highlighted. Mesoderm (shades of blue), germline (magenta), and mesodermal posterior growth zone (8ML) arise from the 4d lineage. b’’ The segmental mesoderm blocks that arise from this lineage are shown with respect to the larval schematic (b’’’) (modified from Özpolat et al. 2017). c–e’ Cell lineage-specific markers (e’ is modified from Wilson 1892, Bastin et al. 2015, Vopalensky et al. 2019) Otx (c,) and fzCRD-1 (c’) expressing cells at 12 hpf were aligned onto the live-imaging movie of a Platynereis embryo used for constructing the anterior cell lineages (d). d’ A subset of the cell lineage from b is shown (). Otx, which patterns neuronal tissues, and fzCRD-1, which labels a pair of kidney-like cells, are differentially expressed in daughter cells from the same founder cells, 1cd1122. The progeny of the otx-expressing daughter cell will continue to divide and form neurons while the fzCRD-1 expressing daughter cell will cease dividing and differentiate into a kidney-like cell. e At 24 hpf, fzCRD-1 is expressed in two elongated cells that correspond to the pair of kidney-like cells originally observed by E. B. Wilson in his foundational work on P. megalops and Nereis cell lineages in 1892 [53 (arrowheads in e’) study focused on the cell lineages forming the head region and revealed the transition from spiral cleav- age to bilaterally symmetric arrangement of founder cells in the head [63]. Here, one bilateral symmetri- cal founder cell divides to give rise to daughter cells with distinct fates: one otx-expressing daughter cell will eventually differentiate into neuronal tissue, and another daughter cell expressing fzCRD-1 (frizzled cysteine-rich domain 1) will terminally differentiate into a kidney-like cell [53, 63, 67] (Fig. 2c–e). Thus, lineage-tracing and gene expression studies are linked to individual differentiated cells and cell types. These studies also allow an evolutionary comparison of cell types across the phylogenetic tree [68]. onic cells, and the use of fluorescent dyes for cell-fate mapping [54–57]. Fertilization, gametogenesis and oocyte maturation have also been extensively studied in P. dumerilii and P. megalops [27, 28, 43, 58, 59]. Linking cell lineages and individual cells to gene expression and cell fate is possible in P. dumerilii [57, 60–63]. Field collection dumerilii: Linking lineage to gene expression and fate cell-by-cell. a Early stages of the P. dumerilii embryo. The mitotic spindles are labeled with anti-alpha-tubulin antibody (green), the actin with rhodamine phalloidin (magenta), and the nucleus with DAPI (blue). Arrows show the direction of cell division. The zygote first divides into AB and CD cells (2-cell stage) and after the second cleavage A, B, C, and D cells are generated. The subsequent spiral cleavages with alternating division axes lead to a 64-cell stage embryo with A, B, C, and D quadrants (described in Hsieh 2020 [70]). b–b’’’ P. dumerilii cell lineage with 4d lineage highlighted. Mesoderm (shades of blue), germline (magenta), and mesodermal posterior growth zone (8ML) arise from the 4d lineage. b’’ The segmental mesoderm blocks that arise from this lineage are shown with respect to the larval schematic (b’’’) (modified from Özpolat et al. 2017). c–e’ Cell lineage-specific markers (e’ is modified from Wilson 1892, Bastin et al. 2015, Vopalensky et al. 2019) Otx (c,) and fzCRD-1 (c’) expressing cells at 12 hpf were aligned onto the live-imaging movie of a Platynereis embryo used for constructing the anterior cell lineages (d). d’ A subset of the cell lineage from b is shown (). Otx, which patterns neuronal tissues, and fzCRD-1, which labels a pair of kidney-like cells, are differentially expressed in daughter cells from the same founder cells, 1cd1122. The progeny of the otx-expressing daughter cell will continue to divide and form neurons while the fzCRD-1 expressing daughter cell will cease dividing and differentiate into a kidney-like cell. e At 24 hpf, fzCRD-1 is expressed in two elongated cells that correspond to the pair of kidney-like cells originally observed by E. B. Wilson in his foundational work on P. megalops and Nereis cell lineages in 1892 [53 (arrowheads in e’) Fig. 2 Spiral embryogenesis in P. dumerilii: Linking lineage to gene expression and fate cell-by-cell. a Early stages of the P. dumerilii embryo. The mitotic spindles are labeled with anti-alpha-tubulin antibody (green), the actin with rhodamine phalloidin (magenta), and the nucleus with DAPI (blue). Arrows show the direction of cell division. The zygote first divides into AB and CD cells (2-cell stage) and after the second cleavage A, B, C, and D cells are generated. Field collection Field collection of Platynereis species at juvenile stages is relatively easy since the worms are associated with shal- low vegetated habitat, as well as in the intertidal zone such as seagrass meadows. While seagrass collection requires SCUBA diving, collection from very shallow hard bottoms and tidal flat habitats can be accomplished via snorkeling. Collection involves removal of algal thalli inside fabric bags and then sorting of the worms in the Özpolat et al. EvoDevo (2021) 12:10 Özpolat et al. EvoDevo Page 5 of 22 Fig. 2 Spiral embryogenesis in P. dumerilii: Linking lineage to gene expression and fate cell-by-cell. a Early stages of the P. dumerilii embryo. The mitotic spindles are labeled with anti-alpha-tubulin antibody (green), the actin with rhodamine phalloidin (magenta), and the nucleus with DAPI (blue). Arrows show the direction of cell division. The zygote first divides into AB and CD cells (2-cell stage) and after the second cleavage A, B, C, and D cells are generated. The subsequent spiral cleavages with alternating division axes lead to a 64-cell stage embryo with A, B, C, and D quadrants (described in Hsieh 2020 [70]). b–b’’’ P. dumerilii cell lineage with 4d lineage highlighted. Mesoderm (shades of blue), germline (magenta), and mesodermal posterior growth zone (8ML) arise from the 4d lineage. b’’ The segmental mesoderm blocks that arise from this lineage are shown with respect to the larval schematic (b’’’) (modified from Özpolat et al. 2017). c–e’ Cell lineage-specific markers (e’ is modified from Wilson 1892, Bastin et al. 2015, Vopalensky et al. 2019) Otx (c,) and fzCRD-1 (c’) expressing cells at 12 hpf were aligned onto the live-imaging movie of a Platynereis embryo used for constructing the anterior cell lineages (d). d’ A subset of the cell lineage from b is shown (). Otx, which patterns neuronal tissues, and fzCRD-1, which labels a pair of kidney-like cells, are differentially expressed in daughter cells from the same founder cells, 1cd1122. The progeny of the otx-expressing daughter cell will continue to divide and form neurons while the fzCRD-1 expressing daughter cell will cease dividing and differentiate into a kidney-like cell. e At 24 hpf, fzCRD-1 is expressed in two elongated cells that correspond to the pair of kidney-like cells originally observed by E. B. Wilson in his foundational work on P. megalops and Nereis cell lineages in 1892 [53 (arrowheads in e’) Fig. 2 Spiral embryogenesis in P. Field collection Embryos are large enough to microin- ject for fluorescent labeling of cell nuclei and mem- brane, they show fast recovery after injection, and are small and transparent enough to image for several days (Additional file 1: Table S1). In addition, vari- ous sequencing approaches for obtaining develop- mental stage-specific transcriptomes have been used [64–66]. These resources allow the identification of transcription factors and signaling pathways and assist in mapping gene expression to cell lineages and cell fates. Two recent studies identified the precise single-cell origins of segmental mesoderm, germline, and the mesodermal posterior growth zone, along with cell cycling dynamics [57, 60] (Fig. 2b). Another One area of interest in spiralian embryos is a deeper understanding of the molecular basis of asymmet- ric cell divisions and the cytoskeletal dynamics of the spiral cleavage mode itself. Currently, little is known about the underlying molecules and biophysi- cal mechanisms, but with the establishment of new Özpolat et al. EvoDevo (2021) 12:10 Özpolat et al. EvoDevo (2021) 12:10 Page 6 of 22 Page 6 of 22 organisms—but lacking in the main model ani- mals—are features that make the P. dumerilii larva a favorable system for investigating the sensory biol- ogy of zooplankton and the neuronal control of cilia. The larva has bands of locomotor cilia in the head, trunk and tail that beat in a tightly regulated pat- tern (Fig. 3a, b). The activity of the ciliary bands is, in turn, a function of the stimuli perceived and the larva’s internal physiological state. Work in P. dumer- ilii is revealing the sensory cues, molecules and neuronal networks involved in the control of cili- ary beating. The sensory cues known to alter ciliary beating include light and water-borne vibrations. For instance, the 1-day-old trochophore changes its swimming direction by differentially altering the beating on each side of the ciliary band [81]. In con- trast, vibrations lead to sudden and transitory ciliary arrests of the entire band [82]. Ciliary band activity shows longer-term changes across the day that are induced by melatonin [83]. Other signaling mole- cules that affect ciliary band activity include neuro- peptides, which alter ciliary beat frequency, ciliary arrests, or both, causing larvae to swim upwards or downwards [84]. The cells controlling ciliary band activity have been identified using a combination of serial electron microscopy and calcium imaging [83, 85]. Field collection Cholinergic, serotonergic and monoamin- ergic neurons form a neuronal network converging on the ciliary band cells that ultimately define their beating pattern [85]. How these cells control this pat- tern and how other molecules such as neuropeptides modulate this network, and therefore the swimming behavior of the larva, are exciting questions still wait- ing to be answered. imaging techniques in P. dumerilii, quantitative stud- ies of the dynamics of the spiralian development are now possible [69, 70]. In recent years, comparative work between spirally cleaving embryos has gained attention [44]. In several spiralians including annelids, the specialized cells of the D-quadrant (named 2d and 4d) (Fig. 2) possess inductive capabilities that affect dorsal–ventral patterning of the embryos (the spiral- ian organizer). Experimental manipulations suggest the existence of so far unknown molecular determi- nants in the zygote that establish the D quadrant in P. dumerilii, including 2d and 4d. The discovery of these determinants is now within reach [71, 72]. Altogether, these data shed light onto which signaling molecules and pathways are operating during spiralian develop- ment, and when and where tissue specification and differentiation take place [63, 64, 67, 73–75] (Fig. 2b’’). Spiralian embryos and larvae constitute a mosaic of stem cells and differentiating cells that continue to divide or stop dividing to form distinct cell types. The stereotypic cell lineages enable systems-level analy- ses as well as the molecular dissection of the intri- cate interplay of cell-cycle regulation, cell division, cell death, cell delamination, cell migration, cell-fate specification, and cell-type differentiation. This paves the way to unravel the cell and developmental biol- ogy of spiralian embryogenesis in unprecedented detail in future studies. 2. Sensory biology of the swimming trochophore larva d Transmission electron microscopy image of ciliary photoreceptor cells located in the larval brain. The sensory cilia (outlined in blue) of one of the two pairs of cells is shown. e Mechanosensory ciliated cells (msCells) in the pygidium labeled using a promoter construct. Acetylated tubulin labels sensory cilia (s.cilia). f EM volume reconstruction of the neurosecretory cells in the apical organ region of a 3 dpf larva. Ae: adult eye; le: larval eye; ans: apical nervous system; cPRCs: ciliary photoreceptor cells. Image in a by J. Berger; image in d by R. Shahidi Fig. 3 Locomotor and sensory cilia in the P. dumerilii larva. a SEM micrograph of a larva at 3 days post-fertilization (dpf) (nectochaete stage). Ciliary bands in the head, trunk and tail (pygidium) propel the larva in the water. Dorsal view. b A 3 dpf larva engages in swimming, as visualized by the movement of microbeads added to the water. c Bright-field image of a 3 dpf larva showing the location of adult and larval eyes. Dorsal view. d Transmission electron microscopy image of ciliary photoreceptor cells located in the larval brain. The sensory cilia (outlined in blue) of one of the two pairs of cells is shown. e Mechanosensory ciliated cells (msCells) in the pygidium labeled using a promoter construct. Acetylated tubulin labels sensory cilia (s.cilia). f EM volume reconstruction of the neurosecretory cells in the apical organ region of a 3 dpf larva. Ae: adult eye; le: larval eye; ans: apical nervous system; cPRCs: ciliary photoreceptor cells. Image in a by J. Berger; image in d by R. Shahidi projection patterns [84, 93]. Transient and stable transgenesis have also allowed the identification of individual sensory cells [82, 89] (Fig. 3e). Some sen- sory cells can also be identified in P. dumerilii larvae during live imaging with the assistance of vital dyes, such as Mitotracker, which was used to identify che- mosensory cells in the larval head [94]. used to identify their role in detecting specific wave- lengths/colors of light thereby regulating swimming depth [88, 90]. A combination of microfluidics and calcium imaging enabled the identification of chem- osensory centers in P. dumerilii larvae, such as the palps, antennae, and nuchal organ [96]. These studies provide the basis for future investigations on how the sensory systems allow the P. 2. Sensory biology of the swimming trochophore larva 2. Sensory biology of the swimming trochophore larva Platynereis dumerilii exhibits a pelago-benthic life cycle with non-feeding planktonic larvae called tro- chophore, followed by a bottom-dwelling stage after larval settlement and metamorphosis (Fig. 1c). The trochophore has many features in common with other ciliated larvae: an apical tuft, ciliary band cells, and a range of ciliated sensory cells. The shared gene repertoire, and morphological and developmental features of the P. dumerilii larva have prompted far- reaching homology hypotheses on the evolution of structures such as the larval apical organ (a sensory structure common among primary larvae [76–78]) and the larval foregut [79]. More recent single-cell RNA-sequencing data from P. dumerilii larvae [62, 82] will enable further comparisons and bring a clearer picture of the cell-type complement of pri- mary larvae, a stage assumed to have existed already early in metazoan evolution [80].h The seemingly small and simple P. dumerilii trocho- phore larva is equipped with a remarkable array of different sensory cell types. Most prominent among these are the two pairs of eyes on the dorsal side (adult eyes) and a pair of eyes on the ventral side (lar- val eyes) (Fig. 3c). The photoreceptor cells in these eyes are rhabdomeric and express multiple r-opsins and a Go-opsin [86–91]. In the center of the brain, two pairs of ciliary photoreceptor cells are recogniza- ble due to their multiple ramified cilia (Fig. 3d). These cells express a ciliary opsin (c-Opsin1) [92]. Other sensory cell types can also be identified by their con- sistent spatial location and distinctive sensory cilia morphology. In P. dumerilii, sensory-neurosecretory cells are abundant in the apical organ [76–78]. The expression of neuropeptides in these sensory-neuro- secretory cells allows immunostaining with antibod- ies to highlight their morphology, including axonal The relatively small size, simple nervous system and the ciliated planktonic stage shared with many other Page 7 of 22 Özpolat et al. EvoDevo (2021) 12:10 Özpolat et al. EvoDevo Fig. 3 Locomotor and sensory cilia in the P. dumerilii larva. a SEM micrograph of a larva at 3 days post-fertilization (dpf) (nectochaete stage). Ciliary bands in the head, trunk and tail (pygidium) propel the larva in the water. Dorsal view. b A 3 dpf larva engages in swimming, as visualized by the movement of microbeads added to the water. c Bright-field image of a 3 dpf larva showing the location of adult and larval eyes. Dorsal view. 2. Sensory biology of the swimming trochophore larva The Nereid young worm as a model for cellular-reso- lution multimodal atlases and connectomics 3. The Nereid young worm as a model for cellular-reso- lution multimodal atlases and connectomics y q y ypy Leveraging these unique advantages, gene-expression atlases for multiple embryonic, larval, and young worm stages have been obtained through profil- ing by image registration (PrImR; [101]) and profil- ing by signal probability mapping (ProSPr; [105]) and currently comprise more than 250 differentially expressed genes [101, 105, 106] (Bertucci et al., in prep.) (Fig. 4). The systematic exploration of gene expression and co-expression patterns in a common reference framework facilitates an unbiased defini- tion of molecular regions and cell types along the whole body [101, 105]. This provides a new rigor in evo-devo studies, which traditionally relied on the comparison of broad spatial expression of only a few select genes [101] 105, 106]. Furthermore, the availa- bility of gene expression atlases for multiple consecu- tive stages gives a dynamic picture of the lineage and molecular composition of cell types through devel- opment and differentiation (Bertucci et al., in prep). The power of multi-stage expression atlases allows, for example, to establish the developmental origin and differentiation path of neurons and glial cells (Bertucci et al., in prep.). This can be complemented and validated by lineage-tracing via targeted photo- conversion of selected cells, and by the gene knock- down or knock-out of lineage-specific marker genes. Ultrastructure of these cells can be further resolved by registering a high resolution volume obtained by focussed ion beam milling combined with scanning electron microscopy (FIB-SEM) of a 6 dpf young worm on the ProSPr atlas. Overall, diverse sensory morphologies including mechanosensory and chem- osensory endings have been and are being molecu- larly and morphologically characterized (Bertucci et al., in prep.). The three-segmented young worm represents another key stage of the Nereid life cycle that has attracted strong interest by evolutionary developmental biolo- gists, comparative anatomists and neurobiologists. As early as 3 dpf, the larva shows a prototype anne- lid body plan with many presumed ancestral features that have homologs across annelids, spiralians, proto- stomes, or even bilaterians. This includes individual cell types such as photoreceptors [92] and muscle cells [99, 100] to whole organs such as the axochord [99, 100] and brain regions such as the mushroom bodies [101]. 2. Sensory biology of the swimming trochophore larva dumerilii larva to detect and respond to other cues such as temperature, pres- sure, and pH, and how the larva integrates different cues to guide behavior in their natural habitat. Although most of the sensory cells in the larva await functional characterization, work on P. dumerilii has already shown how a combination of behavior, genet- ics and ecology can be harnessed to study sensory system function. The best-studied sensory modality in P. dumerilii larvae is light detection. Cell ablation, electron microscopy reconstruction and behavioral tracking in P. dumerilii have been used to understand different phototactic mechanisms in zooplankton. In the early larva, direct innervation of the ciliary band cells by the larval eyespot and spiral swimming account for light-directed swimming [81]. Older larvae use a more complex visual circuit that allows the detection of very small changes in light intensity through a feedback circuit motif [81, 95]. Targeted genome editing of Go-Opsin and c-Opsin1 was g The larval stage of P. dumerilii ends with settlement onto the sea floor. This habitat transition requires the larva to use its sensory capacities to navigate the benthic habitat, find a beneficial site to settle and begin feeding and tube-building. Previous stud- ies discovered that the neuropeptide myoinhibitory peptide (MIP) plays a role in the regulation of lar- val settlement and the initiation of feeding [94, 97]. Some MIP-expressing neurons with chemosensory morphology are found in the apical organ, an area implicated in the detection of environmental cues for larval settlement in aquatic larvae [77, 98] (Fig. 3f). Özpolat et al. EvoDevo (2021) 12:10 Özpolat et al. EvoDevo (2021) 12:10 Page 8 of 22 Page 8 of 22 Cells of the apical organ region have cilia with diverse morphologies, suggestive of several different sensory modalities [79] (Fig. 3f). Further studies are required to understand the specific function of the cells that populate the P. dumerilii larval apical organ and the different neuropeptidergic signaling pathways that may synergize with MIP to guide larval settlement. depends on time and temperature) and which are very similar in cell number, morphology and arrange- ment. This striking overall reproducibility of develop- ment and differentiation makes P. dumerilii ideal for creating multimodal cellular-resolution atlases for the entire body (Fig. 4). Beyond 6 dpf, young worms start feeding, which affects their developmental syn- chrony and, consequently, their stereotypy. 3. 2. Sensory biology of the swimming trochophore larva Equipped with a complex rope-ladder-like nerv- ous system, the three-segmented young worm moves around, changes substrate and explores the environ- ment. At 5–6 dpf, when the young worm finally set- tles, the nervous system is almost fully differenti- ated—with all major parts of the ventral nerve cord, peripheral nervous system and brain in place, includ- ing rudimentary mushroom body stalks and calyces [101, 102]. Still, at 6 dpf, the entire body contains no more than 12,000 cells comprising hundreds of cell types [103], which is a huge advantage relative to other model systems with cell counts in the millions for sim- ilar differentiated stages. For instance, the low number of cells per cell type allows simultaneous and complete capture of all body cell types in single-cell sequencing experiments; and the few neurons representing each neuron type permit tracing their connections for the entire nervous system [61, 85, 104]. i Ultrastructure of these cells can be further resolved by registering a high resolution volume obtained by focussed ion beam milling combined with scanning electron microscopy (FIB-SEM) of a 6 dpf young worm on the ProSPr atlas. Overall, diverse sensory morphologies including mechanosensory and chem- osensory endings have been and are being molecu- larly and morphologically characterized (Bertucci et al., in prep.). The high degree of developmental stereotypy that is characteristic for the embryonic spiral cleavage stages, manifests not only in the trochophore larva, but also in the nervous and muscular systems of the three-segmented young worm. This goes in concert with highly complex cellular arrangements and mor- phologies and the relatively low number of cells that force the bulk of cells into stereotypical and repro- ducible locations. The unique combination of mass reproduction, synchronicity and stereotypy allows obtaining thousands of individuals from the same batch at the same developmental stage (that only Following the same principle, the PrImR and ProSPr molecular atlases have been integrated at different levels with datasets of other modalities to obtain a more comprehensive picture of the Nereid cell types. Using the atlas gene set as a spatial reference system, it is possible to infer the anatomical origin of cells in single-cell RNA-Seq experiments [61, 104]. This allows the recovery of the original position of cells and complements the molecular information of the Özpolat et al. EvoDevo Özpolat et al. EvoDevo (2021) 12:10 Page 9 of 22 Fig. 4 P. dumerilii multimodal atlases. 2. Sensory biology of the swimming trochophore larva On the top row, five illustrative examples for the ProSPr gene expression atlases at different stages, showing the expression for the genes pax6 in red and gata123 in green. Their co-expression is shown in black. These are sum-projections for the full body from a ventral view (anterior up). On the bottom row, in left–right order: representation of single-cell rna sequencing and spatial mapping to the 48hpf gene expression atlas [61]; reconstructed neurons in the 3dpf P. dumerilii connectome, colored by cell type (the background is a graph representation of such connectome, where nodes represent cells and edges represent synaptic connections) [77, 95]; 2D section of the multimodal cellular atlas for the 6dpf P. dumerilii larva [102] showing the expression of the same genes as the top row, the segmentation of the muscles in cyan, and the segmentation of the chromatin in each nuclei Fig. 4 P. dumerilii multimodal atlases. On the top row, five illustrative examples for the ProSPr gene expression atlases at different stages, showing the expression for the genes pax6 in red and gata123 in green. Their co-expression is shown in black. These are sum-projections for the full body from a ventral view (anterior up). On the bottom row, in left–right order: representation of single-cell rna sequencing and spatial mapping to the 48hpf gene expression atlas [61]; reconstructed neurons in the 3dpf P. dumerilii connectome, colored by cell type (the background is a graph representation of such connectome, where nodes represent cells and edges represent synaptic connections) [77, 95]; 2D section of the multimodal cellular atlas for the 6dpf P. dumerilii larva [102] showing the expression of the same genes as the top row, the segmentation of the muscles in cyan, and the segmentation of the chromatin in each nuclei Fig. 4 P. dumerilii multimodal atlases. On the top row, five illustrative examples for the ProSPr gene expression atlases at different stages, showing the expression for the genes pax6 in red and gata123 in green. Their co-expression is shown in black. These are sum-projections for the full body from a ventral view (anterior up). On the bottom row, in left–right order: representation of single-cell rna sequencing and spatial mapping to the 48hpf gene expression atlas [61]; reconstructed neurons in the 3dpf P. 4. Platynereis as a model for chronobiology for multiple days under constant darkness even after decapitation [111]. In mature animals, recent work has revealed a role of the ~ 24-h clock in set- ting the time of the nuptial dance. The clock displays a remarkable plasticity in response to naturalistic moon light: its periodicity changes when the moon phase changes from moonlit to darkness. This modu- lation of a “circadian-circalunidian” oscillator results in shifting the onset of the nuptial dance to the dark- est hours of the night, a temporal niche changing over the course of the month (Fig. 5, middle panel). Functional interplay between the Cryptochrome L-Cry and the melanopsin ortholog r-Opsin1 is required for this precise timing [112]. A further exciting feature of P. dumerilii is its chronobiology, as this worm integrates both solar and lunar cues into its life cycle. Like most animals, P. dumerilii possesses an inner oscillator with a period length of ~ 24 h, trainable by daylight. Behavioral and molecular data suggest that already the 2-day-old planktonic larvae undergo day-to-day changes [83]. Whether these changes are also maintained under constant conditions, and are therefore controlled by a circadian clock, remains to be tested. This represents a particularly interesting aspect, as it might help to further functionally dissect the role of daily timing mechanisms in diel-vertical migrations [81], a wide- spread and crucial phenomenon for marine ecosys- tems [109, 110]. Furthermore, besides the “circadian-circalunid- ian” ~ 24 h clock, P. dumerilii also possesses an inner “calendar” (monthly oscillator) whose main function is the synchronization of reproductive events to spe- cific days of the month across the population. Moon- controlled monthly or semi-monthly reproductive cycles are fundamental and widespread in the marine environment, yet, mechanistically they are poorly understood [113]. This monthly rhythm in P. dumer- ilii is under the control of an endogenous oscillator, During the worm’s immature and pre-mature life, a circadian oscillator runs in both head and periph- eral tissues of the trunk [20, 111]. The head circa- dian oscillator is essential for rhythmic locomotion and trunk gene expression. Meanwhile, the circadian expansion and contraction of the worm’s peripheral pigmented cells (chromatophores) occur robustly Fig. 5 Platynereis dumerilii chronobiology. Sun and moon provide different cues for the rhythms that govern the worms’ biology. L-Cry functions as a valence detector, as well as a detector for the full moon phase. 2. Sensory biology of the swimming trochophore larva EvoDevo (2021) 12:10 Page 10 of 22 Özpolat et al. EvoDevo (2021) 12:10 Page 10 of 22 2. Sensory biology of the swimming trochophore larva dumerilii connectome, colored by cell type (the background is a graph representation of such connectome, where nodes represent cells and edges represent synaptic connections) [77, 95]; 2D section of the multimodal cellular atlas for the 6dpf P. dumerilii larva [102] showing the expression of the same genes as the top row, the segmentation of the muscles in cyan, and the segmentation of the chromatin in each nuclei been registered to the ProSPr expression atlas, result- ing in a full whole-body cellular-resolution alignment of gene expression with subcellular structures [102]. Together with the tracing of hundreds of randomly selected brain cells, this allows the exploration of cel- lular phenotypes and gene expression for the entire animal, and has enabled the unbiased characteriza- tion of morphologically defined tissues (Fig. 4). atlas in an unbiased manner (Fig. 4). This approach has revealed for example that the pre-metamorphic larva is composed of five distinct families of cell types that are likely conserved across animals [61, 104], and similar efforts are underway for the three-segmented young worm (Arendt laboratory, unpublished). The small size of the three-segmented larval stages (~ 300 µm) also enables the acquisition and compre- hensive analysis of nanometer-resolution whole-body serial electron microscopy datasets for the 3 dpf [95, 107] and the 6 dpf young worm [102]. In the 3 dpf worm, the complete morphological cell-type comple- ment and neuronal connectome have been mapped, revealing 90 non-neuronal and 180 neuronal cell types [103] (Fig. 4). Transient transgenesis with cell type-specific regulatory sequences and immuno- gold EM have also been used to map neurotrans- mitter and neuropeptide content to the connectome resource [85, 108] providing a link to the gene expres- sion atlas. For the 6 dpf young worm, a full serial blockface electron microscopy (SBEM) volume has g yi g The gene content of the P. dumerilii gene atlas so far reflects the focus of the community on specific organ systems (e.g., nervous system and muscula- ture). Other systems are still underrepresented (e.g., nephridia, heart, midgut). In the near future, any bias will be removed by the mapping of single cell data to the atlas. Furthermore, expanded multimodal atlases will serve as a link between subcellular ultrastructure and connectomics, and various kinds of single-cell multi-omics data, generating ultrastructurally and molecularly complete whole-body reference frame- works that will enable a new dimension of cellular- resolution linking of genotype and phenotype. Özpolat et al. 4. Platynereis as a model for chronobiology The general principle of posterior growth is shared by distantly related organisms including vertebrates, arthropods, annelids, and hemichordates, segmented or not, and pertains to the main clades of bilaterians [118–121]. P. dumerilii posterior growth has been studied in much detail and, it appears to rely on a ring-shaped growth zone in juveniles, anteriorly bor- dering the pygidium [118, 122]. Labeling of S-phase cells by 5-ethynyl-2′-deoxyuridine (EdU) pulse and chase experiments has shown the presence of pro- liferative cells in this growth zone, and cell divisions sustain tissue production and body elongation [62, 122]. Molecular characterization of the growth zone cells revealed the presence of ectodermal and meso- dermal cells exhibiting a largely overlapping molecu- lar signature [122]. These molecular signatures con- tain some hox genes [122, 123] but also genes such as piwi and vasa that are a part of the molecular signa- ture of P. dumerilii germ cells [25, 62, 122, 124]. This similarity of putative somatic stem cells and germ cells is consistent with the hypothesis of a common molecular signature of multipotency, also referred to as the Germline/Multipotency Program (GMP) [122, 125–129]. the functions of which are maintained even in the absence of the oscillations of the conventional core circadian clock [20]. Systematic transcriptomic and proteomic data further support the notion that circa- dian and circalunar oscillators are distinct [114]. The Cryptochrome L-Cry is also required for the proper monthly oscillator function via its ability to discrimi- nate between sun- and moonlight valence. Its specific activation kinetics by moonlight result in a fully acti- vated state only when exposed to moon phases close to the full moon [115]. This provides a first mecha- nistic understanding of how animals can set an inner calendar specifically to the full moon phase [115]. As a laboratory species with significant functional tools and resources, P. dumerilii is one of the best- accessible animal models to disentangle the cellular and molecular mechanisms of monthly oscillators. This is paralleled by efforts to obtain detailed knowl- edge on the light spectra and intensities in P. dumer- ilii’s natural habitats. An almost year-long daylight dataset from 10 m depth and additional informa- tion from 4 m have been collected in a Mediterra- nean habitat [116]. These datasets revealed that short wavelengths, particularly in the UVA/deep violet light range, carry annual time information, as their intensity changes are shifted relative to the photoperiod. 4. Platynereis as a model for chronobiology The photoreceptor c-Opsin1 helps convey this informa- tion in adult worms to adjust neuropeptide and neuro- transmitter dynamics, as well as behavior (Fig. 5) [116, 117]. As UVA/deep violet light is typically not present in standard laboratory lighting, this research illustrates the opportunities from integrating environmental parameters into lab research. In turn, it suggests that there may be other behavioral or molecular paradigms that might be modulated under more natural condi- tions. It will be important in the future to test for these modulations. Furthermore, it is clear that additional light receptors are important to convey the (full)moon signals to the different oscillators of the animal. The nature of these receptors, as well as the molecular and cellular pathways downstream will certainly be impor- tant aspects of future research. Platynereis dumerilii also harbors extensive regen- erative capabilities during the extended juvenile phase [130]. Annelids are known as one of the highly regenerative metazoan taxa [129, 131, 132]. P. dumer- ilii, like other Nereididae, can regenerate complex body structures. Upon amputation of the posterior body (Fig. 6a), both differentiated structures of the pygidium and putative stem cells involved in pos- terior growth are regenerated. In 3- and 4-month- old worms, this process is a rapid event that fol- lows a highly reproducible path and timeline, going through five specific stages in 5 days (Fig. 6b) [133]. Posterior regeneration relies on blastema formation and requires cell proliferation to proceed normally. Blastema cells express the GMP signature and likely mainly derive from a dedifferentiation process of cells next to the amputation plane [133]. Such extensive reprogramming of differentiated cells into prolifer- ating progenitors or stem cells during regeneration may rely on epigenetic mechanisms such as DNA methylation and histone modifications [134] (Schen- kelaars et al., in prep.). Posterior regeneration is then followed by post-regenerative posterior growth during which new segments bearing parapodia are formed [122, 133, 135]. In addition to posterior regeneration, parapodia regeneration following para- podia injury is known to be successful in P. dumerilii 5. Stem cell-based posterior growth and regeneration in Platynereis 4. Platynereis as a model for chronobiology r-Ops1 biochemically cannot discriminate between sun or moonlight, but its high sensitivity allows it to precisely determine moonrise. While the ~ 24-h-long clock relies on the “conventional” circadian transcription factor network, the nature of the circalunar (monthly) clock still remains unknown. It also remains to be tested if the annual changes are just direct responses to environmental changes or controlled by an endogenous annual oscillator Fig. 5 Platynereis dumerilii chronobiology. Sun and moon provide different cues for the rhythms that govern the worms’ biology. L-Cry functions as a valence detector, as well as a detector for the full moon phase. r-Ops1 biochemically cannot discriminate between sun or moonlight, but its high sensitivity allows it to precisely determine moonrise. While the ~ 24-h-long clock relies on the “conventional” circadian transcription factor network, the nature of the circalunar (monthly) clock still remains unknown. It also remains to be tested if the annual changes are just direct responses to environmental changes or controlled by an endogenous annual oscillator Özpolat et al. EvoDevo (2021) 12:10 Page 11 of 22 Page 11 of 22 Page 11 of 22 known as posterior growth (or posterior elongation). The general principle of posterior growth is shared by distantly related organisms including vertebrates, arthropods, annelids, and hemichordates, segmented or not, and pertains to the main clades of bilaterians [118–121]. P. dumerilii posterior growth has been studied in much detail and, it appears to rely on a ring-shaped growth zone in juveniles, anteriorly bor- dering the pygidium [118, 122]. Labeling of S-phase cells by 5-ethynyl-2′-deoxyuridine (EdU) pulse and chase experiments has shown the presence of pro- liferative cells in this growth zone, and cell divisions sustain tissue production and body elongation [62, 122]. Molecular characterization of the growth zone cells revealed the presence of ectodermal and meso- dermal cells exhibiting a largely overlapping molecu- lar signature [122]. These molecular signatures con- tain some hox genes [122, 123] but also genes such as piwi and vasa that are a part of the molecular signa- ture of P. dumerilii germ cells [25, 62, 122, 124]. This similarity of putative somatic stem cells and germ cells is consistent with the hypothesis of a common molecular signature of multipotency, also referred to as the Germline/Multipotency Program (GMP) [122, 125–129]. known as posterior growth (or posterior elongation). 5. Stem cell-based posterior growth and regeneration in Platynereis Once the three-segmented young worm stage ends, P. dumerilii juvenile worms grow during an extended phase of their life cycle, until sexual maturation occurs. Growth relies on the sequential addition of segments in an anterior-to-posterior temporal progression in the posterior body region, a process Özpolat et al. EvoDevo (2021) 12:10 Page 12 of 22 Özpolat et al. EvoDevo Fig. 6 Posterior regeneration as a paradigm for studying regenerative mechanisms and their regulation. a Experimental amputation of the posterior part of the body; b key stages (st.1–st.5) of posterior regeneration, re-establishing the growth zone (red), as well as molecular and morphological segment boundaries (dashed lines), reminiscent of the arrangements in posterior growth (right scheme). c Experimental paradigm establishing that transplanted juvenile heads produce a “brain hormone” endowing decerebrated individuals with the capacity to regenerate. d Exemplary results of brain grafts, charting the production of brain hormone (blue) to a medial region of the posterior brain. b Modified after Planques et al. 2019; d modified after Hofmann, 1976 Fig. 6 Posterior regeneration as a paradigm for studying regenerative mechanisms and their regulation. a Experimental amputation of the posterior part of the body; b key stages (st.1–st.5) of posterior regeneration, re-establishing the growth zone (red), as well as molecular and morphological segment boundaries (dashed lines), reminiscent of the arrangements in posterior growth (right scheme). c Experimental paradigm establishing that transplanted juvenile heads produce a “brain hormone” endowing decerebrated individuals with the capacity to regenerate. d Exemplary results of brain grafts, charting the production of brain hormone (blue) to a medial region of the posterior brain. b Modified after Planques et al. 2019; d modified after Hofmann, 1976 (Velasquillo et al., in prep.). Future work will need to investigate the precise staging of this process, as well as the origin and contribution of cells. As posterior and parapodia regeneration include regeneration of many tissue and cell types such as stem cells, neu- rons, muscle, gut, and the germline, and thanks to the availability of transgenic tools (Additional file 1: Table S1 and Additional file 2: Table S2), P. dumer- ilii lends itself to be a powerful model to dissect the molecular and cellular mechanisms of regeneration. instead promotes sexual maturation. Biochemical fractionation of P. dumerilii head extracts revealed methylfarnesoate as a key component of the brain hormone [30]. The possibility to combine biochemi- cal and genetic approaches makes P. 5. Stem cell-based posterior growth and regeneration in Platynereis dumerilii an attractive model for future research into the molecu- lar mechanisms responsible for the modulation and gradual loss of regenerative capacity. In turn, these analyses will also open up possibilities to investigate broader aspects of age-dependent development, senescence, and ageing (Additional file 1: Table S1). g Across animal phyla, regenerative capacities typically decline with developmental progress and age [136]. However, the underlying molecules and mechanisms remain largely unknown. Building on concepts devel- oped in earthworms [137], the decline of regenerative capacity in P. dumerilii has been linked to changes in the levels of endocrine factors. Systematic trans- plantations (Fig. 6c) of brain pieces from donor indi- viduals to decapitated hosts (unable to regenerate on their own) established that the responsible endocrine factors are produced in a distinct region of the P. dumerilii brain (Fig. 6d) [130]. These endocrine fac- tors are collectively referred to as the annelid “brain hormone”. Decline of brain hormone levels with age, or experimental decerebration of worms, abrogates regenerative capacity and posterior growth, and 7. The Platynereis genome Platynereis dumerilii has 14 pairs of chromosomes (2n = 28) [142, 155]. The current best estimates of the P. dumerilii genome lie between ~ 1 and 2 Gbp [142, 155–157]. Recent assemblies agree with this estimate and suggest ~ 1.4 Gbp. The P. dumerilii genome shows considerable polymorphism, heterogeneity [156], and repeat content (~ 43% of total nucleotides) with a CG ratio of 33.47% [158]. The presence of various hap- lotypes in a multitude of habitats make it an attrac- tive model system to explore genotype–phenotype relationships and standing genetic variation. Cur- rent community sequencing efforts and advanced sequencing techniques will survey P. dumerilii genomic variability. Furthermore, short reads can also be mapped to a reasonable reference genome. This will be an exciting endeavor in the near future, given the interest to characterize the polymorphisms and heterogeneity to exploration of the genetics and genomics of not only P. dumerilii but also other Platynereis species. The mitochondrial genome of P. dumerilii has been sequenced [159]. Mitochondrial genome data is also available for other Platynereis spe- cies, as a basis for Platynereis species trees [159, 160]. Studies are also underway for sequencing genomes of sister Platynereis species such as P. massiliensis and P. megalops. Beyond the genome, various transcrip- tomic resources are available for specific developmen- tal and life-cycle stages, body regions, and physiologi- cal phenomena [65, 114, 161, 162]. Reproduction Prior to reproduction, P. dumerilii undergoes metamorphosis into the reproductive form (the heteronereis) that dies after the reproduc- tive event (semelparity) with gametes shed into the water. Gametes in the water only remain fertilizable for a few minutes [143]. This requirement for highly synchronized reproduction makes P. dumerilii an ideal system to research the mechanisms under- pinning the timing of the reproductive events as discussed in the chronobiology section [144, 145], including the use of chemosensory signals and the sex pheromones [144–146]. Understanding the complex timing of the reproductive mass-spawning events in the field requires studies into the environ- mental factors coordinating hormonal control of maturation [30, 147], and a mode of metamorphosis induction [148] that ensures a significant part of a population to be mature and available for a once in a lifetime spawning event. Timed by moon phase, time of night, and even weather conditions [144, 149], these worms commit to a single reproductive event at the water surface. 6. Platynereis gametogenesis and reproduction Gametogenesis Platynereis dumerilii has 4 primor- dial germ cells (PGCs) that arise from the 4d lineage during embryogenesis (Fig. 2). The 4 PGCs migrate anteriorly at the end of larval development, to the segment behind the jaws. These cells then start prolif- erating in this anterior region, a process that appears to be coupled with growth state and the number of total segments that the individuals have reached [25, 124, 138, 139]. These cells are then thought to give rise to numerous gonial clusters that are found in the coelom and parapodia across the worm’s trunk [26– 28, 124, 138–140]. Initially, the gonial clusters are Özpolat et al. EvoDevo (2021) 12:10 Özpolat et al. EvoDevo (2021) 12:10 Page 13 of 22 Page 13 of 22 not obviously different between males and females, but as gametogenesis progresses, maturing oocytes and sperm can be detected, even before the individu- als start showing external morphological changes [26–28]. Thus, some cellular and biochemical aspects of oogenesis like vitellogenesis and spermatogenesis have been studied in more detail. As gametes grow floating in the coelomic body cavity, coelomic pro- teins have been one research focus—especially the production of yolk protein (vitellogenin) by a spe- cialized coelomic cell type, the eleocytes. Other pro- teins such as lipoproteins, and gamete metabolism in general, remain less explored [141]. Similarly, the sex determination mechanisms are largely unknown in P. dumerilii. Empirical observations from lab cultures suggest genetic rather than environmental determi- nation of sex. However, karyotyping has not revealed specific sex chromosomes [142]. as species specific: exposure to gamete-containing coelomic fluid of a large number of Nereidids induces heterospecific responses [152] with environmental timing of the reproductive events [153]. These chem- ical cues have also been shown to be used for aggres- sive mimicry by cone snails, which produce similar compounds to attract polychaete worms including Platynereis species as prey [154]. Future research directions will include the interconnection of gonadal maturation and precise reproductive timing synchro- nization within a population of worms, as well as the feedback regulation between maturation and regen- eration. 7. The Platynereis genome The release of sex pheromones coordinates the reproductive behavior, includ- ing nuptial dance and the gamete release, as well as sperm–egg interaction cues [144]. Volatile ketones (5-methyl-3-heptanone) function as distance cues inducing rapid swimming that culminates in meet- ing of the sexual partners [146], albeit trail com- pounds such as the peptide cues in the related spe- cies Alitta (Nereis) succinea have not been identified in P. dumerilii [146]. The release of gametes follows pheromonal stimulation [150] through uric acid in males [146] and l-Ovothiol-A [151] in females. This last crucial step of the reproductive behavior is not With the broad range of animal genomes available in the era of comparative genomics, it has become increasingly apparent that some animal genomes are unusual and distinctive, with plenty of lineage- specific gene gains/losses and rearrangements, while other genomes have evolved in a much more conservative fashion: they are less derived from an ancestral state such as the urbilaterian [163–167]. Based on current available data it appears that the Özpolat et al. EvoDevo (2021) 12:10 Özpolat et al. EvoDevo (2021) 12:10 Page 14 of 22 signaling via 17-β-estradiol [181] or complements of immune-related genes [162]. genome of P. dumerilii falls into this latter less- derived category. As such, this annelid has been, and will likely continue to be, a member of an important group of species that will enable us to infer major features of ancient ancestral states that became the starting points for the evolution of large swathes of the animal kingdom. As one would expect, there has also been some evo- lution of the genome structure and content along the lineage to P. dumerilii relative to the bilaterian ances- tor, such as the initial stages of dispersal of the Hox and ParaHox gene clusters, in which single genes have broken away in both cases [169, 182]. As a high- quality assembly of the P. dumerilii genome sequence becomes available with chromosome-level resolu- tion, further aspects of genome organization and evolution will be revealed, helping with a broader understanding of the general features of animal genome evolution and organization versus lineage- specific features. Specific examples of this conservative genome evolu- tion include the greater similarity of intron locations and abundance between P. dumerilii and vertebrates than was seen for more traditional invertebrate model species such as D. melanogaster and C. ele- gans [168]. 7. The Platynereis genome Also, gene linkage, or synteny, appears relatively less derived from the urbilaterian condi- tion than in fly and nematode, at least as indicated by the Antennapedia (ANTP)-class homeobox-con- taining genes, with their largely similar organization relative to chordates [169]. Similarly, P. dumerilii gene sequences appear to be relatively ‘short branch’ [168] and transcriptomes show a higher similar- ity of homologues with deuterostomes rather than ecdysozoans [64]. In addition, complements of gene families and composition of pathways show high lev- els of similarity to what is inferred for the bilaterian ancestor (or even earlier). P. dumerilii possesses 12 of the 13 ancient Wnt family members [74, 170] and a full complement of the frizzled-related genes that were inferred to be present in the urbilaterian [67]. P. dumerilii has both Retinoic Acid and Retinoid X Receptors (RAR and RXR) and the rest of the basic RA signaling pathway machinery [171, 172]. Both the Notch receptor and many Delta and Jagged ligands, as well as the full signaling pathway machinery [173] and an extensive complement of putative target genes of the Hes superfamily [174] are also present. P. dumerilii has an ancestral complement of miRs that are deployed in various tissues [175] as well as neu- rogenic genes (including NeuroD and Churchill genes that are important in vertebrates but do not exist in Drosophila), neuropeptides, particularly G-protein- coupled receptors and neurotransmitter recep- tor pathways [161, 176–180], which have tended to become derived via secondary gene losses in other protostomes as well as some deuterostomes. As with several of these examples above, examining genetic mechanisms in P. dumerilii has led to revisions in our understanding of processes that at one time were thought to be specific to vertebrates but can now be inferred to have been present in the last common ancestor of vertebrates and annelids, such as com- plements of opsins in photoreception [92], estradiol 8. Platynereis as a model for ecology and toxicology 8), with different cryptic species associated to each unique vent’s systems [3] (Gambi M.C., unpublished) (Fig. 7). These findings open questions about the taxonomic identity of both sibling species and on speciation within them, due to unique environmental conditions, such as those occurring at the hydrothermal vents. Only stud- ies that included individuals bred in the laboratory or using metamorphosed heteronereids enable the dis- tinction between the sibling species. Nevertheless, both sibling species of Platynereis seem to be ideal models to understand the potential for acclimation/adaptation to climate stressors, also throughout multigenerational studies. The use of a well-studied model species such as P. dumerilii represents an ideal opportunity to link the mechanistic understanding of the impacts and physi- ological costs of changing climate, including changes in water carbonate chemistry, upon functional traits with ecological endpoints including survival, fitness and reproductive success. For example, ocean acidification is known to alter animal behavior [203], cause changes to signaling molecules [204] and alter neurotransmis- sion [205]. Studies using P. dumerilii could shed light on both the mechanisms and consequences including cost- liness of adaptation to environmental change. sites have largely been utilized as a natural laboratory to study the effects of ocean acidification on the ben- thic biota, since the pH and carbonate chemistry con- ditions are simulating the further acidification scenar- ios of the global oceans for the end of the century [202]. Analyses showed the occurrence of multiple differen- tial genotypes, suggesting the presence of other cryptic species, for both siblings P. dumerilii and P. massiliensis [3] (Fig. 8). P. massiliensis was also found associated to the hydrothermal vents off the islands of Vulcano and Panarea (Aeolian Archipelago, north Sicily) (Fig. 8), with different cryptic species associated to each unique vent’s systems [3] (Gambi M.C., unpublished) (Fig. 7).hi Fig. 7 A female specimen of Platynereis cf massiliensis in reproduction, with the development of juveniles, from the population of the Vulcano island (Aeolian Archipelago, North Sicily) hydrothermal vent’s system (from Waege et al., 2017, modified) These findings open questions about the taxonomic identity of both sibling species and on speciation within them, due to unique environmental conditions, such as those occurring at the hydrothermal vents. Only stud- ies that included individuals bred in the laboratory or using metamorphosed heteronereids enable the dis- tinction between the sibling species. 8. Platynereis as a model for ecology and toxicology Nevertheless, both sibling species of Platynereis seem to be ideal models to understand the potential for acclimation/adaptation to climate stressors, also throughout multigenerational studies. The use of a well-studied model species such as P. dumerilii represents an ideal opportunity to link the mechanistic understanding of the impacts and physi- ological costs of changing climate, including changes in water carbonate chemistry, upon functional traits with ecological endpoints including survival, fitness and reproductive success. For example, ocean acidification is known to alter animal behavior [203], cause changes to signaling molecules [204] and alter neurotransmis- sion [205]. Studies using P. dumerilii could shed light on both the mechanisms and consequences including cost- liness of adaptation to environmental change. Fig. 7 A female specimen of Platynereis cf massiliensis in reproduction, with the development of juveniles, from the population of the Vulcano island (Aeolian Archipelago, North Sicily) hydrothermal vent’s system (from Waege et al., 2017, modified) (See figure on next page.) Fig. 8 The phylogeographic tree of Platynereis dumerilii and P. massiliensis based on COI analyses (From Waege et al. 2017, modified). Clade 1 and clade 2 represent siblings of P. massiliensis at Ischia (blue dot) and Vulcano (orange dots) vents; clade 3 and clade 4 represent siblings of P. dumerilii, grouped without a clear geographic pattern 8. Platynereis as a model for ecology and toxicology Platynereis dumerilii is particularly abundant under organic pollution, therefore it is considered as a use- ful bioindicator species [183]. Based on the extensive use of Nereid polychaetes in ecotoxicological stud- ies, Hutchinson et al. [184] evaluated P. dumerilii for its potential as an ecotoxicology model species to evaluate hazardous materials in the marine environ- ment, and examined acute toxicity impacts of phe- nolic compounds on P. dumerilii larvae [185]. The genotoxicity of sewage effluents using Comet assays have also been studied but showed limited impacts of settled sewage upon larval stages [186]. Recent stud- ies building on this demonstrated negative effects of halogenated and brominated disinfection byproducts found in sewage treatment works effluents [187]. In addition, toxicity of silver nanoparticles on early life stages, especially larvae, have been established [188]. Sublethal effects of volatile organic carbon from fuel oil induced male worms to shed gametes [189], high- lighting the sublethal impacts of toxicants upon ani- mal behavior [190]. Despite the ease of culturing and availability of the molecular, biochemical, and ecological data, P. dumerilii is still underused for ecotoxicology. In field studies, a problem related to the true distribution and ecology of this species arises by the fact that P. dumerilii, has a sibling species, P. massiliensis [37, 191] that is characterized by having a non-planktonic larval development (Fig. 7). Due to their similar mor- phologies at the juvenile and adult stages, only P. dumerilii is reported and P. massiliensis is not pre- sent in most ecological surveys in the Mediterranean Sea [192, 193]. Therefore, this sibling species has been largely overlooked [194], at least in the Medi- terranean Sea, while it is better reported in northern Europe [195]. Page 15 of 22 Page 15 of 22 Özpolat et al. EvoDevo (2021) 12:10 Özpolat et al. EvoDevo sites have largely been utilized as a natural laboratory to study the effects of ocean acidification on the ben- thic biota, since the pH and carbonate chemistry con- ditions are simulating the further acidification scenar- ios of the global oceans for the end of the century [202]. Analyses showed the occurrence of multiple differen- tial genotypes, suggesting the presence of other cryptic species, for both siblings P. dumerilii and P. massiliensis [3] (Fig. 8). P. massiliensis was also found associated to the hydrothermal vents off the islands of Vulcano and Panarea (Aeolian Archipelago, north Sicily) (Fig. Acknowledgements We thank the reviewers and editors for their feedback and comments that improved this manuscript. Availability of data and materials Availability of data and materials Not applicable. Not applicable. Experimental approaches From genome editing and stable transgenesis to bioinfor- matics, single-cell RNA-sequencing, single cell atlases, and behavioral assays, P. dumerilii has many fundamen- tal and cutting-edge techniques available and established. Application examples for many techniques are covered above in the context of the scientific questions addressed. In addition, we also provide a comprehensive table listing methods, relevant example references, noteworthy limi- tations, efficiencies and possible future directions (Addi- tional file 1: Table S1). The techniques are discussed in detail in the referenced literature. Recently, both sibling species, P. dumerilii and P. massiliensis, have been found with high abundance in the unique CO2 vent systems off the island of Ischia, where CO2 bubbling from the bottom is naturally acid- ifying the surrounding water [196–199]. Worms from this habitat show specific eco-physiological adapta- tions, including distinct responses to chemical stimuli mediated by ocean acidification [196, 200, 201]. These Özpolat et al. EvoDevo (2021) 12:10 Özpolat et al. EvoDevo Page 16 of 22 Fig. 8 (See legend on previous page.) Fig. 8 (See legend on previous page.) Özpolat et al. EvoDevo (2021) 12:10 Özpolat et al. EvoDevo (2021) 12:10 Page 17 of 22 Page 17 of 22 Multiple transgenic and inbred lines are hosted by indi- vidual labs and are shared within the community (Addi- tional file 2: Table S2). Additional resources include the sequenced genome and transcriptomes (Additional file 1: Table S1) [64, 65]. Furthermore, a cellular gene expres- sion atlas and a whole-body connectome with synaptic resolution are accessible via the Platybrowser [206] and CATMAID [207], respectively. Competing interests Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13227-021-00180-3. Additional file 1: Table S1. List of techniques. Additional file 2: Table S2. List of available wild-type and mutant strains. The authors declare that they have no competing interests. The online version contains supplementary material available at https://doi. org/10.1186/s13227-021-00180-3. Supplementary Information Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s13227-021-00180-3. Additional file 1: Table S1. List of techniques. Additional file 2: Table S2. List of available wild-type and mutant strains. Funding Funding resources are shown after author initials. EAW: BBSRC David Phillips Fellowship BB/T00990X/1. BDÖ: NIH NIGMS MIRA 1R35GM138008-01; NSF-EDGE Award no 1923429; Hibbitt Startup Funds. GJ, LABC, CH: Wellcome Trust Investigator Award 214337/Z/18/Z. KNM: Marie Sklodowska-Curie fellow supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 766053, project EvoCELL. NR: European Union Horizon 2020, Marie Skłodowska-Curie Grant No 838225. MCG: Stazione Zoologica A. Dohrn (Napoli) and the Ischia Marine Center technical staff; Open University PhD programme; ASSEMBLE; PON-MODO project (Campania Region, Italy), RITMARE - Flag project, Italy; MARES Consortium. Thanks to the ECCSEL - NatLab Italy facilities, man‑ aged by the OGS (Trieste), to support collection at Panarea and Vulcano islands. JDH: NERC award NE/T001577/1. MHT: Deutsche Forschungsge‑ meinschaft (DFG), Grant Number TO563/7-1. EG and MV: Labex ‘Who Am I?’ (No. ANR-11-LABX-0071) funded by the French Government through its ‘Investments for the Future’ program operated by the ANR under Grant No. ANR-11-IDEX-0005-01, Centre National de la Recherche Scientifique (DBM Grant), Université de Paris (IDEX Emergence grant 2020), Agence Nationale de la Recherche (Grant TELOBLAST no. ANR-16-CE91-0007; Grant STEM No. ANR-19-CE27-0027-02), the «Association pour la Recherche sur le Cancer» (Grant PJA 20191209482), and the «Ligue Nationale Contre le Cancer» (Grant RS20/75-20). SQS: NSF (US) Award IOS-1455185, MOST (TW) 108-2311-B-001- 002-MY3, Academia Sinica Career Development Award AS-CDA-110-L02, and the Institute of Cellular and Organismic Biology (ICOB) of Academia Sinica (TW). YWH: Deutsche Forschungsgemeinschaft (DFG), grant number TO563/7-1 (to Pavel Tomancak). OS: Austrian Science Fund Grant P32190. GB: The Balavoine Lab was funded by the CNRS, the Université de Paris and grants from the ANR (TELOBLAST no. ANR-16-CE91-0007) and from the ARC (PJA 20181208248). FR and KTR: The research leading to these results has received funding from the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007–2013)/ERC Grant Agreement 260304 (F.R.) and ERC Grant Agreement 337011 (K.T.-R.); the Horizon 2020 Programme ERC Grant Agreement 81995 (K.T.-R.); the research platforms ‘Rhythms of Life’ (K.T.-R., F.R.) and “Single-cell genomics of stem cells” (F.R.) of the University of Vienna; the Austrian Science Fund (FWF) START award project Y413 (K.T.-R.); the Austrian Science Fund (FWF) projects P28970 (K.T.-R.) and I2972 (F.R.); the Austrian Science Fund (FWF) grant F78 (K.T.-R., F.R.). DA and PB ERC Advanced grant NeuralCellTypeEvo #788921. Consent for publication Consent for publication Not applicable. Not applicable. Author details 1 Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543, USA. 2 Department of Zool‑ ogy, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK. 3 Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, UK. 4 Living Systems Institute, University of Exeter, Stocker Road, Exeter, UK. 5 Max Perutz Labs, University of Vienna, Dr. Bohr‑Gasse 9/4, 1030 Vienna, Austria. 6 Institut Jacques Monod, University of Paris/CNRS, 15 rue Hélène Brion, 75013 Paris, France. 7 European Molecular Biology Laboratory, Developmental Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany. 8 Gatty Marine Laboratory, The Scottish Oceans Institute, University of St Andrews, East Sands, St Andrews, Fife KY16 8LB, UK. 9 National Institute of Oceanography and Applied Geophysics – OGS, Trieste, Italy. 10 Max Planck Institute of Molecu‑ lar Cell Biology and Genetics, Pfotenhauerstraße 108, 01307 Dresden, Germany. 11 Department of Biological & Marine Sciences, Hull University, Cot‑ tingham Road, Hull HU67RX, UK. 12 School of Life Sciences, Simon F.S. Li Marine Research community and resourcesh The Platynereis community has been growing over the last few decades. Currently, about 15 Platynereis labs are studying a broad range of research topics, including development, evolution, regeneration, chronobiology, and neurobiology. Last year, the first Platynereis confer- ence was organized, resulting in the formation of several working groups to address challenges and encourage col- laborations in the community. One outcome has been the community website [208], which includes key infor- mation about the Platynereis community and existing resources. The growing and interactive community of the Nereid welcomes other scientists to join us in the exciting jour- ney of exploring the biology of this animal. Platynereis is now a mature experimental system with transgenesis, knockouts, genomic resources, gene-expression atlases and a full connectome, and progress can be made rapidly to gain mechanistic and molecular insights into devel- opmental, physiological or neuronal processes and also ecological interactions. There are many open questions ranging from evo-devo through neurobiology, to bio- physics and endless possibilities for technology develop- ment. In this review, we tried to give an overview of the broad and often unique biological questions that can now be addressed in Platynereis and the increasing experi- mental power of the system. The time is ripe to switch to the Nereid. Declarations Ethics approval and consent to participate Not applicable. p y 1101/2021.04.22.439825v1. 4. Kisseleva MI. 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Ophelia. 1973;11(1):1–507. • 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. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • 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. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • 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. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: 196. p y 1101/2021.04.22.439825v1. Effects of the volatile fraction of crude oil on spawning behaviour of nereids (Annelida, Polychaeta). Mar Environ Res. 1995;40(3):267–76. 210. Kinberg JGH. Annulata nova. 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Adaptation and acclimatization to ocean acidification in marine ectotherms: an in situ transplant experiment with polychaetes at a shallow CO2 vent system. Philos Trans R Soc Lond B Biol Sci. 2013;368(1627):20120444. 197. Lucey NM, Lombardi C, DeMarchi L, Schulze A, Gambi MC, Calosi P. To brood or not to brood: are marine invertebrates that protect their offspring more resilient to ocean acidification? Sci Rep. 2015;9(5):12009.
https://openalex.org/W4231661669	https://europepmc.org/articles/pmc3820897?pdf=render	English	null	The age lipid A2E and mitochondrial dysfunction synergistically impair phagocytosis by retinal pigment epithelial cells.	Journal of biological chemistry/The Journal of biological chemistry	2,013	cc-by	141	ADDITIONS AND CORRECTIONS THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 45, p. 32639, November 8, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 45, p. 32639, November 8, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. The age lipid A2E and mitochondrial dysfunction synergistically impair phagocytosis by retinal pigment epithelial cells. Arash K. Shirazi has changed his name to Ashton K. Shiraz. The new author line appears above. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts. JOURNAL OF BIOLOGICAL CHEMISTRY 32639 JOURNAL OF BIOLOGICAL CHEMISTRY 32639 NOVEMBER 8, 2013•VOLUME 288•NUMBER 45
https://openalex.org/W2897322543	https://aeji.journals.ekb.eg/article_16067_18ff58b241f5baa570bba5358e180391.pdf	English	null	Image Case:Extraction of an Impacted Coin in Upper Esophagus in 3 Years Old Female	Afro-Egyptian Journal of Infectious and Endemic Diseases/Afro-Egyptian Journal of Infectious and Endemic Diseases	2,012	cc-by	205	Image Case 132 Image Case 132 Corresponding author: Mohamed I Radwam email: mradwan@zu.edu.eg mobile:002010688872 85 Received :11 / 6 /2012 Accepted after revision: 30 /6 /2012 Image Case:Extraction of an Impacted Coin in Upper Esophagus in 3 Years Old Female Mohamed I Radwan, Mohamed H Emara Tropical Medicine Department, Faculty of Medicine, Zagazig University, Egypt Mohamed I Radwan, Mohamed H Emara Tropical Medicine Department, Faculty of Medicine, Zagazig University, Egypt coin impacted in the upper esophagus. Extraction under general anesthesia was done 5 days after swallowing. Areas of pressure necrosis at the site of impaction, as well as changes in the coin were noticed. A 3-year-old female swallowed a large metallic coin (one Egyptian pound; the largest coin in Egypt). Laryngeal examination failed to demonstrate the coin and serial X ray films showed the Corresponding author: Mohamed I Radwam email: mradwan@zu.edu.eg mobile:002010688872 85 Received :11 / 6 /2012 Accepted after revision: 30 /6 /2012 Corresponding author: Mohamed I Radwam email: mradwan@zu.edu.eg Figure (1): X ray showing the coin impacted in upper esophagus. Figure (1): X ray showing the coin impacted in upper esophagus. Figure (2): The extracted coin. Figure (2): The extracted coin. Radwan and Emara, Afro-Egypt J Infect Endem Dis 2012; 2(3): 132 www.mis.zu.edu.eg/ajied/home.aspx
https://openalex.org/W4242350286	https://pure.eur.nl/ws/files/48316741/Repub_128823_O-A.pdf	English	null	What methods are used to promote patient and family involvement in healthcare regulation? A case study across four countries	Research Square (Research Square)	2,020	cc-by	15,848	Wiig et al. BMC Health Services Research (2020) 20:616 https://doi.org/10.1186/s12913-020-05471-4 Wiig et al. BMC Health Services Research (2020) 20:616 https://doi.org/10.1186/s12913-020-05471-4 Open Access What methods are used to promote patient and family involvement in healthcare regulation? A multiple case study across four countries Siri Wiig1, Suzanne Rutz2,3, Alan Boyd4, Kate Churruca5, Sophia Kleefstra3, Cecilie Haraldseid-Driftland1, Jeffrey Braithwaite5, Jane O’Hara6 and Hester van de Bovenkamp2 © 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. Abstract Background: In the regulation of healthcare, the subject of patient and family involvement figures increasingly prominently on the agenda. However, the literature on involving patients and families in regulation is still in its infancy. A systematic analysis of how patient and family involvement in regulation is accomplished across different health systems is lacking. We provide such an overview by mapping and classifying methods of patient and family involvement in regulatory practice in four countries; Norway, England, the Netherlands, and Australia. We thus provide a knowledge base that enables discussions about possible types of involvement, and advantages and difficulties of involvement encountered in practice. Methods: The research design was a multiple case study of patient and family involvement in regulation in four countries. The authors collected 1) academic literature if available and 2) documents of regulators that describe user involvement. Based on the data collected, the authors from each country completed a pre-agreed template to describe the involvement methods. The following information was extracted and included where available: 1) Method of involvement, 2) Type of regulatory activity, 3) Purpose of involvement, 4) Who is involved and 5) Lessons learnt. Results: Our mapping of involvement strategies showed a range of methods being used in regulation, which we classified into four categories: individual proactive, individual reactive, collective proactive, and collective reactive methods. Reported advantages included: increased quality of regulation, increased legitimacy, perceived justice for those affected, and empowerment. Difficulties were also reported concerning: how to incorporate the input of users in decisions, the fact that not all users want to be involved, time and costs required, organizational procedures standing in the way of involvement, and dealing with emotions. (Continued on next page) Correspondence: siri.wiig@uis.no 1SHARE-Centre for Resilience in Healthcare, Faculty of Health Sciences, University of Stavanger, Stavanger, Norway Full list of author information is available at the end of the article * Correspondence: siri.wiig@uis.no 1SHARE-Centre for Resilience in Healthcare, Faculty of Health Sciences, University of Stavanger, Stavanger, Norway Full list of author information is available at the end of the article Background In many countries, patient and family involvement is high on the health policy agenda. Efforts to increase in- volvement can be seen at multiple levels of decision- making: the individual, the organizational and the policy level. The rationale for involving patients and families is two-fold. First, the expectation is that it will lead to bet- ter quality decisions across levels and thereby to better quality, and person-centred care. Second, as patients are the ones affected by decisions, they should have the op- portunity to influence decisions [1–8]. In the regulation of healthcare, the subject of patient and family involvement also figures increasingly promin- ently on the agenda [1, 9]. User involvement in regula- tion can be varied in nature. It can include providing individuals with information about a regulator; mobiliz- ing users and patients as sources of information; and reviewing whether health service providers involve users in service delivery and planning [5, 6]. This means that, on the one hand, involvement of patients and families is incorporated in regulatory standards and expectations directed at healthcare service providers [1, 5, 10–12]. On the other hand, involvement figures increasingly in the work of healthcare regulators themselves [5, 6, 13, 14]. In this paper, our main focus is on involvement initiated by regulators, although we recognize that this is interre- lated with regulatory standards addressing involvement in service provision. The following research questions guided our study: 1. What kind of methods for patient and family involvement in regulatory practice do regulators use in different countries? 2. What are the reported benefits and challenges of involving patients and family members? The paper proceeds as follows. First, we describe the methods used in our study. Second, in the results section we categorize and describe the methods of involvement that we found in the selected countries and present available evaluations of these. In the discussion, we re- flect on our findings and relate them to the literature in order to identify lessons learnt for involvement in regu- lation, alongside topics for future research. There is a growing literature on involving patients and families in their own care, especially in service provision [3, 4]. In comparison, the literature on involving patient and families in regulation remains in its infancy [5]. Background There are exploratory studies reporting experiments with involvement in different aspects of regulation, such as including patients in inspection teams, theme-based inspections, the investigation of sentinel events, using them as mystery guests and involving patients and fam- ilies in developing inspection assessment criteria [5, 6, 9, 12, 15–17]. However, despite these examples of specific cases in the literature, a more systematic analysis of how patient and family involvement in regulation is accom- plished across different health systems is lacking. (Continued from previous page) (Continued from previous page) (Continued from previous page) Conclusions: Our mapping of user involvement strategies establishes a broad variety of ways to involve patients and families. The four categories can serve as inspiration to regulators in healthcare. The paper shows that stimulating involvement in regulation is a challenging and complex task. The fact that regulators are experimenting with different methods can be viewed positively in this regard. Keywords: Patient involvement, Family involvement, Healthcare regulation, Participation, Decision-making, Norway, England, the Netherlands, Australia involvement in regulatory practice in four countries; Norway, England, the Netherlands, and Australia. We do so by analyzing the literature and other relevant sources such as the grey literature, websites, and documents of regulators that describe user involvement. By mapping methods of involvement and the use of patient and fam- ily experiences in the regulation of healthcare organiza- tions, we provide a knowledge base that enables discussions about possible types of involvement, and ad- vantages and difficulties of involvement, encountered in practice. Such an analysis can help regulators, and also shape the agenda for future research. © 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 15 Wiig et al. BMC Health Services Research (2020) 20:616 Research design and case selection The research design was a multiple case study of patient and family involvement in regulation in four countries. A case was defined as a country and the methods identi- fied for patient and family involvement in organizational regulation of quality and safety in healthcare [18]. The scope of our cases was limited to the regulation and regulatory practice related to organizations such as hos- pitals, nursing homes, youth care and home care. This means that involvement in regulation of individual healthcare professionals, such as involvement in deci- sions on disciplining or striking off doctors and nurses from their licenses and registries, was omitted. The regu- latory bodies included in our country cases are the In this paper, we provide such an overview by map- ping and classifying methods of patient and family Wiig et al. BMC Health Services Research (2020) 20:616 Page 3 of 15 Page 3 of 15 Wiig et al. BMC Health Services Research (2020) 20:616 Norwegian Board of Health Supervision (NBHS) and County Governors in Norway, the Care Quality Com- mission (CQC) in England, and the Health and Youth Care Inspectorate (HYCI) in the Netherlands. In Australia, the Australian Commission on Safety and Quality in Health Care (ACSQHC), which developed that country’s accreditation standards, was included as the key stakeholder in this particular regulatory system (see Table 1 for details about the country regulatory contexts and references to official web pages). in native language where native language search words were used. To identify grey literature and information on the web pages of each country’s regulatory body, the researchers used similar search words as in the literature searches. Moreover, some of the regulatory bodies had gathered information about user involvement and pub- lished reports on the topic on their web pages. This helped the researchers to identify relevant projects and reports. In addition, due to the limited literature identi- fied in data bases, some of the research team used Twit- ter to ask if someone knew of published papers or reports covering the topic of patient and family involve- ment in healthcare regulation or discussed this question with experts in the field. We aimed to sample a broad range of empirical mater- ial from different nations and healthcare systems to illu- minate the research questions from several angles [18, 28, 29]. We purposively selected countries with different types of healthcare systems. Research design and case selection Notwithstanding this, all are high-income countries where one could expect a devel- oped approach to user involvement at all system levels, including the regulatory level. The rationale for conduct- ing cross-country studies can be for comparative pur- poses, however the main reason for our case selection was not comparison, but rather to provide a broad- ranging overview. Our case selection enabled access to data on methods of involvement from a variety of con- texts. This served as a basis for developing an overview of existing methods and how countries approach user involvement in regulation. The authors from each country completed a pre- agreed template to describe the involvement methods found. The following information was extracted and in- cluded if available: 1) Method of involvement, 2) Type of regulatory activity, 3) Purpose of involvement, 4) Who is involved, and 5) Lessons learnt. In addition, researchers from each country provided a short description of their regulatory system, the actors, and their roles and respon- sibilities in order to provide contextual understanding of the different healthcare systems from which the data was collected. During the data collection, questions from the re- searchers were handled by authors SW and HvB, in order to clarify and align the methodological approach across the research team. The discussions were import- ant to ensure a similar approach and trustworthiness of the results. The completed templates were submitted to authors SW and HvB who led the cross-country analysis. During the cross-country analyses, results were synthe- sized [30, 31], which enabled us to identify existing methods of involvement, commonalities and differences, and clusters of similar types of involvement activities. Table 1 Description of regulatory context per country Country Description of regulatory context The supervision authority in England is the Care Quality Commission (CQC) [19]. CQC was established in 2009 as an independent non-departmental body, which is at arm’s length from government ministers. The CQC is held accountable to Parliament through the Health and Social Care Select Committee and to the Department of Health and Social Care through quarterly accountability review meetings. The CQC has a unitary board with a majority of non-executive members, which holds public and private meetings. The purpose of the CQC is to make sure health and social care services provide people with safe, effective, compassionate, high-quality care and to encourage care to improve. The CQC regulates the activities of health and social care organizations at almost 50,000 locations, serving the 55 million people in England. The main functions of the CQC are: • Register: Maintain a register of who is legally able to deliver regulated activities. Organizations are required to show they can meet standards of care set out in regulations in order to join the register. Subsequently they must notify the CQC of deaths and other incidents affecting service users, such as deaths not attributable to their illness, injuries, abuse, insufficient staff, and interruption of basic services such as gas and electricity. • Monitor, inspect and rate: Monitor the quality of care by gathering and analyzing data, including from people who use services, providers and other stakeholders. Monitoring informs the inspection of services to make sure they are providing care that is safe, effective, caring, responsive and well-led. Inspection findings are published, including for many services a rating of the quality of care. The CQC protects people by taking enforcement action to address poor care. • Independent voice: Publish reports on major national and regional quality topics, while also highlighting good practice. The CQC does not routinely have a role in investigating adverse incidents or complaints. Healthwatch England, the national consumer champion for users of health and social care services, is a statutory committee of the CQC’s Board. The CQC has a duty in law to take account of the views and experiences of local Healthwatch. (Source: [19]) Australia Regulation of the Australian healthcare system is complex and fragmentary. Responsibilities are shared among a network of national, six state and two territory departments of health, in addition to other government bodies [21]. Accreditation against standards is one of the major strategies for assuring the quality and safety of healthcare organizations. Before the federal government became involved, Australian hospitals were early-adopters of this approach, dating from the 1970s via the Australian Council on Healthcare Standards [22]. Accreditation was initially a voluntary activity. Due to growing scrutiny of hospital adverse events in the 1990s, Australian states passed laws mandating their hospitals take part in accreditation [23]. From 2000, the Council of Australian Governments passed reforms that subsequently established the Australian Commission on Safety and Quality in Health Care (ACSQHC) [24] and made accreditation against the national standards mandatory for all public and private hospitals [23]. However, the accreditation of general practices is still voluntary [25], and national accreditation standards have only recently been made mandatory for government subsidized residential aged care facilities [26]. y g g Unlike other health systems discussed in this paper, in Australia, there is no national regulatory body conducting inspections to ensure health services are delivered safely and according to the law. Rather, the National Safety and Quality Health Service Standards, developed by the ACSQHC, are used by independent organizations who are contracted by healthcare organizations to conduct their accreditation surveys, usually on a 3–4 yearly basis [24]. If any of the standards are ‘not met’, hospitals have up to 3 months to resolve the issue, depending on the risks associated with the issue. At state and territory levels, there are also departments of health, and a range of commissions and divisions that play a role in monitoring and improving the quality and safety of healthcare organizations, such as the Clinical Excellence Commission in New South Wales (Australia’s most populous state). When a standard is not met during an accreditation survey, state and territory health departments are notified and may take other regulatory action or provide support to health services as they address the issue [24]. (Source: [24–26]) Data collection and analysis However, the accreditation of general practices is still voluntary [25], and national accreditation standards have only recently been made mandatory for government subsidized residential aged care facilities [26]. Unlike other health systems discussed in this paper, in Australia, there is no national regulatory body conducting inspections to ensure health services are delivered safely and according to the law. Rather, the National Safety and Quality Health Service Standards, developed by the ACSQHC, are used by independent organizations who are contracted by healthcare organizations to conduct their accreditation surveys, usually on a 3–4 yearly basis [24]. If any of the standards are ‘not met’, hospitals have up to 3 months to resolve the issue, depending on the risks Table 1 Description of regulatory context per country Country Description of regulatory context Data collection and analysis The data collection was conducted between February and April 2019 according to a template designed by the authors centred on the research questions. The authors based in each country collected 1) academic literature if available and 2) documents of regulators that describe user involvement. These documents included: grey lit- erature, project reports, policy documents, and projects conducted by healthcare regulatory bodies described on their web pages. We felt it important to incorporate the grey literature, as involvement projects in regulation are often not designed as research projects, and therefore not published in peer reviewed journals. We conducted a broad search, as the main aim was to map existing methods of involvement in addition to possible existing evaluative data. yp In order to find a meaningful way to categorize the in- volvement methods and to learn from the diversity and similarities, the cross-country synthesis sought to categorize involvement methods inspired by Tritter’s [1] framework from two dimensions: individual vs collective, and proactive vs reactive involvement. This synthesis re- sulted in four categories of involvement methods, de- scribed as 1) individual and proactive 2) individual and reactive, 3) collective and proactive, and 4) collective and reactive (see Tables 2, 3, 4 and 5). The results were categorized as individual if they related to individual pa- tients/user/family experiences with their own care in specific situations. Results were categorized as collective if they related to general aspects where involvement was not related to a specific patient’s own case or treatment, but those involved were expected to represent a group of interests or inform regulatory inspectors on specific topics based on their experiences. We categorized In each country the researchers identified relevant arti- cles and reports based on their expertise of the subject. In addition, they searched academic data bases such as Medline, PubMed, Cinahl, and Google Scholar with search words covering user involvement, patient partici- pation, family involvement, regulation, supervision, in- spection, healthcare, quality, quality improvement and patient safety in combination with the names of the countries. We used this approach to identify country specific academic literature. In addition, the Norwegian and the Dutch data collection covered relevant journals Page 4 of 15 Wiig et al. BMC Health Services Research (2020) 20:616 Table 1 Description of regulatory context per country Country Description of regulatory context England The supervision authority in England is the Care Quality Commission (CQC) [19]. Data collection and analysis CQC was established in 2009 as an independent non-departmental body, which is at arm’s length from government ministers. The CQC is held accountable to Parliament through the Health and Social Care Select Committee and to the Department of Health and Social Care through quarterly accountability review meetings. The CQC has a unitary board with a majority of non-executive members, which holds public and private meetings. The purpose of the CQC is to make sure health and social care services provide people with safe, effective, compassionate, high-quality care and to encourage care to improve. The CQC regulates the activities of health and social care organizations at almost 50,000 locations, serving the 55 million people in England. The main functions of the CQC are: • Register: Maintain a register of who is legally able to deliver regulated activities. Organizations are required to show they can meet standards of care set out in regulations in order to join the register. Subsequently they must notify the CQC of deaths and other incidents affecting service users, such as deaths not attributable to their illness, injuries, abuse, insufficient staff, and interruption of basic services such as gas and electricity. • Monitor, inspect and rate: Monitor the quality of care by gathering and analyzing data, including from people who use services, providers and other stakeholders. Monitoring informs the inspection of services to make sure they are providing care that is safe, effective, caring, responsive and well-led. Inspection findings are published, including for many services a rating of the quality of care. The CQC protects people by taking enforcement action to address poor care. • Independent voice: Publish reports on major national and regional quality topics, while also highlighting good practice. The CQC does not routinely have a role in investigating adverse incidents or complaints. Healthwatch England, the national consumer champion for users of health and social care services, is a statutory committee of the CQC’s Board. The CQC has a duty in law to take account of the views and experiences of local Healthwatch. (Source: [19]) The Netherlands The regulatory authority in the Netherlands is the Health and Youth Care Inspectorate (HYCI) (Inspectie voor de Gezondheidszorg en Jeugd, IGJ) [20], which is part of the Ministry of Health, Welfare and Sport. This inspectorate regulates and promotes good and safe care. It’s regulatory activities are partly risk-based, thematic and incident based. Data collection and analysis The work of the inspectorate is based on different national acts focusing on, amongst others, quality of care in different sectors and the governance thereof, individual professionals, complaint procedures, and medication safety. One of the current focus points of the HYCI is person-centred care as it is considered an important condition for providing good and safe care. The HYCI distinguishes between the perspective of the public and the patient. Including both perspectives in its work is high on the regulatory agenda and mentioned specifically in multi-annual policy plans of ‘16-‘19 and ‘20-‘23. A vision document ‘Public and patient’s perspective in regulation’ was also written. The HYCI installed a Coordination Group Public and Patient Perspective in Regulation in May 2018 to collect information, advice and coordinate activities aimed at stimulating the inclusion of the public and patients perspective in regulation. The National Healthcare Report Centre, where citizens can ask questions and report complaints about the quality of care, is part of HYCI. In the Netherlands, healthcare organizations mostly conduct their own investigations in response to incidents. The HYCI requires that they involve patients or family members in these investigations. In addition, the HYCI has conducted many experiments to involve citizens in its inspections (more on this below). The HYCI works together with a number of universities in an academic collaborative where research is conducted into all kinds of aspects of regulatory work. This includes the subject of public/patient participation. (Source: [20]) Australia Regulation of the Australian healthcare system is complex and fragmentary. Responsibilities are shared among a network of national, six state and two territory departments of health, in addition to other government bodies [21]. Accreditation against standards is one of the major strategies for assuring the quality and safety of healthcare organizations. Before the federal government became involved, Australian hospitals were early-adopters of this approach, dating from the 1970s via the Australian Council on Healthcare Standards [22]. Accreditation was initially a voluntary activity. Due to growing scrutiny of hospital adverse events in the 1990s, Australian states passed laws mandating their hospitals take part in accreditation [23]. From 2000, the Council of Australian Governments passed reforms that subsequently established the Australian Commission on Safety and Quality in Health Care (ACSQHC) [24] and made accreditation against the national standards mandatory for all public and private hospitals [23]. The Netherlands The regulatory authority in the Netherlands is the Health and Youth Care Inspectorate (HYCI) (Inspectie voor de Gezondheidszorg en Jeugd, IGJ) [20], which is part of the Ministry of Health, Welfare and Sport. This inspectorate regulates and promotes good and safe care. It’s regulatory activities are partly risk-based, thematic and incident based. The work of the inspectorate is based on different national acts focusing on, amongst others, quality of care in different sectors and the governance thereof, individual professionals, complaint procedures, and medication safety. One of the current focus points of the HYCI is person-centred care as it is considered an important condition for providing good and safe care. The HYCI distinguishes between the perspective of the public and the patient. Including both perspectives in its work is high on the regulatory agenda and mentioned specifically in multi-annual policy plans of ‘16-‘19 and ‘20-‘23. A vision document ‘Public and patient’s perspective in regulation’ was also written. The HYCI installed a Coordination Group Public and Patient Perspective in Regulation in May 2018 to collect information, advice and coordinate activities aimed at stimulating the inclusion of the public and patients perspective in regulation. The National Healthcare Report Centre, where citizens can ask questions and report complaints about the quality of care, is part of HYCI. p In the Netherlands, healthcare organizations mostly conduct their own investigations in response to incidents. The HYCI requires that they involve patients or family members in these investigations. In addition, the HYCI has conducted many experiments to involve citizens in its inspections (more on this below). The HYCI works together with a number of universities in an academic collaborative where research is conducted into all kinds of aspects of regulatory work. This includes the subject of public/patient participation. 1The Tables 2, 3, 4 and 5 may be empty or sparse because of how regulation and governance of healthcare is organized. For example, in England, one reactive category in the country table is empty because CQC does not routinely have a role in investigating adverse incidents, and most complaints are handled by other organizations. We would also note that analyses of complaints and incident reports occur at various levels, but not necessarily as a direct response or mechanism of regulatory agencies, or with patients and families involved. Individual proactive methods of involvement p Individual proactive methods refer to involvement of in- dividuals with the purpose of collaboration and use of information for setting the future regulatory agenda and planning regulatory activities. The results in this cat- egory showed that the regulators in Norway, England, and Australia used some kind of patient and user sur- veys to collect information as part of setting the regula- tory agenda and informing inspection activities. National surveys are a way of collecting information about experi- ences and outcomes of the health services from broad groups of patients and users. We also identified more targeted surveys. In such cases, surveys were designed for specific groups, such as next of kin, and used to in- form future system audits and make these more context- specific and relevant for the target group of regulation (e.g., children). Questionnaires were also utilized as part Methods of involvement in regulation across countries Methods of involvement in regulation across countries The data synthesis identified a wide variety of methods for user involvement in regulatory practice. In the fol- lowing, we present the four categories of activities within these. A summary of these results per country can be found in Tables 2, 3, 4 and 5. (To ease readability of Ta- bles 2, 3, 4 and 5 and the four involvement methods cat- egories, we present this text without references. All references are included when we present the reported benefits and difficulties of involvement identified in re- search studies or in the published grey literature). Norway The supervision authorities are the Norwegian Board of Health Supervision (NBHS) (the central office), and the Offices of the County Governors (regional offices). The NBHS [27] is a national public institution organized under the Ministry of Health and Care Services. The overall aim of public supervision is to ensure that health and social services are provided according to national acts and regulations. In Norway, there is comprehensive legislation regarding child welfare, health and social services that: Page 5 of 15 Wiig et al. BMC Health Services Research (2020) 20:616 Table 1 Description of regulatory context per country (Continued) Country Description of regulatory context Table 1 Description of regulatory context per country (Continued) Country Description of regulatory context • constitute requirements about the services that shall be offered to the population; • constitute requirements about the quality of services; • regulate the work of health care personnel who have authorization; • give users of the services rights, for example, according to the Patients’ Rights Act. Supervision applies to all statutory services, irrespective of whether they are provided by municipalities, private businesses, publicly owned hospitals or health care personnel who run their own practice. Regulatory activities vary from area surveillance, proactive and planned supervision, and reactive event based after adverse events or deficiencies in services. At the level of the counties, supervision is carried out by the Offices of the County Governors. The NBHS has a special Department that can conduct onsite inspections in cases of the most severe adverse events. Most inspection activities are conducted by the Offices of County Governors. (Source: [27]) challenges of involvement based on the evaluations, if available, of experiments included in the study. involvement as proactive if the involvement was about collecting information or involving patients/users/fam- ilies as part of planning future inspections, setting the regulatory agenda or standards, or conducting routine, planned inspections. We characterized results as reactive if the involvement was related to follow up or handling of issues, for example, adverse events, deviances from standards or regulations, or complaints. The results of this synthesis are presented in Tables 2, 3, 4 and 5.1 We also analyzed the results in light of the reported benefits and challenges present in the material. Regulatory context Regulatory context In Table 1 we provide a brief overview of the regulatory context of Norway, England, the Netherlands, and Australia as a backdrop for this paper. Some countries use the term regulation, while others use supervision or inspection about their role and activity. This is reflected in the context description in Table 1. All authors are experienced researchers in the field of health services research, regulation, health policy and user involvement. Among the authors, there are also members with a background as regulatory inspectors (SW) or in a current main position as members of an in- spectorate (authors SK, SR) in addition to being re- searchers. This experience in the author team was beneficial in helping to identify important cases, chal- lenges, possible literature, projects, and to suggest pos- sible recommendations of relevance for both the research community and for other regulatory bodies. In England, a senior manager within the CQC was also consulted, to ensure thoroughness in our search, and clarification of the breadth of available involvement methods. Collective/proactive • Awareness campaign using social media, digital marketing, charity communications channels and other CQC communication channels to encourage patients and families to share their experiences with CQC • Collaboration with national charities to collect information about user experiences via their helplines • Collaboration with Healthwatch to produce guidance for local Healthwatch and Inspectors on working with CQC to promote involvement in reviews, on a general basis, and encourage members to submit user experiences to CQC • Collaboration with Healthwatch to produce guidance for local Healthwatch and Inspectors on working with CQC to promote involvement in reviews, on a general basis, and encourage members to submit user experiences to CQC p • Public online community for involvement in health policy and service design – includes both paid vouchers and self-selected groups • Experts-by-experience involvement in thematic reviews (involvement varies depending on topic) • Expert advisory group set up as part of inspection approach - including experts-by-experience members • Establish user panels and advisory groups (children, mental health) • Experts-by-experience used for speaking to people using services, families and organizations, that support them g pp • Experts-by-experience assist in registration, thematic reviews, local systems reviews, co-production, advisory groups, promotion of CQC work, and training of inspectors g p • Co-production of events by seldom heard communities y • “Mystery shoppers” and hidden cameras – CQC has considered this approach but has decided not to conduct covert surveillance • User panels (mental health, children and young) • Commissioned research with users • Data sharing partnership with other websites collecting intelligence from users from users Individual/reactive Individual/reactive Collective/proactive • Patient and user panel established for the regulatory body over time to inform all regulatory activity (national and regional level) • Next of kin as co-investigators in system audit • User involvement in inspection team • Organize seminars with adolescent user organization to inform system audit of child protective services • Develop national regulatory recommendations for user involvement in regulatory practice • Organize co-investigator experience workshop • Patient and user panel established for the regulatory body over time to inform all regulatory activity (national and regional level) • Next of kin as co-investigators in system audit • User involvement in inspection team • Organize seminars with adolescent user organization to inform system audit of child protective services • Develop national regulatory recommendations for user involvement in regulatory practice • Organize co-investigator experience workshop • Interview with young social care users to inform system audit • Interview with disabled users to inform system audit • Questionnaire to next of kin to inform system audit • Questionnaire to young service users in system audit • Meeting with next of kin to inform system audit • Interviews with service users during system audit • Develop digital tool for communication with children under 13 in child protection services • Including interpreter in system audit of under aged refugees in child protective service institutions Results First, we present a short introduction to the regulatory context of the four countries, before presenting the over- all findings relating to involvement methods across countries. We then describe the reported benefits and Page 6 of 15 Wiig et al. BMC Health Services Research (2020) 20:616 • Patients share experience with services in an online form to CQC • Annual user surveys to collect user experiences of particular service of interest • Inspectors and Experts by Experience speak with children, young people, parents, families, and carers during inspections. • National surveys • CQC commission community groups and charities to collect user experiences of particular pathways or types of care • CQC carries out research and focus groups to collect user experiences of particular pathways or types of care Table 3 England Individual/proactive Collective/proactive • Awareness campaign using social media, digital marketing, charity communications channels and other CQC communication channels to encourage patients and families to share their experiences with CQC • Collaboration with national charities to collect information about user experiences via their helplines • Collaboration with Healthwatch to produce guidance for local Healthwatch and Inspectors on working with CQC to promote involvement in reviews, on a general basis, and encourage members to submit user experiences to CQC • Public online community for involvement in health policy and service design – includes both paid vouchers and self-selected groups • Experts-by-experience involvement in thematic reviews (involvement varies depending on topic) • Expert advisory group set up as part of inspection approach - including experts-by-experience members • Establish user panels and advisory groups (children, mental health) • Experts-by-experience used for speaking to people using services, families and organizations, that support them • Experts-by-experience assist in registration, thematic reviews, local systems reviews, co-production, advisory groups, promotion of CQC work, and training of inspectors • Co-production of events by seldom heard communities • “Mystery shoppers” and hidden cameras – CQC has considered this approach but has decided not to conduct covert surveillance • User panels (mental health, children and young) • Commissioned research with users • Data sharing partnership with other websites collecting intelligence from users Collective/proactive Individual/reactive • Meeting between inspectors and next of kin in investigation of deaths • Regulator organizes meeting with patient and healthcare professionals in complaints cases or investigation of adverse events • Regulated right for patient/family to a meeting with service provider after a severe adverse event/death • Regulator contacts/consults with next of kin, informs about the process of investigation of the most severe adverse events conducted at the national level inspectorate investigation unit • Patient and user complaints can be sent to regulator users, and frail older service users. We found these methods were part of planned inspections (not initiated due to an ad- verse event) in Norway, England, and the Netherlands. Regu- lators collected information based on users’ experiences with the services and used this information to assess if services were provided according to standards and regulation. of a regulatory activity to collect information from a broader audience of users in planned inspections. The regulators also made use of qualitative methods such as individual interviews and meetings with service users. Reg- ulators spoke with children, adolescents, parents, families, next of kin, social and mental healthcare users, disabled Individual/reactive Collective/proactive • Patient survey at the state level about recent experiences and outcome of care • REACH – Recognize, Engage, Act, Call, Help is on its way: state initiative to empower consumers to ‘speak up for safety’, engage with their nurses or medical team, and request clinical review within 30 min Collective/reactive Collective/proactive • Interview patients or family during planned inspections in all care sectors • Collect experiences from adolescents and other patient groups (such as people living in poverty) in theme based inspections • Interview frail older service users to inform theme based inspection • Search social media to collect information from individual patients as signals in risk based supervision • User panels on specific regulatory topics • User involvement in multi annual policy plan • Advisory board of children (Children’s Council) • User involvement in designing supervision frameworks • Sharing information about the inspectorate to the public • Use patients as a source of information for planning and conducting theme based inspections • Interview adolescents represented in a youth council as part of theme based inspection • Young peer-inspectors in inspection team to interview young people • Experts-by-experiences (with learning disabilities) involved in the entire inspection process of theme based inspection • Experts-by-experience were trained and involved in inspections in elderly care homes • Mystery-guests with learning disabilities used in review of access of services (experts by experience show up in institutions under-cover and inform inspectors about their experiences) • Mystery guests used in reviews of elderly home care • Search social media to collect information about organizations as signal in risk based supervision • The ratings and reviews of an independent patient rating website are used by inspectors in identifying risks or themes or to prioritize their visits • User panels on specific regulatory topics • User involvement in multi annual policy plan • User involvement in multi annual policy plan • Advisory board of children (Children’s Council) • Mystery-guests with learning disabilities used in review of access of services (experts by experience show up in institutions under-cover and inform inspectors about their experiences) p p • Mystery guests used in reviews of elderly home care • Search social media to collect information about organizations as signal in risk based supervision • The ratings and reviews of an independent patient rating website are used by inspectors in identifying risks or themes or to prioritize their visits Collective/reactive • Analyses of complaints and concerns from various sources as part of its risk-based supervision of providers, called “intelligent monitoring” • Analyses of complaints and concerns from various sources as part of its risk-based supervision of providers, called “intelligent monitoring” Page 7 of 15 Wiig et al. BMC Health Services Research (2020) 20:616 Table 4 The Netherlands Collective/reactive • Patient complaints • Regulatory requirement for involvement of patient/next of kin in incident investigation • Contact next of kin after sentinel events to identify if they have been involved in the required investigation by the healthcare organization • Patient complaints • Regulatory requirement for involvement of patient/next of kin in incident investigation • Contact next of kin after sentinel events to identify if they have been involved in the required investigation by the healthcare organization • Patient complaints • Regulatory requirement for involvement of patient/next of kin in incident investigation • Contact next of kin after sentinel events to identify if they have been involved in the required investigation by the healthcare organization • Inspecting of how healthcare organizations involve patient and next of kin in incident investigation • Inspecting of how healthcare organizations involve patient and next of kin in incident investigation g • Aggregated information from complaints on sector and themes is used in risk based supervision for agenda setting and prioritization • Text mining research is performed to find relevant topics in complaints g • Aggregated information from complaints on sector and themes is used in risk based supervision for agenda setting and prioritization • Text mining research is performed to find relevant topics in complaints with autism, people with a migrant background, elderly and people with learning disabilities are tar- geted through qualitative methods. Such methods included the development of digital tools for com- munication with children under 13, and experi- menting with including interpreters in system audits of under aged refugees in child protective service institutions. The synthesis identified specific efforts among the regulators to involve and get in contact with vul- nerable groups with increased risk of not being heard through routine approaches. For example, in some Australian states, surveys are specifically di- rected at ‘hard to reach’ groups (with low response rates, however). In other countries, hard to reach groups such as young asylum seekers, young people Table 5 Australia Individual/proactive Table 5 Australia Individual/proactive Collective/proactive • Patient survey at the state level about recent experiences and outcome of care • REACH – Recognize, Engage, Act, Call, Help is on its way: state initiative to empower consumers to ‘speak up for safety’, engage with their nurses or medical team, and request clinical review within 30 min • Consumers members of clinical governance committees • Involvement of consumers in accreditation standard development • Accreditation standard requires healthcare organizations to partner with consumers in planning, designing, measuring, delivery and evaluation of care • Co-surveyors where consumer are involved in the accreditation process as team members • Consumer involvement in regulation of healthcare research – 2 members are laypersons in all Human Research Ethics committees • Collaborative Pair Program – National program to promote meeting of accreditation standards requiring patient and consumer involvement. Support clinician-patient approaches. Individual/reactive Collective/reactive • Regulatory requirement for investigation most severe adverse events by a formal root cause analysis (RCA). Interviews with patients and next of kin may be part of the RCA. ◦Apology to patient and family as part of open disclosure in RCA process • Consumer, patients, families can submit complaints to ombudsman Collective proactive methods of involvement We identified varia- tions on the expert-by-experience approach in different fields. Examples included adolescents involved in the in- spection team to interview adolescents in thematic in- spections, co-designing regulatory frameworks, or involvement of people with learning disabilities and next of kin in an investigation process. Moreover, the synthe- sis uncovered examples of older people being trained and included in the inspection process in elderly care homes. Similarly, the “mystery guest” method was ap- plied in the Netherlands. Mystery guests are a subset of the experts-by-experience approach, where potential ser- vice users visited service providers “undercover” and evaluated aspects of the services given. For example, people with learning disabilities assessed the accessibility of services as mystery guests. Another example of the ways in which mystery guests were used, was consider- ing whether the information they received from the ser- vice provider would be easy to understand for the group they represented. Based on these experiences the people with learning disabilities, service providers and inspec- tors discussed the accessibility of the services, what in- formation they wanted to keep and what could be improved. In this particular example, managers were also later involved. The regulator used this information in the assessment of the service provider. g In this category, we also found methods of involvement in the regulatory investigation of adverse events. A regula- tory investigation may be initiated based on patient com- plaints, or by mandatory reporting of adverse events from the service providers to the regulator. In Norway, results showed several examples of individual involvement methods in the investigation process. In the most severe cases (e.g., deaths), investigated by the NBHS at the na- tional level, the regulator always consults with the family, informs them about the progress and collects their views on the event. Moreover, there were examples at the county level in Norway of regulators organizing formal face-to-face meetings between regulatory inspectors and next of kin to collect information about the adverse event leading to patient death from the next of kin’s perspective. In the Netherlands and Australia, we identified regulatory requirements for service providers to conduct investiga- tions of adverse events, but the regulator does not usually investigate these. Collective proactive methods of involvement Collective proactive methods of involvement Individual reactive methods of involvement refer to how regulators involve individuals (patient, user, or next of kin), when they have experienced either an adverse event or filed a complaint about the service provision to the regulator. The Netherlands, Australia and Norway have regulation enabling users, or next of kin, to file com- plaints to the regulator or an ombudsman, or both. This legal right is one way of involvement in itself, but we also sought to identify if, and how, people were involved in the regulatory process after filing a formal complaint. In the included countries, a complaint can relate to both lack of service provision and being denied a service. In addition, complaints can include reporting experiences of adverse events, near misses, or patient harm. We found examples from Norway where the regulator estab- lished meeting arenas between regulatory inspectors, service providers, and users or next of kin. The purpose was to gather the involved parties and through a dialogue-based approach, try to solve each complaint case so it did not proceed to a formal and often long- lasting written information exchange process between the parties before reaching a conclusion. In collective proactive methods of involvement, the in- volvement is not so much focused on a patient’s specific case or treatment, but more generally. Users involved are expected to represent a group of interests. The pur- pose is to inform the future regulatory agenda or specific inspections. The collective and proactive methods of in- volvement demonstrated a wide repertoire in all the in- cluded countries. England and the Netherlands were at the forefront in utilizing a range of approaches. Methods identified in this category related to involvement of user panels and user organizations, campaigns, expert-by- experience and mystery guests, data sharing, and commissioning of research. Across countries, the most common method of this type is using experts-by-experience, peer-inspectors (in inspection processes) or co-surveyors (in accreditation processes). These methods consider users, patients and family members to be experts on care, which warrants them being a part of planned inspection activities (or ac- creditation surveys in Australia). The degree of involve- ment varied from being involved in an inspection planning meeting, to being part of the inspection team as co-investigator on site and in the analysis of results. Experts-by-experience were reported to be involved in thematic reviews and system audits. Individual/reactive • Consumer, patients, families can submit complaints to ombudsman Page 8 of 15 Wiig et al. BMC Health Services Research (2020) 20:616 Page 8 of 15 Wiig et al. BMC Health Services Research (2020) 20:616 Collective proactive methods of involvement However, since both countries require service providers to involve patients and the families in the investigations, this implies that one way of prompting user involvement is by regulating and requiring the service providers themselves to involve patients and family in investigations (as described in the introduction of this paper). In addition, Norway has recently made it mandatory for health service providers to invite patients and users to a meeting after a severe adverse event. In addition, in long-term care, the Dutch inspectorate experi- ments with checking directly with next of kin whether the service provider has involved them in the investigation process. Our synthesis found that user panels and user advisory groups were common in the regulatory bodies across countries. In England, the CQC has user panels to Page 9 of 15 Wiig et al. BMC Health Services Research (2020) 20:616 Page 9 of 15 Wiig et al. BMC Health Services Research (2020) 20:616 provide input and advice regarding the regulation of children and mental healthcare services. In Norway, the NBHS recently established a user panel at the national level to inform all regulatory activity. Several Norwegian regional regulatory offices have established user panels. The Dutch inspectorate organizes user panels on specific regulatory topics and is investigating the possibility of a structural user advisory board. Furthermore, the Dutch inspectorate involved users in developing their multi an- nual policy plan ‘20–23’, and the Youth Care department of this inspectorate has been advised by a Children’s Council in 2019. Similar methods that focus on involve- ment in committees and panels are human research ethic committees and clinical governance committees which are established in Australia. In England, the CQC commissioned research with users. strategic effort to improve user involvement in regula- tion by different means, such as funding innovation pro- jects to test new ways of approaching user involvement in regulation. As a result of the four-year involvement program, the NBHS developed national recommenda- tions for user involvement in regulation. Lessons learnt and strategic actions were summarized and the recom- mendations related to methods, experiences, and how regulators can and should involve users in their own regulatory activities and their training of inspectors. The recommendations underline patient and user involve- ment as a core value for health service provision and for regulatory bodies. Collective reactive methods of involvement This category relates to collective involvement after healthcare has failed, in terms of adverse events or com- plaints, but implies that it is not related to an individ- ual’s own specific case. The collective reactive methods repertoire was limited in all countries. Nevertheless, we note some direct examples whereby the Dutch inspect- orate aggregated the information from complaints col- lected at the National Healthcare Report Centre by sector and by theme. Also, the Dutch inspectorate per- formed an explorative pilot on text mining the content of the complaints for relevant topics. Similarly, in England, the CQC analyses complaints and concerns from various sources. This information is used as a signal in risk-based regulation for agenda setting and prioritization in both countries. The regulators also collaborated and organized semi- nars with user groups, councils, charities, and organiza- tions to collect information and experiences as part of the planning of inspection activities. Some of these activ- ities were co-produced by the user organizations and the regulatory bodies. We also found examples of organizing co-investigator workshops to collect experiences and les- sons learnt from the experts-by-experience who had been involved in some kind of regulatory activity. g y y In this category, we also identified virtual involvement initiatives and campaigns using social media, digital mar- keting, and other online platforms to encourage patients and families to share their knowledge and experiences with the service providers. Other initiatives online re- lated to disseminating general information to user groups about the regulators’ role and responsibility. In the Netherlands, the ratings and reviews of an independ- ent patient rating website are used by inspectors to iden- tify risks and themes for theme based regulation and to prioritize their visits. The Dutch inspectorate also searches social media for signals from users about the quality of care in the health services they visit. Further- more, we identified an English initiative where CQC had established a public online community for involvement in health policy and service design. This included both a representative panel and self-selected groups. The CQC additionally took advantage of a data sharing partnership with other websites collecting intelligence from users that is then used by the regulator. Reported difficulties concerning participation This argument is not only used to support the active involvement of patients and families, but also to send information through, for ex- ample social media, about the work of the regulator to the public [55, 56]. Third, involvement is a way of achieving justice for those affected. This especially applies to the involvement of patients and families during incident investigations as shown in Norway, the Netherlands and Australia. Here their involvement is a way of regaining trust and restor- ing the therapeutic relationship, including the opportun- ity to apologize [13, 14, 32, 48]. Moreover, involvement allows for the provision of information to the patient and family, as well as space to share emotions and pro- vide aftercare [13, 14, 16, 32, 33, 40, 48, 59, 60]. Also, some reports mentioned that through involvement escal- ation of issues to legal claims could be avoided [16, 40]. The need for justice for those affected by poor care can also be found in relation to other methods. For example, in a case of conducting interviews with the elderly, it was shown that participants appreciated the genuine interest in their perspective and felt it was positive to be heard [61]. This was also found in relation to involve- ment of next of kin in the regulatory investigation of ad- verse event when patients had died. Next of kin expected to be involved and evaluation showed that in- volvement could have a therapeutic effect [13]. The regulatory inspectors in this case found involvement to be in accordance with overall political expectation [14]. Fourth, involvement can be a way for users to em- power themselves and learn new skills [12, 17, 41, 49, 50, 54]. For example, experts-by-experience learn a lot Second, not all patients or family members want to be involved or are easy to involve. For example, in case of incident investigations it has been reported that partici- pation is too burdensome as the incident can have a large impact on patients and families [16, 32, 33, 48]. In other cases, it is difficult to do justice to the diversity of patients, as some groups are more inclined to participate than others. This leads to questions concerning the rep- resentativeness of those involved and for whom they can speak [5, 48]. This is also an important difficulty at- tached to professionalization attempts of participants. For example, experts-by-experience are often trained for their task. Reported difficulties concerning participation p g p p Along with the possible benefits reported above, the re- sults also show a number of difficulties experienced by regulators when putting involvement into practice. First, it can prove difficult to incorporate the input of patients and families into the decisions or reports of the regula- tor [6, 15, 16, 32, 40, 48]. This has partly to do with the perceived lack of legitimacy of patient or family input. It was regularly argued that patients lack the necessary knowledge to contribute, and less weight is given to their input than, for instance, the input of professionals (e.g., in case of incident investigations, theme based inspec- tions or experts-by-experience) [6, 12–14, 16, 32, 35, 48, 51]. As described earlier, the main argument for involve- ment is that patients and families provide additional in- formation. However, in cases of conflicting input (e.g., different views on what an incident is, what should be the focus of the investigation or what should be consid- ered good quality care) the contribution of patients and family appears to be difficult to incorporate [6, 16, 48]. As a result, there is a danger that involvement becomes tokenistic [44, 45]. A recent example from Dutch re- search tackles this conflict by making the client perspec- tive the starting point for regulation. Inspectors followed the client’s perspective and judgements throughout the whole inspection process [54]. However, possible con- flicts are likely to persist, and the question remains “how will inspectors make informed judgements in such cases in order to do justice to the complexities of regulatory practice”? Second, involvement is said to legitimize decision- making of the regulator by using information gathered from patients and the family in the regulatory assess- ment, but also by co-producing inspection criteria [5, 6, 54]. This relates to the goal of democratic decision- making. For example, when patients and the families support the findings of the regulator this is added to regulatory reports [6, 12, 54]. Patient and family involve- ment is also related to being transparent about the work of the regulator, and connecting with patients or the public more generally improves the image of, or trust in, the regulator [5, 13, 14, 48]. Reported benefits of involvement Also, by analyzing reviews written on an independent patient rating website and social media posts, the regulator may identify risks from the pa- tient’s perspective [35–37, 55–58]. Reported benefits of involvement The above synthesis shows the varied nature of user par- ticipation in regulation in terms of the type of regulation and the type of involvement. Not all the initiatives men- tioned above have been evaluated. Evaluation practices differ between countries. Comparatively, most practices have been evaluated in the Netherlands where the in- spectorate works with researchers in an academic collab- orative. The practices that have been evaluated do offer important insights into the main reported benefits and challenges of involvement practice. First, the primary espoused reason for involvement is improving regulatory work, and by consequence improv- ing the quality and safety of care [5, 16, 17, 32–47]. In this view, patients and families are seen as an additional information source. In case of incident investigations for example, patients and families are able to put incidents into a broader perspective and offer a more holistic un- derstanding of what happened [13, 14, 16, 32, 33, 48]. The espoused reasons are similar for the input they pro- vide during thematic inspections [17]. Similarly, experts- by-experience can provide important knowledge as they are more able to tap into experiences of patients, The results show collective user involvement for pro- active purposes in Australia in accreditation requiring healthcare organizations to partner with consumers in planning, designing, measuring, delivering and evaluat- ing care. A final example in this category is the national recommendations for user involvement in regulations in Norway. Results from Norway suggest a large emphasis on user involvement in regulation over the previous four-year period (2014–2018), where the NBHS made a Wiig et al. BMC Health Services Research (2020) 20:616 Page 10 of 15 Wiig et al. BMC Health Services Research (2020) 20:616 about a specific subject (e.g., elderly care, alcohol mis- use) and the work of the inspectorate [17, 49, 52]. about a specific subject (e.g., elderly care, alcohol mis- use) and the work of the inspectorate [17, 49, 52]. especially in the case of sensitive subjects such as alcohol misuse amongst adolescents [12, 15, 49, 50]. Experts-by- experience and related methods also offer another view on quality of care by focusing more on relations and ‘softer’ aspects of service provision (such as qual- ity of the food, time spent outside, decorative aspects of living facilities) rather than concentrating on safety [15, 51–54]. Unpacking the landscape of user involvement in regulation As studies into patient and family involvement in regula- tion are scarce (e.g., [5, 9]) one could question whether regulatory bodies have been part of the general trend in healthcare to involve patients and family members in decision-making. Our study reveals that regulators are very much part of this development. They are in fact experimenting with a wide variety of user, patient, and family involvement methods. Although the context, way of organizing and regulatory environment varied by country, and some regulators are experimenting more intensively with involvement than others, we saw a var- iety of methods in each of our cases. Regulators used methods in all four categories identified in the paper. g Fourth, organizational procedures can stand in the way of involvement. This includes the language used by inspectors, and is also due to certain protocols [6, 16, 32]. For example, incident investigations need to happen in a specific timeframe, the deadline of which can be too soon for the patients or family to be able to participate [16, 32, 33]. Also, the regulatory context can prohibit taking the input of patients on board. The case of youth involvement in a thematic inspection on care for chil- dren growing up poor by the Dutch inspectorate is a case in point. The interviewed youths stated that they felt their privacy was very important and therefore pro- fessionals should not share information about them with each other. In this case part of this conflict was influ- enced by the regulatory context as policy makers and regulators put much emphasis on sharing information in response to fatal incidents, which were analyzed as resulting from a lack of sharing information [6]. The study shows that the benefits of involvement are multi-faceted. Reported advantages from the published empirical research include for example increased quality of regulatory practice – because it is informed by the unique experiences and insight of people who use care services. Moreover, patient and family involvement in- creased legitimacy, empowerment, and contributed to justice done to those affected by adverse events (see also [13, 14, 54]). We thus found that involvement can serve multiple purposes – such as in strengthening the quality of regulatory practice, and being beneficial for the users, patients and families involved [13, 14]. Unpacking the landscape of user involvement in regulation In addition, in- volvement can be used as an instrument to prevent fur- ther escalation of problems, through formal regulatory investigation processes or legal claims [11, 16]. A final difficulty was identified as dealing with the emotions of those involved. This not only applies to the emotions of patients and family members, but of the professionals and regulators themselves, who can also be affected by participation. Emotions can be felt especially keenly during incident investigations [13, 14, 16, 48]. For the next of kin who have lost a close relative, it can be a considerable mental strain to be part of the entire inves- tigation process when they are grieving and sometimes traumatized. The investigation process can repeatedly remind them of the event leading to the death. However, the interpersonal skills of the inspectors may help to re- duce this emotional burden. From the inspectors’ point of view, it can be emotionally challenging to involve next of kin in investigation meetings, because the inspectors can worry about attending beforehand and might con- tinue to dwell on things after the meeting, especially in severe cases. Some inspectors have a legal professional background where they were not trained for face-to-face meetings with people in grief, as inspectors with a healthcare background may have been. At the same time, inspectors report that it is a positive experience to offer support to the next of kin and clarify any misun- derstandings or questions [13, 14, 48]. In other cases emotions can play a role as, for example, a project Despite this reported added value, our study also iden- tified challenges of user involvement in regulation. It is not easy to develop a regulatory culture where involve- ment is meant to be integrated into work practice. Also, it is not clear how to use information from experts-by- experience. This is especially important if involvement is conducted to comply with political expectations and not as a way of improving the quality of regulatory activities [40, 62]. In many cases, information provided by users introduces an additional, and different, perspective, but this also means that it can clash with the perspective of, for instance, professionals or regulators [6, 16, 48, 63]. Our findings suggest that the input from users has sometimes been put aside by questioning its legitimacy. Reported difficulties concerning participation This may ultimately diminish the value of the authentic perspective that they can provide [12]. This leads us to the third difficulty we identified, that participation involves time and costs [5, 13, 14, 33, 34, 40, 48, 54]. This is especially true for practices directed at so-called ‘hard to reach’ groups because the regulator must expend significant time and effort to come into contact with them, and attune participation to their needs. User participation in general is also expensive. Page 11 of 15 Wiig et al. BMC Health Services Research (2020) 20:616 Wiig et al. BMC Health Services Research (2020) 20:616 For example, the CQC’s Experts By Experience program has cost an average of £4 million annually in recent years (CQC news). Inspectors in a pilot project in Norway sometimes found it time consuming to involve next of kin due to difficulties in scheduling meetings and the need for additional follow up contacts to collect new information from, for example, actors newly implicated in contributing to the cause of an adverse event. Inspec- tors did not recommend continuing this involvement method unless it was compensated properly in the work schedule and incorporated in the organization [48]. involving mystery guests, where civil servants providing services to people with learning disabilities reported feel- ing left out as their perspective was not sufficiently taken into account [54]. Strengths and limitations Several researchers participated in the data collection, which made it possible to include information from vari- ous countries and sources. This, however, may also have created variation in data sources. This limitation was moderated by using the template and having numerous discussions between authors. There are also ways to include users, patient, and fam- ily perspectives based on existing information which can be exploited [5]. Examples of such involvement are 1) to use social media as a way to trace problems in health- care, 2) to aggregate information from patient com- plaints, or 3) to perform text mining from patient complaints to search for new types of risks and topics for future inspections [35–38, 57, 58, 70–72]. While uniquely representing quality of care from a patient’s point of view, however, this requires further reflection on methodological issues such as the reliability and val- idity of these (public) resources. The regulatory regime is complex [74] and the avail- able data in each country varied, especially in terms of the number of published empirical studies. Most re- search on involvement in regulation has been conducted in the Dutch setting. We cannot be sure that this is a complete mapping, since involvement methods can be used in regulatory practice without being published or even identified as ‘regulatory practice’. We used data from different sources, such as internet websites, evalu- ation reports, and peer reviewed papers. This broad ap- proach was important in providing an overview of methods available, in addition to documenting possible advantages and disadvantages and lessons learnt that are relevant to other regulators. Another lesson is that involvement needs an embed- ding strategy. Regulators should think about how to sup- port involvement in their activities, including valuing the financial and time investments [69]. There is also a need to train inspectors such that they can reflect on how to judge different perspectives on quality and how to deal with the emotional burden that can accompany involved users [13, 14]. Good judgement on how and when to in- corporate the input of patients is also needed, if that in- put is to be productive. This might warrant changes in procedures and regulatory frameworks as patients and regulators can have different values and perspectives (e.g., what counts as an incident, how quality of care should be judged) [6, 16, 54, 63]. Unpacking the landscape of user involvement in regulation If this happens, it equates to epistemic injustice [64, 65] – especially if the knowledge base of users, patients, and family members is considered of less value than those of other actors. This can clearly occur if these extra Wiig et al. BMC Health Services Research (2020) 20:616 Page 12 of 15 Page 12 of 15 learning [2, 32]. An important question to reflect on is how to conduct training of users, patients, family mem- bers, and inspectors to succeed with involvement in regulation [12]. The danger is that such training may lead to the professionalization of patient input, which can distance the participants from their experiences as patients [62]. This is problematic because tapping into these experiences is often considered the most import- ant reason for involvement [2, 73]. perspectives do not fit with longstanding regulatory pro- cedures, or with views held strongly by professionals, managers and regulators [66–68]. Other challenges include that not all patients and fam- ily members want to be involved, involvement costs time and money, and it can pose an emotional burden on those involved (see also [13, 14, 69]). The emotional bur- den is especially pertinent to the individual reactive in- volvement category discussed above [5, 13, 14]. If user involvement in regulation is a question of morality or logic [8] then how regulators deal with the challenge of emotionality, or epistemic injustice [64], should be fur- ther investigated to understand the rationale and experi- ences of regulatory bodies. Finally, it is important to make participation user- friendly and to ensure inclusiveness. In England, much effort is used in making forms and websites accessible, and in building relationships and trust with people from vulnerable groups. Our findings indicate that involving ‘hard to reach’ groups might be challenging, but it can be done [74]. All-in-all, further investigation is needed to evaluate targeted involvement methods of hard to reach groups. There are a number of lessons for regulatory practice. A clear lesson is to target involvement activities and to take advantage of existing opportunities [5, 6, 54]. Since participation is time-consuming for all involved, it is im- portant to consider for which situations it is most im- portant, and to identify the best ways to go about it. The case of using mystery guests with learning disabilities in the Netherlands is a good example in this regard [17, 54]. Unpacking the landscape of user involvement in regulation The mystery guests were asked to assess the access to municipal services for people with learning disabil- ities, an assessment which can only be adequately made through experience. Strengths and limitations In this regard, it should be emphasized that participation on its own does not ne- cessarily lead to increased patient-centeredness or We categorized methods according to the proactive- reactive and individual-collective dimensions inspired by Tritter’s [1] published framework. We used the dimen- sions as a heuristic tool to gain insight into the variety of methods. Tritter’s [1] original work on reactive and pro- active involvement distinguished between whether par- ticipation is responding to a pre-existing agenda, in our case set by a regulator (reactive), or if the participants are helping to shape it (proactive). We have added to this conceptualization by including: 1) if participants have experienced an adverse event and responded ac- cording to the regulators’ procedures for follow up (re- active), or 2) if the participants have not experienced an adverse event, but provide information to the regulator Page 13 of 15 Page 13 of 15 Wiig et al. BMC Health Services Research (2020) 20:616 Wiig et al. BMC Health Services Research (2020) 20:616 based on their experiences, to influence future regulatory activities (proactive). On the one hand, this illustrates the strengths, showing that with a few changes, Tritter’s [1] framework may be broadly applicable, including in the regulatory context. On the other hand, the slight de- viance from the original version could be considered a limitation. instance via the European Partnership for Supervisory Organisations in Health Services and Social Care (EPSO) or the Supervision and regulation Innovation Network for Care (SINC) that inspectorates from various coun- tries established recently. Further development of similar collaboration arenas should be stimulated. As the issue of user involvement is high on the agenda of many regu- lators, it underscores the importance of this collaborative research agenda. based on their experiences, to influence future regulatory activities (proactive). On the one hand, this illustrates the strengths, showing that with a few changes, Tritter’s [1] framework may be broadly applicable, including in the regulatory context. On the other hand, the slight de- viance from the original version could be considered a limitation. We do not know whether the diversity we found in methods relates to country characteristics, regulatory re- gimes or other factors. Further studies might continue investigations along these lines. Examining a number of different countries with diverse regulation systems was a strength as it enabled us to identify a variety of ways of involving users. Acknowledgements The authors would like to thank Jill Morrell, Head of Public Engagement at the Care Quality Commission (CQC), for helping the research to identify and understand the range of involvement methods used by CQC. The views expressed in this paper are those of the authors, and are not necessarily those of Jill, or of the Care Quality Commission. Any errors are solely the responsibility of the authors. Strengths and limitations However, it also made it difficult to de- termine to what extent differences in user involvement were the result of the different countries’ regulatory systems. Conclusion Our mapping of user involvement methods brought into focus a broad variety of methods. These can serve as in- spiration to regulators in healthcare. Based on our map- ping exercise we suggest that future regulatory practice continues to develop and pilot new types of user in- volvement methods including individual and collective, and proactive and reactive activities. The paper shows that making involvement in regulation successful is a challenging and complex task. Our findings suggest that it is not easy to reach the goals of increased involvement and democratic decision-making. The fact that regula- tors are experimenting with different methods can be valued positively in this regard. As a result, regulators have room to innovate and evaluate work involving new kinds of groups (e.g., older people, youths, people with learning disabilities); new topics and areas of inspections (e.g., social care, elderly care, care transition); different degrees of involvement (e.g., involved in a meeting vs part of inspection team during inspection processes); and new ways of engaging with users and service provision (e.g., mystery guests). Experimenting with these methods means that lessons can be drawn to im- prove involvement practices, such as how the perspec- tive of users can be incorporated in the judgements that regulators make or in accessing hard to reach groups. For this, further research into these programs is recommended. Authors would like to thank the two reviewers for valuable input to improve the paper. Finally, we would like to thank Kate Gibbons, PhD, Centre for Healthcare Resilience and implementation Science, Australian Institute of Health Innovation, Macquarie University, Australia for her help with proof reading and language editing. Abbreviations ACSQHC A ACSQHC: Australian Commission on Safety and Quality in Health Care; CQC: Care Quality Commission; EPSO: European Partnership for Supervisory Organisations in Health Services and Social Care; HYCI: Dutch Health and Youth Care Inspectorate; NBHS: Norwegian Board of Health Supervision; RCA: Root Cause Analysis; SINC: Supervision and regulation Innovation Network for Care ACSQHC: Australian Commission on Safety and Quality in Health Care; CQC: Care Quality Commission; EPSO: European Partnership for Supervisory Organisations in Health Services and Social Care; HYCI: Dutch Health and Youth Care Inspectorate; NBHS: Norwegian Board of Health Supervision; RCA: Root Cause Analysis; SINC: Supervision and regulation Innovation Network for Care Authors’ contributions SW and HvB had the idea, designed the study and the data collection framework, contributed to data collection, led the analysis and drafted the first version of the manuscript. Authors SR, SK, CHD, JO, AB, KC, and JB all contributed to data collection, analysis, and had significant contributions and commented on the manuscript in several iterations. All authors have approved the final version. Funding h d The study did not receive funding. The two research projects SAFE-LEAD primary care (grant agreement no 256681) and Resilience in Healthcare (grant agreement no 275367), funded by the Research Council of Norway, contributed to fund two research visits for author SW to Erasmus School of Health Policy and Management, Erasmus University, Rotterdam, the Netherlands (January–May 2019), and the Australian Institute of Health Innovation, Macquarie University, Sydney Australia (July–October 2019). The paper was drafted during these visits. JO is supported by funding from the National Institute for Health Research Yorkshire and Humber Patient Safety Translational Research Centre (NIHR Yorkshire and Humber PSTRC). JB reports funding from the National Health and Medical Research Council for grants: NHMRC Partnership Grant for Health Systems Sustainability (ID: 9100002); NHMRC Centre of Research Excellence grant (1135048); NHMRC Project grant (ID: 1143223) and NHMRC Investigator grant (ID: 1176620). AB reports funding from the Care Quality Commission to investigate CQC’s impact on the quality of care (ID: CQC PSO 140). Most research is seen in the Dutch context. However, these studies suggest that the promotion of collaboration between regulatory bodies and research groups to build a network for research-based-regulation and regulation- based-research, may help stimulate better research, regu- lation, and educational programs for future regulators. More international collaborations between researchers and inspectorates could provide further impetus, for Availability of data and materials All data are publicly available online and cited in the manuscript and in the reference list. References 24. 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https://openalex.org/W2990378819	https://www.frontiersin.org/articles/10.3389/fpls.2019.01485/pdf	English	null	Modeling Stem Water Potential by Separating the Effects of Soil Water Availability and Climatic Conditions on Water Status in Grapevine (Vitis vinifera L.)	Frontiers in plant science	2,019	cc-by	10,150	Keywords: grapevine, water status, stem water potential, predawn leaf water potential, maximum air temperature, modeling Original Research doi: 10.3389/fpls.2019.01485 published: 22 November 2019 Edited by: Eddo Rugini, University of Tuscia, Italy Reviewed by: Elena Brunori, The Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), Italy Hipólito Medrano, University of the Balearic Islands, Spain Reviewed by: Elena Brunori, The Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), Italy Hipólito Medrano, University of the Balearic Islands, Spain Correspondence: Cornelis Van Leeuwen vanleeuwen@agro-bordeaux.fr Correspondence: Cornelis Van Leeuwen vanleeuwen@agro-bordeaux.fr Specialty section: This article was submitted to Crop and Product Physiology, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Crop and Product Physiology, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Crop and Product Physiology, a section of the journal Frontiers in Plant Science Received: 04 September 2019 Accepted: 25 October 2019 Published: 22 November 2019 Modeling Stem Water Potential by Separating the Effects of Soil Water Availability and Climatic Conditions on Water Status in Grapevine (Vitis vinifera L.) Measuring seasonal plant water status is critical in choosing appropriate management strategies to ensure yields and quality of agricultural products, particularly in a context of climate change. Water status of grapevines is known to be a key factor for yield, grape composition, and wine quality. Predawn leaf water potential (PLWP) and stem water potential (SWP) proved to be simple and precise indicators for assessing grapevine water status and subsequent same-day spatial comparisons. A drawback of SWP is that it does not allow for temporal comparisons, because the measured value is impacted both by soil water availability and climatic conditions on the day of measurement. The objectives of this study are i) to provide a model that separates the effect of soil water content from the effect of climatic conditions on the SWP value and ii) to standardize the SWP value to a value under predefined reference climatic conditions in order to compare SWP values collected under different climatic conditions. SWP and PLWP were temporally assessed on three soil types in Saint-Émilion (Bordeaux, France) in 2015 and on five soil types in Margaux (Bordeaux, France) in 2018 using a pressure chamber. SWP measurements on two consecutive days with contrasting climatic conditions allowed to assess the impact of these conditions on SWP values. A large portion of the variability in SWP values was explained by PLWP. Model selection further showed that the addition of maximum air temperature and seasonality explained a significant amount of the remaining variability in SWP values. SWP values could be successfully standardized to a theoretical value under reference climatic conditions, which allows for temporal comparisons of SWP values. A plant-based measurement, such as the water potential, can be considered as the most straightforward indicator of plant water status as it integrates the effects of soil, plant, and atmospheric conditions. More precise interpretation of SWP values provides winegrowers with a tool to more adequately implement short- and long-term management strategies to adapt to drought in order to ensure yield and grape quality. INTRODUCTION measurement, is not only necessary when soil water is readily available (as in the baseline approach), but also under limiting soil water conditions. Fine-tuning the interpretation of SWP values across a wide range of water deficit would help growers in the short term to adapt to drought by means of irrigation, adjustment of the canopy size, or vineyard floor management, and in the long term by choosing appropriate plant material (rootstocks and cultivars) and planting densities (Chaves et al., 2007; Van Leeuwen and Destrac-Irvine, 2017; Van Leeuwen et al., 2019).i Climate change will result in an increase in temperature and an intensification of drought in many regions across the globe (IPCC, 2014). Measuring seasonal plant water status is an essential step in choosing appropriate adaptations in management strategies to ensure yield and quality of agricultural products in these changing conditions. The water status of grapevines is known to be a key factor for yield, grape composition at ripeness, and wine quality (Van Leeuwen et al., 2009). q y Several techniques have been developed to measure water status in plants. Among these, predawn leaf water potential (PLWP) and stem water potential (SWP) have proven to be simple and precise indicators for assessing plant water status (Choné et al., 2001; Williams and Araujo, 2002; Shackel, 2011; Santesteban et al., 2019). However, PLWP tends to overestimate soil water availability in conditions of heterogeneous soil humidity. Under these conditions, PLWP will equilibrate with the most humid soil layer and is therefore not related to the mean water status of the soil in the root zone (Améglio et al., 1999). Another drawback of PLWP measurements is that they must be measured before dawn, which does not make it practical to implement. SWP measurements overcome this problem as they are generally measured in the early morning, at midday or in the early afternoon. SWP measured in the early afternoon is more easily implemented as there is more time available to take readings under stable conditions than at early morning when environmental conditions change more quickly (Intrigliolo and Castel, 2010). The relation between SWP and PLWP in grapevine has been shown to follow a linear function under both irrigated (Williams and Araujo, 2002; Williams and Trout, 2005) and rainfed (Zufferey and Murisier, 2007) conditions. INTRODUCTION At the same time, SWP is known to decrease with increasing vapor maximum deficit (VPD), but this relationship has been demonstrated to change over the growing season (Olivo et al., 2009) and to be different among and within species (McCutchan and Shackel, 1992; De Swaef et al., 2009; Rogiers et al., 2009; Marchin et al., 2016), and irrigation treatments (Williams and Baeza, 2007; Gálvez et al., 2014).fi Grape cultivars have been classified as (near-)isohydric or (near-)anisohydric in accordance with their type of stomatal response (Chaves et al., 2010). Isohydric plants close their stomata when they sense a decrease in soil water potential or an increase in evaporative demand, while anisohydric plants allow leaf water potential to decrease with increasing VPD in order to continue gas exchange (Tardieu and Simonneau, 1998). Distinctions between the two strategies within grape cultivars, however, are often not easy to assess (Domec and Johnson, 2012). Working with previously reported (near-)anisohydry of Cabernet franc, Cabernet-Sauvignon, and Merlot (Chaves et al., 2010; Lovisolo et al., 2010; Domec and Johnson, 2012), we hypothesized the following: SWP is expected to decline with an increasingly warm and dry climate under non-limiting soil water conditions, and to become less responsive to high temperature and high VPD when soil water is limiting. Stomata close under water deficit conditions to maintain water potential above a critical minimum threshold (Hogg and Hurdle, 1997). As stomata progressively close under increasing water deficit, the impact of climatic variables on SWP values is expected to decrease.h The aims of this work are i) to provide a model that separates the effect of soil water availability from the effect of the climatic conditions on SWP value and ii) to correct the SWP value to a value under standard climatic conditions. The second objective yields a corrected SWP value that better reflects soil water availability. This value will be easier to use for strategic management decisions as it reduces the variability caused by the climatic conditions on the day of measurement. In order to more efficiently manage irrigation, non-stressed baselines of SWP as a function of VPD were established for prune, almond, and grapevine (McCutchan and Shackel, 1992; Williams and Baeza, 2007; Olivo et al., 2009). These baselines represent the upper boundary for steady-state water transport when soil water is readily available (Sperry et al., 2002). INTRODUCTION These baselines allow to define at which SWP values water uptake can be considered as non-limiting across a range of climatic conditions. Shackel et al. (2000) showed that this approach can reduce the use of water for irrigation while maintaining productivity in prune trees under non-limiting soil water conditions. These baselines, however, are less useful for water management in grapevines, because significant areas of grapevines are dry-farmed and a water deficit is considered favorable to wine quality, as long as it remains moderate (Van Leeuwen et al., 2009). For the same reason, even irrigated grapevines are often maintained at moderate water deficit (Matthews and Anderson, 1988). Hence, interpretation of SWP, while considering the climatic conditions at the day of Citation: Suter B, Triolo R, Pernet D, Dai Z and Van Leeuwen C (2019) Modeling Stem Water Potential by Separating the Effects of Soil Water Availability and Climatic Conditions on Water Status in Grapevine (Vitis vinifera L.). Front. Plant Sci. 10:1485. doi: 10.3389/fpls.2019.01485 November 2019 \| Volume 10 \| Article 1485 1 Frontiers in Plant Science \| www.frontiersin.org Modeling Stem Water Potential Suter et al. Frontiers in Plant Science \| www.frontiersin.org Experimental Set-Up Field experiments were carried out in 2015 (from 8 June to 9 September) in the Saint-Émilion appellation and in 2018 (from 20 June to 14 September) in the Margaux appellation in the Bordeaux area, France. Experimental plots were planted with Vitis vinifera (L.) cv. Merlot, Cabernet franc, and Cabernet-Sauvignon in dry-farmed commercial vineyards. Grapevines were Guyot pruned and trained with a vertical shoot positioned trellis. Plots were chosen to ensure a large range of grapevine water uptake conditions. A soil pit study allowed for the characterization of soils and grapevine-rooting profiles. In Saint-Émilion (plots M to R), soils were highly variable in texture and coarse elements content (Table 1). The sandy soils had a water table within the reach of the roots. At each measurement, leaves were sampled from eight adjacent grapevines. The 2015 water potential values November 2019 \| Volume 10 \| Article 1485 Frontiers in Plant Science \| www.frontiersin.org 2 Modeling Stem Water Potential Suter et al. are the average from these eight grapevines. In Margaux, plots had been selected based on historical SWP measurements from 2009 to 2017 (data not shown). The data could be distributed into three SWP thresholds, as proposed by Van Leeuwen et al. (2009). Within these thresholds three soils were chosen for the experiment. Five grapevines were selected in each plot being at least five grapevines away from the border. Measurements were always executed on the same grapevines for as long as there were enough primary leaves. Later in the 2018 season plots J, K, and L were added in order to obtain a larger range of PLWP potential values. Consequently, less measurements have been performed on these plots. The maximum distance between the experimental plots in Saint-Émilion and Margaux were 500 and 1,700 m respectively. Given the small differences in altitude and the absence of slopes, all plots were considered as located in homogeneous climatic conditions. Plant and soil properties of each plot are presented in Table 1. Appellation Plot Cultivar1 Rootstock Year of planting Grapevines per ha (spacing in m) Row orientation Depth (cm) Clay (%) Silt (%) Sand (%) Gravel (%) CEC (Metson Cmol (+)/kg) No. Experimental Set-Up of measurements Margaux A CS SO4 + 3309 C 1969 10,000 (1 × 1) NE-SW 0–100 21.9 21.5 56.6 24.5 7.89 92 Margaux B M 161–49 C 1959 10,000 (1 × 1) N-S 0–125 14.3 12.7 73.0 53.1 4.52 92 Margaux C M 101–14 MG 1944 10,000 (1 × 1) N-S 0–120 8.4 8.5 83.2 58.6 4.10 92 Margaux D CS 101–14 MG 2002 9,091 (1 × 1.1) E-W 0–130 11.4 12.6 76.0 59.4 5.38 91 Margaux E CS 101–14 MG 1966 10,000 (1 × 1) N-S 0–100 21.3 24.6 54.0 5.0 8.03 91 Margaux F CS RGM 1986 10,000 (1 × 1) N-S 0–75 33.4 47.2 19.4 8.1 10.74 92 Margaux G CS NA2 NA 10,000 (1 × 1) N-S 0–70 4.7 14.6 80.7 26.4 3.46 92 Margaux H CS 101–14 MG 1999 10,000 (1 × 1) E-W 0–125 15.0 11.0 74.0 43.1 3.76 91 Margaux I M SO4 1974 10,000 (1 × 1) E-W 0–100 21.3 24.6 54.0 5.0 8.03 92 Margaux J M SO4 1972 10,000 (1 × 1) N-S 0–125 14.3 12.7 73.0 53.1 4.52 40 Margaux K M 101–14 MG 1992 10,000 (1 × 1) E-W 0–125 15.0 11.0 74.0 43.1 3.76 50 Margaux L CS 101–14 MG 1999 10,000 (1 × 1) E-W 0–60 8.1 17.1 74.8 53.6 8.37 50 Saint-Émilion M CF NA 1940 6,410 (1.2 × 1.3) N-S 0–100 42.0 30.6 27.4 5.6 14.90 16 Saint-Émilion N CF RGM 1997 5,830 (1.2 × 1.4) N-S 0–100 9.0 19.6 71.5 55.4 6.78 16 Saint-Émilion O CF 101–14 MG 1963 5,830 (1.2 × 1.4) N-S 0–100 5.4 10.2 84.4 0.0 3.99 16 Saint-Émilion P M RGM 1989 5,830 (1.2 × 1.4) N-S 0–100 35.5 27.1 36.7 5.8 15.58 16 Saint-Émilion Q M 420A MG 2012 7,890 (1.0 × 1.3) N-S 0–100 6.55 12.6 81.3 46.4 3.60 16 Saint-Émilion R M RGM 1989 5,830 (1.2 × 1.4) N-S 0–100 15.1 20.8 63.3 4.6 7.08 16 Climatic Data Air temperature, relative humidity, global radiation, average wind speed, and rainfall were recorded during both experiments by the nearest meteorological stations (Table S1). The weather stations in Saint-Émilion and Margaux were within 500 and 1,460 m from the experimental plots, respectively. The weather station of Météo-France in Saint-Émilion recorded all required data. The weather station in Margaux recorded only temperature, relative humidity, and rainfall. Global radiation data was retrieved from a weather station in Saint-Julien (distance from plots < 17.7 km) and average wind speed data from a weather station (La Crosse Technology, WS 2355, France) in Arsac (distance from plots < 5.1 km). The weather stations in Margaux and Saint-Julien were part of the CIMEL automated DEMETER network. The Tmax data from the weather stations in Saint-Julien and Arsac correlated well with that in Margaux and followed a 1:1 line over the whole 2018 season and the sampling days in particular (r2 = 0.98, r2 = 0.94, r2 = 0.99, and r2 = 0.99, respectively). The weather stations in Saint-Julien and Arsac were located in an area with a very similar climate and grapevine phenology compared to that of Margaux (Bois et al., 2018). Reference evapotranspiration (ET0) was calculated according to the FAO-56 Penman-Monteith equation (Zotarelli et al., 2010). Growing degree days (GDD) were calculated with a base 0°C and summed starting from 1 April (around budbreak). The maximum VPD (VPDmax) was calculated using the equations described by Abtew and Melesse (2013), using the variables Tmax and minimum relative humidity. Statistical Analysis y Relationships between SWP, PLWP, and climatic variables were estimated via nonlinear modeling. All analyses were performed in R (R Core Team, 2017). Nonlinear models were fitted using the R function nls and models were compared using Akaike’s Information Criterion (AIC) and the Bayesian Information Criterion (BIC). The performance of each model was evaluated by the calculation of the root-mean-square error (RMSE). RMSE is the distance, on average, of a data point from a fitted line, measured along a vertical projection. A cross-validation method was applied to assess the predictive accuracy (in RMSE) of the models. A single plot was retained as the validation data for testing the models, and the remaining plots were used as training data. This procedure was repeated eighteen times (each of the eighteen plots was retained once). The assumptions of normality and equal variance were checked by quantile plots and plotting standardized model residuals against fitted values, respectively. The standardized residual plots of the models were found to be homoscedastic. Multicollinearity did not occur in any of the models (variance inflation factors < 1.2). FIGURE 1 \| Seasonal pattern of stem water potential and predawn leaf water potential on the primary axis, and precipitation and Tmax on the secondary axis in (A) Saint-Émilion in 2015 and (B) Margaux in 2018. Tmax at the days of sampling are specifically represented by the gray circles. Vertical arrows represent dates of mid-veraison for Merlot (30/07) and Cabernet franc (08/08) in 2015, and Merlot and Cabernet Sauvignon (08/08) in 2018. Values are averages over plots as described in Table 1. Error bars indicate standard deviations. FIGURE 1 \| Seasonal pattern of stem water potential and predawn leaf water potential on the primary axis, and precipitation and Tmax on the secondary axis in (A) Saint-Émilion in 2015 and (B) Margaux in 2018. Tmax at the days of sampling are specifically represented by the gray circles. Vertical arrows represent dates of mid-veraison for Merlot (30/07) and Cabernet franc (08/08) in 2015, and Merlot and Cabernet Sauvignon (08/08) in 2018. Values are averages over plots as described in Table 1. Error bars indicate standard deviations. recovered (Figure 1A). From mid-August 2015 water deficit started to increase again. In Margaux in early June 2018 all plots were at or close to field capacity and as the season progressed both SWP and PLWP decreased (Figure 1B). Water Potential Measurements SWP values were measured with the pressure chamber (SAM Précis 2000, 33170 Gradignan, France) technique on sun- exposed and fully expanded leaves which were enclosed in an opaque plastic bag for more than 1 h to prevent transpiration and allow to reach an equilibrium with water potentials in stems (Choné et al., 2001). SWP was measured on leaves up to the sixth internode at solar noon until the early afternoon (13h30 to 17h00 local time). The operator was the same throughout the experiment to reduce human error (Levin, 2019). In this study, November 2019 \| Volume 10 \| Article 1485 3 Modeling Stem Water Potential Suter et al. FIGURE 1 \| Seasonal pattern of stem water potential and predawn leaf water potential on the primary axis, and precipitation and Tmax on the secondary axis in (A) Saint-Émilion in 2015 and (B) Margaux in 2018. Tmax at the days of sampling are specifically represented by the gray circles. Vertical arrows represent dates of mid-veraison for Merlot (30/07) and Cabernet franc (08/08) in 2015, and Merlot and Cabernet Sauvignon (08/08) in 2018. Values are averages over plots as described in Table 1. Error bars indicate standard deviations. the PLWP measurement was considered a proxy for the soil matrix potential experienced by the roots. Despite the limitations of PLWP as reported by Améglio et al. (1999), no other easy to measure estimator to assess soil water availability exists to date for deep rooting species like grapevine. PLWP was measured prior to sunrise (between 02h00 to 06h00 local time). PLWP measurements were started no earlier than 04h00 when average PLWP was lower than −0.2 MPa. For each measurement, one leaf per grapevine was sampled. Water potentials were collected between day of year (DOY) 159–257 (from fruit set until maturity) at several occasions on two consecutive days, with SWP measurements carried out on day 1 and 2 and PLWP during the night between day 1 and 2. The underlying idea was that climatic conditions varied between day 1 and 2, while variations in soil water were minimal over two consecutive days. Hence, PLWP was considered representative for soil water availability during both days. This approach allowed creating a dataset (n=1,061) from which the effect of soil and climate on SWP values could be separated by an appropriate modeling approach. Statistical Analysis Over the course of both seasons the difference between SWP values and PLWP values generally increased. The average difference between SWP values and PLWP values at the beginning and the end of the 2015 season equaled 0.51 and 0.86 MPa, respectively. The average difference at the beginning and the end of the 2018 season equaled 0.30 and 0.94 MPa, respectively. Minimum values of PLWP and SWP for Cabernet franc, Cabernet-Sauvignon, and Merlot were, respectively, −0.61 MPa and −1.59 MPa, −0.90 MPa and −1.74 MPa, and −0.87 MPa and −1.85 MPa, respectively. SWP measured on eight leaves on five vines outside the experiment in plot A showed that SWP could on average vary by 0.18 MPa within the same grapevine (Table S2).h Modeling of Stem Water Potential It firmly held extracting urrence of er content ng of plots vere water d gravelly described considered ty in SWP er at weak moderate n the next as also an max on SWP For a same predictive ability of SWP models also depended on the type of climatic variable included. The predictive ability of the climatic variables is as follows (in increasing order of AIC and BIC): wn leaf water potential (PLWP) along a temperature gradient [Tmax, from 23.8°C (blue) to 38.9°C mental plot (n=1,061). The letters correspond to the plots as specified in Table 1. The vertical FIGURE 3 \| Stem water potential over the course of the 2015 and 2018 growing season along a temperature gradient [Tmax, from 23.8°C (blue) to 38.9°C (red)], where the size of the points corresponds to predawn leaf water potential (PLWP) categories in steps of 0.1 MPa. Data was averaged for each plot within each PLWP category. DOY, day of the year. FIGURE 3 \| Stem water potential over the course of the 2015 and 2018 growing season along a temperature gradient [Tmax, from 23.8°C (blue) to 38.9°C (red)], where the size of the points corresponds to predawn leaf water potential (PLWP) categories in steps of 0.1 MPa. Data was averaged for each plot within each PLWP category. DOY, day of the year. FIGURE 3 \| Stem water potential over the course of the 2015 and 2018 growing season along a temperature gradient [Tmax, from 23.8°C (blue) to 38.9°C (red)], where the size of the points corresponds to predawn leaf water potential (PLWP) categories in steps of 0.1 MPa. Data was averaged for each plot within each PLWP category. DOY, day of the year. FIGURE 3 \| Stem water potential over the course of the 2015 and 2018 growing season along a temperature gradient [Tmax, from 23.8°C (blue) to 38.9°C (red)], where the size of the points corresponds to predawn leaf water potential (PLWP) categories in steps of 0.1 MPa. Data was averaged for each plot within each PLWP category. DOY, day of the year. In addition to the apparent effects of PLWP and Tmax on SWP variability, there also appeared to be a seasonal effect. Predawn Leaf Water Potential, Stem Water Potential, and Climate From June Through September in 2015 and 2018 PLWP values ranged from −0.06 to −0.86 MPa in 2015 and from −0.01 to −0.90 MPa in 2018 (Figure 1). SWP values ranged from −0.33 to −1.69 MPa in 2015 and from −0.12 to −1.85 MPa in 2018. Severe water deficit with SWP values below −1.4 MPa and PLWP values below −0.8 MPa were recorded in both Saint- Émilion in 2015 and in Margaux in 2018. Based on the SWP values, grapevines in Saint-Émilion experienced moderate water deficit until the end of July 2015, after which periods of precipitation replenished the soil and both SWP and PLWP The average daily temperature from June until September in 2015 (20.7°C) and 2018 (21.6°C) were slightly higher than the 10-year average daily temperature of the Bordeaux area (20.3°C). In both seasons VPDmax ranged from approximately 1.2 to 5.6 kPa, and ET0 ranged from approximately 2 to 8 mm. Global radiation exceeded 9.74 MJ m−2 at all measurement days in both seasons. Total rainfall from June through September in 2015 and 2018 were very similar (176 and 165 mm, respectively), and lower November 2019 \| Volume 10 \| Article 1485 Frontiers in Plant Science \| www.frontiersin.org 4 Modeling Stem Water Potential Suter et al. given PLWP and Tmax, SWP values became more negative as the season progressed (Figure 3). than the 10-year average total rainfall in the Bordeaux area (194 mm). Using the vintage classification for drought presented in Van Leeuwen and Darriet (2016), and based on water balance modeling according to Lebon et al. (2003), the 2015 season turned out to be the second driest in Bordeaux since 1952 (after 2005), and 2018 was the ninth driest. Modeling of Stem Water Potential g PLWP explained 66% of the variability in SWP (model 1, Table 2). Model selection further showed that adding climatic variables explained a significant amount of the remaining variability in SWP values (Table 2). Furthermore, the improvement of the predictive ability of SWP models also depended on the type of climatic variable included. The predictive ability of the climatic variables is as follows (in increasing order of AIC and BIC): Despite 2015 and 2018 being dry years in the Bordeaux area, grapevines on the experimental plots showed substantial differences in water status (Figure 2). In all plots SWP decreased with more negative PLWP. Plots could roughly be separated in two groups. The first group consisted of plots A, D, E, F, H, I, K, M, O, P, and R where the grapevines were rarely exposed to PLWP values lower than −0.3 MPa. The soils of plots A, E, F, I, M, P, and R contained important clay fractions (Table 1). Plots O and R were known to have a water table accessible to the root system. Plots M and P have a very high clay content (up to 60% in some of the soil layers) and the clay fractions are mostly composed of montmorillonite (swelling clay). It has been shown that in these soils the water is so firmly held by the clay that the grapevines have difficulties in extracting the water from the clay, which explains the occurrence of moderate to severe water deficit despite high soil water content (Tramontini et al., 2013). The second group, consisting of plots B, C, G, J, L, N, and Q, experienced moderate to severe water deficit and could be identified mostly as sandy and gravelly soils with low cation exchange capacity (Table 1). As described in the next section, among the climatic variables considered Tmax explained the next largest part of the variability in SWP after PLWP. The effect of Tmax on SWP was stronger at weak to moderate water deficit and became weaker at moderate to severe water deficit (Figure 2). As described in the next section, the DOY (duration through the season) was also an important factor.f decreased parated in E, F, H, I, y exposed plots A, E, (Table 1). cessible to ay content y fractions g clay). Modeling of Stem Water Potential The correlation among Tmax and VPDmax was stronger than these variables with either ET0 or global radiation. The AIC of model 1 improved by 90.5 (36.3%) when adding VPDmax (model 2) and 139.2 (55.8%) when adding Tmax (model 3). Modeling was therefore continued with the sole inclusion of Tmax. The model including PLWP and Tmax (model 3) only explained part of the large variability in SWP (−0.25 down to −1.40 MPa) at PLWP between 0 and −0.25 MPa (Figure 2). When model 2 was fitted separately for Cabernet franc, Cabernet-Sauvignon, and Merlot, RMSE could be improved by 0.019 compared to the model containing all data, which is only a marginal gain (data not shown). This seems to indicate that the proposed models can be applied across Vitis vinifera varieties. Careful consideration is, however, needed since it is based on an unbalanced design and a limited number of varieties. Any of the models that contained PLWP with one of the climatic variables (VPDmax or Tmax) substantially improved when DOY (day of the Modeling of Stem Water Potential Models were cross-validated by retaining one SWP, stem water potential; PLWP, predawn leaf water potential; VPDmax, maximum vapor pressure deficit at the day of measurement; Tmax, maximum air temperature at the day of measurement; DOY, day of the year of measurement; AIC, Akaike’s Information Criterion; BIC, Bayesian Information Criterion; RMSE, root-mean-square error (MPa), ± standard deviation. year), i.e., seasonality, was included. PLWP correlated moderately negative with DOY (r = −0.40, P < 0.001). The inclusion of DOY to models 2 and 3 further reduced both AIC and BIC on average by 436.7 and 431.7, respectively. The coefficient for DOY in model 5 was 0.00543 (Table 2). This means that over the 86-day measurement period (from 20 June until 14 September) seasonality accounted for an additional 0.47 MPa decrease in SWP on top of the effect of Tmax (Figure 3). In other words, for a same given Tmax and PLWP, SWP becomes substantially more negative as the season progresses. The effect of soil moisture (PLWP), climatic conditions (Tmax), and seasonality (DOY) explained 80% of the variance in SWP (model 5). GDD were also tested in this study. When DOY was replaced by GDD in models 4 and 5, AIC was increased by 1.55 and 4.88, respectively. Tmax > VPDmax. Initially, ET0 was considered as well but it yielded positive estimates, meaning that SWP would be more negative with lower evaporative demand. This artifact was caused by high ET0 recorded during the first two couples of measurements by the end of June 2018 at concurrently weakly negative PLWP and SWP. ET0 was shown to be strongly correlated to global radiation (r = 0.82; Table 3), and to decrease with DOY (r = −0.79; Table 3). For the climatic variables used in the models it was decided to use daily maximum values. Instantaneous T, VPD, and ΣET0 have also been tested, but yielded slightly higher AIC (by 4.5, 23.0, and 11.3 respectively) compared to their equivalent daily maximum values. The daily maximum versus instantaneous values of T, VPD, and ET0 were found to be strongly correlated (r = 0.99, 0.98, and 0.94, respectively). Hence, the use of instantaneous climatic variables is not justified, because their utilization to run the models is much more constraining and they do not improve the models. The climatic variables Tmax, VPDmax, and ET0 of the measurement days were all significantly correlated (Table 3). Modeling of Stem Water Potential For a same FIGURE 2 \| Relationship between stem water potential and predawn leaf water potential (PLWP) along a temperature gradient [Tmax, from 23.8°C (blue) to 38.9°C (red)] collected during the 2015 and 2018 seasons for each experimental plot (n=1,061). The letters correspond to the plots as specified in Table 1. The vertical dotted line is drawn for reference at −0.3 MPa PLWP. FIGURE 2 \| Relationship between stem water potential and predawn leaf water potential (PLWP) along a temperature gradient [Tmax, from 23.8°C (blue) to 38.9°C (red)] collected during the 2015 and 2018 seasons for each experimental plot (n=1,061). The letters correspond to the plots as specified in Table 1. The vertical dotted line is drawn for reference at −0.3 MPa PLWP. November 2019 \| Volume 10 \| Article 1485 5 Frontiers in Plant Science \| www.frontiersin.org Modeling Stem Water Potential Suter et al. TABLE 2 \| Comparison of goodness-of-fit and predictive power of models for SWP. Models were cross-validated by retaining one plot at a time as a validation dataset. No. Models AIC BIC r2 RMSE training RMSE cross validated 1 SWP = 1.243 · e4.011 · PLWP − 1.616 −249.42 −229.55 0.659 0.214 0.216 ± 0.038 2 SWP = 1.518 · e4.063 · PLWP · VPDmax −0.233 − 1.614 −339.94 −315.10 0.688 0.205 0.210 ± 0.038 3 SWP = 20.164 · e3.890 · PLWP · Tmax −0.819 − 1.628 −388.95 −363.76 0.702 0.201 0.204 ± 0.037 4 SWP = 1.479 · e2.304 · PLWP · VPDmax −0.318 − 0.00580 · DOY − 0.444 −792.71 −762.91 0.796 0.166 0.165 ± 0.035 5 SWP = 24.789 · e2.144 · PLWP · Tmax −0.896 − 0.00543 · DOY − 0.579 −809.20 −779.40 0.800 0.164 0.165 ± 0.040 SWP, stem water potential; PLWP, predawn leaf water potential; VPDmax, maximum vapor pressure deficit at the day of measurement; Tmax, maximum air temperature at the day of measurement; DOY, day of the year of measurement; AIC, Akaike’s Information Criterion; BIC, Bayesian Information Criterion; RMSE, root-mean-square error (MPa), ± standard deviation. predictive power of models for SWP. Models were cross-validated by retaining one plot at a time as a validation dataset. TABLE 2 \| Comparison of goodness-of-fit and predictive power of models for SWP. DISCUSSION In order to also test model 5 with the seasonality (DOY) term, only data was selected where the difference between PLWP values within a plot did not exceed 0.005 MPa and measurement days were at least a week apart. PLWP was assumed to be the same between the two data points and would thus allow for the standardization of the SWP value by Tmax and DOY. Model 5 can be rearranged to obtain: The effect of soil water availability (PLWP), climatic conditions (Tmax), and seasonality (DOY) explained 80% of the variance in SWP (model 5), where 66% of the variability in SWP could be explained by PLWP through an exponential term. In line with our results, Abrisqueta et al. (2015) found that for peach 61% of the variability in SWP could be explained by soil water availability. In their modeling exercise they were able to explain 72% of the total variability in SWP by also including VPD and seasonality (as accumulated growing-degree-hours).h SWP SWP DOY T T DOY s max s s = + ⋅ +     − ⋅ − − 0 00543 0 579 0 00543 0 896 . . . . 0 579 . (2) SWP SWP DOY T T DOY s max s s = + ⋅ +     − ⋅ − − 0 00543 0 579 0 00543 0 896 . . . . 0 579 . (2) The relationships between SWP and Tmax and VPDmax were best captured by power functions. In line with our results, previous research has already shown that VPD was nonlinearly related to SWP in both grapevines (Gálvez et al., 2014) and apple trees (De Swaef et al., 2009). The same nonlinearity has been shown for Tmax and SWP in lemon trees (Ortuño et al., 2006; Ortuño et al., 2009), although not yet in grapevine. It is surprising that Tmax explained more of the variability in SWP than did ET0 and VPDmax, since the latter two are estimates of evaporative demand. Santesteban et al. (2011) found similar results, where SWP of Tempranillo was better estimated by T than by VPD. They reported a correlation between T and VPD of 0.82 (versus 0.83 in this study) and justified their result by the fact that the range of VPD values was quite narrow in their study. Standardization of the Stem Water Potential Valueh The nature of the dataset, with measurements performed on two consecutive days in both experimental years, allowed testing the accuracy of standardization. For this purpose, the SWP value measured on day 2 was standardized to the Tmax on day 1, with the assumption that PLWP was maintained at the same value between day 1 and day 2. This standardized SWP (SWPs) could then be compared to the SWP value measured on day 1. In order to obtain SWP values under standardized climatic conditions (in this case the Tmax of day 2, but this could be any Tmax of interest), model 3 was algebraically rearranged. Model 3 (Table 2) can be rearranged as following: SWP SWP T T s max s = +     − − 1 628 1 628 0 819 . . . (1) (1) 6 TABLE 3 \| Pearson correlation matrix of daily values of climatic variables of the measurement days (2015 and 2018). Tmax (˚C) VPDmax (kPa) ET0 (mm) Global radiation (MJ/m2) DOY Tmax 1 VPDmax 0.83 1 ET0 0.40 0.63 1 Global radiation 0.40 0.58 0.82 1 DOY ns* ns −0.79 −0.66 1 Correlations shown are highly significant (P < 0.001). *ns, not significant. Frontiers in Plant Science \| www.frontiersin.org November 2019 \| Volume 10 \| Article 1485 DOY Modeling Stem Water Potential Suter et al. DISCUSSION In the maritime climate of Bordeaux maximum VPD is reached mid-July, after which it decreases. In this study, ET0 was shown not to be a good estimator, possibly because of a large effect of global radiation early in the season on ET0 values.h (2) where, Tmax represents the maximum air temperature at the day of measurement, Ts the temperature to which the measured SWP needs to be standardized to and DOYs the DOY to which it needs to be standardized to. SWP values measured on day 2 standardized to Tmax of day 1 according to Eqn. 1 improved r2 and reduced RMSE (Figure 4B) compared to the unstandardized SWP values on day 2 (Figure 4A). The improvement in RMSE between the observed SWP and standardized SWP by Eqn. 1 was almost 31%. When Eqn. 2 was used on the same (2-day) data the improvement was only 0.008 MPa, which was in line with the inherently low estimated coefficient (0.00543) for DOY in model 5 (Table 2).h fi There were 385 observed SWP values within the plots that did have PLWP values that were almost the same or did not differ more than 0.005 MPa. The number of days between these observations could range from 1 week up to 11 weeks. Eqn. 2 showed to improve r2 and reduce RMSE (Figure 5B), compared to unstandardized SWP values (Figure 5A). The improvement in RMSE between the observed SWP and standardized SWP by Eqn. 2 was almost 24%. The use of instantaneous climatic data did not appear to produce better modeling results than daily climatic data (ΔAIC > 4.5). Strong correlations existed between instantaneous and daily maximum values of T, VPD, and ET0 (r > 0.94). The average variability in air temperature for the part of the days FIGURE 4 \| Comparison of (A) the observed stem water potential (SWP) on day 1 versus the observed SWP on day 2 and (B) the observed SWP on day 1 versus the observed SWP on day 2 standardized to Tmax on day 1 as per Eqn. 1. The solid line represents the 1:1 line and the dotted line represents the linear regression (n = 512). DISCUSSION FIGURE 4 \| Comparison of (A) the observed stem water potential (SWP) on day 1 versus the observed SWP on day 2 and (B) the observed SWP on day 1 versus the observed SWP on day 2 standardized to Tmax on day 1 as per Eqn. 1. The solid line represents the 1:1 line and the dotted line represents the linear regression (n = 512). FIGURE 4 \| Comparison of (A) the observed stem water potential (SWP) on day 1 versus the observed SWP on day 2 and (B) the observed SWP on day 1 versus the observed SWP on day 2 standardized to Tmax on day 1 as per Eqn. 1. The solid line represents the 1:1 line and the dotted line represents the linear regression (n = 512) November 2019 \| Volume 10 \| Article 1485 7 Frontiers in Plant Science \| www.frontiersin.org Modeling Stem Water Potential Suter et al. FIGURE 5 \| Comparison of (A) the observed stem water potential (SWP) on DOYn versus the observed SWP on DOYn>x, where x is at least greater than 2 days and has a predawn leaf water potential almost equal to the observed SWP on DOYn and (B) the observed SWP on DOYn versus the observed SWP at that later date standardized according to Eqn. 2. The solid line represents the 1:1 line and the dotted line represents the linear regression (n = 385). FIGURE 5 \| Comparison of (A) the observed stem water potential (SWP) on DOYn versus the observed SWP on DOYn>x, where x is at least greater than 2 days and has a predawn leaf water potential almost equal to the observed SWP on DOYn and (B) the observed SWP on DOYn versus the observed SWP at that later date standardized according to Eqn. 2. The solid line represents the 1:1 line and the dotted line represents the linear regression (n = 385). growing-degree-hours (r = −0.67, P < 0.001). Adding GDD instead of DOY improved the models, albeit marginally. Vine phenology is, however, not only determined by GDD, but also by temperature requirements of cultivars (Parker et al., 2013). It is out of the scope of this study to produce cultivar-specific models. DOY was therefore preferred to GDD. Olivo et al. (2009) demonstrated seasonal sensitivity of SWP to VPD in a 3-year field experiment in grapevine. DISCUSSION They argued that differences between SWP in irrigation treatments at various phenological stages could be explained by an imbalance between water supply and canopy demand. However, such an imbalance caused by differential canopy demand was unlikely to be of great influence as canopy size was strictly controlled by hedging in this study. Grapevines therefore have other ways to adjust their water use to soil water availability. wherein SWP measurements were performed equaled only 1.06°C. This degree of variability might have been too small to detect any change in SWP. Additionally, the increase in precision might have been offset by within-vine variability of SWP (0.18 MPa; Table S2). From a practical point of view, the advantage of Tmax is that it is easy to acquire from basic field weather stations. Moreover, these are rarely equipped with sensors to measure climatic variables such as ET0 or relative humidity, which makes Tmax an ideal variable for SWP modeling. Temperature and rainfall data were collected very close to the experimental blocks in Saint-Émilion in 2015 (less than 500 m). Hence, climate data can be considered as highly accurate for this dataset. In 2018, the distance from the blocks to the weather station ranged from 0,710 to 1,460 km. This distance has very little impact on the accuracy of the temperature data, because the region is flat and temperatures are not very variable over short distances (Van Leeuwen et al., 2014; Bois et al., 2018). Rainfall can, however, vary over short distances and there can potentially be some discrepancy between rainfall data collected in the weather station and the actual amount of rainfall received on the block where the SWP were measured. Although grapevine cultivars are known to respond very distinctly to water deficits (Chaves et al., 2010; Santesteban et al., 2019), model 2 was only marginally improved when fitted separately for Cabernet franc, Cabernet-Sauvignon, and Merlot. It has been reported that these three cultivars show anisohydric behavior. Further investigation is needed to test if the models proposed here do apply with the same accuracy to cultivars with (near-)isohydric behavior. Vine size is a factor potentially impacting water potentials which has not been taken into account in the models. Leaf area was measured in 2015 on the experimental blocks and varied from 1.36 to 2.30 m2 when fully established at the end of the season. Frontiers in Plant Science \| www.frontiersin.org DISCUSSION Primary leaf area varied much less than secondary leaf area (0.67 to 0.98 and 0.53 to 1.57 m2, respectively). No data is available for leaf area in the experimental blocks in 2018. Vine size is likely to have an impact on SWP, which is measured during the day when vines are potentially transpiring. PLWP is measured at dawn when vines are not transpiring and should be less impacted by leaf area, because at dawn water potentials in the plant adjust with water potentials in the soil. Generally, vines with a bigger canopy also have a more developed root system. Although root to shoot ratio may vary under changing light conditions or nitrogen availability, it is rather stable for a given set of environmental conditions (Grechi et al., 2007). As long as shoot to root ratio remains unchanged, vines with bigger canopies also have access to greater water reserves and the impact of leaf area on SWP can thus be supposed to remain In addition to PLWP and Tmax, seasonality (DOY) was found to explain a large part of the remaining variability in SWP. The seasonality in this study correlated moderately high with SWP (r = −0.64, P < 0.001) and is in line with that found by Abrisqueta et al. (2015), where seasonality was expressed as accumulated November 2019 \| Volume 10 \| Article 1485 Frontiers in Plant Science \| www.frontiersin.org 8 Modeling Stem Water Potential Suter et al. limited. Including vine-specific leaf area could, however, improve the models presented here and can be an area of further investigation. Vine specific leaf area is not very easy to measure (Trégoat et al., 2001) and including this parameter in the models would restrain their application in a production context. a wide range of water deficit would help growers to adapt management strategies to drought, both in the short term by means of irrigation, adjustment of the canopy size or vineyard floor management, or in the long term, by choosing appropriate plant material (rootstocks and cultivars) and planting densities. More research, however, is needed to better understand the effect of seasonality on the relations between climatic conditions and SWP over a wide range of PLWP values. Improvements to the model may be investigated by improving predictability of PLWP under heterogeneous soil humidity as well as accounting for acclimation to water deficit. DATA AVAILABILITY STATEMENT The datasets generated for this study are available on request to the corresponding author. The standardization exercises following the proposed models allow for the comparison of SWP values obtained across dates with variable climatic conditions and different soil water availability. The added value, compared to the baseline approach, is that growers will be able to correct SWP values at levels below those of the baseline established for non-limiting water uptake conditions. This is of significant interest since a moderate water deficit is considered favorable to wine quality (Van Leeuwen et al., 2009). The advantage of this standardization over the use of PLWP is that it does not require the actual measurement of that PLWP. The PLWP measurement remains, however, useful as it represents a value close to the soil matrix potential.h DISCUSSION The performance of the models developed in this study would still need to be assessed in (extremely) wet years, given that they were parameterized using data collected in two dry years. Should PLWP stay between 0 and −0.1 MPa over the course of a season, then the seasonality term in model 5 (Table 2) could underestimate the SWP value and result in positive SWP values. This would be the case if observed SWP is higher than −0.47 MPa at mid-September. In our dataset, a single grapevine in plot F attained a SWP of −0.42 MPa on 14 September 2018, attesting the necessity for recalibration of our model in wet years.h CONCLUSIONS PLWP, Tmax, and DOY were main contributors to estimating SWP. Nonlinear models based on these variables were able to explain 80% of the variance in SWP for Merlot, Cabernet- Sauvignon, and Cabernet franc in field conditions. The algebraic rearrangement of the proposed models allows for standardization of SWP values to a value under predefined reference climatic conditions, in order to better reflect soil water availability. This allows for comparison of SWP values under different climatic conditions. A fine-tuned interpretation of SWP values across ACKNOWLEDGMENTS We thank the two commercial vineyards for making available their plots for experimentation. This study was executed as part of the ClimWaterModel project. We are grateful to Xavier Choné for providing insights in the experimental set-up and Kévin Berteaud for preliminary work for this study and for collecting water potential data in Saint-Émilion in 2015. FUNDING The aim of this study was to provide models that separate effects of soil moisture and climatic conditions on the SWP value. Although the models explain a large part of the variability in SWP values, some of the variance remains unexplained. Part of this variability may be due to the inability of PLWP to represent plant water status when only a small part of the soil contains readily available water while transpiration demand is high (Améglio et al., 1999). Another part of the remaining variance may be explained by grapevines acclimating to water deficit by modifying hydraulic properties and gas exchange (Hochberg et al., 2017). Drought-acclimated grapevines could maintain higher gas exchange under drought, resulting in more negative SWP values compared to vines that were not previously exposed to water deficit. These two leads may be starting points to further improving the models proposed in this study. The study has been carried out with financial support from the French National Research Agency (ANR) in the frame of the Investments for the future Program, within the Cluster of Excellence COTE (ANR-10-LABX-45). It has not been formally reviewed by ANR. The views expressed in this publication are solely those of the authors, and ANR does not endorse any products or commercial services mentioned in this publication. This work was supported by the metaprogramme Adaptation of Agriculture and Forests to Climate Change (AAFCC) of the French National Institute for Agricultural Research (INRA), especially through the Laccave 2.21 project. The study has been carried out with financial support from the French National Research Agency (ANR) in the frame of the Investments for the future Program, within the Cluster of Excellence COTE (ANR-10-LABX-45). It has not been formally reviewed by ANR. The views expressed in this publication are solely those of the authors, and ANR does not endorse any products or commercial services mentioned in this publication. This work was supported by the metaprogramme Adaptation of Agriculture and Forests to Climate Change (AAFCC) of the French National Institute for Agricultural Research (INRA), especially through the Laccave 2.21 project. AUTHOR CONTRIBUTIONS DP, CvL, and RT contributed conception and design of the study. BS organized the database. 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SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2019.01485/ full#supplementary-material November 2019 \| Volume 10 \| Article 1485 Frontiers in Plant Science \| www.frontiersin.org 9 Modeling Stem Water Potential Suter et al. REFERENCES Impact of soil texture and water availability on the hydraulic control of plant and grape-berry development. Plant Soil 368, 215–230. doi: 10.1007/s11104-012-1507-x g y Lebon, E., Dumas, V., Pieri, P., and Schultz, H. R. (2003). Modelling the seasonal dynamics of the soil water balance of vineyards. Funct. Plant Biol. 30, 699–710. doi: 10.1071/FP02222 Van Leeuwen, C., and Darriet, P. (2016). The Impact of Climate Change on Viticulture and Wine Quality. J. Wine Econ. 11, 150–167. doi: 10.1017/ jwe.2015.21 Levin, A. D. (2019). Re-evaluating pressure chamber methods of water status determination in field-grown grapevine (Vitis spp.). Agric. 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Vin. 53, 129–146. doi: 10.20870/oeno-one.2019.53.2.2420 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. Williams, L. E., and Araujo, F. REFERENCES J. (2002). Correlations among predawn leaf, midday leaf, and midday stem water potential and their correlations with other measures of soil and plant water status in Vitis vinifera. J. Am. Soc. Hortic. Sci. 127, 448–454. doi: 10.21273/JASHS.127.3.448 Copyright © 2019 Suter, Triolo, Pernet, Dai and Van Leeuwen. 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 © 2019 Suter, Triolo, Pernet, Dai and Van Leeuwen. 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. Williams, L. E., and Baeza, P. (2007). Relationships among ambient temperature and vapor pressure deficit and leaf and stem water potentials of fully irrigated, field-grown grapevines. Am. J. Enol. Vitic. 58, 173–181. i Williams, L. E., and Trout, T. J. (2005). Relationships among vine- and soil-based measures of water status in a thompson seedless vineyard in response to high- frequency drip irrigation. Am. J. Enol. Vitic. 56, 357–366. November 2019 \| Volume 10 \| Article 1485 Frontiers in Plant Science \| www.frontiersin.org 11
https://openalex.org/W3035938748	https://gfzpublic.gfz-potsdam.de/pubman/item/item_5003225_3/component/file_5003272/5003225.pdf	English	null	The 2019 Eruption Dynamics and Morphology at Ebeko Volcano Monitored by Unoccupied Aircraft Systems (UAS) and Field Stations	Remote sensing	2,020	cc-by	16,134	Received: 20 May 2020; Accepted: 16 June 2020; Published: 18 June 2020 Abstract: Vulcanian explosions are hazardous and are often spontaneous and direct observations are therefore challenging. Ebeko is an active volcano on Paramushir Island, northern Kuril Islands, showing characteristic Vulcanian-type activity. In 2019, we started a comprehensive survey using a combination of ﬁeld station records and repeated unoccupied aircraft system (UAS) surveys to describe the geomorphological features of the ediﬁce and its evolution during ongoing activity. Seismic data revealed the activity of the volcano and were complemented by monitoring cameras, showing a mean explosion interval of 34 min. Digital terrain data generated from UAS quadcopter photographs allowed for the identiﬁcation of the dimensions of the craters, a structural architecture and the tephra deposition at cm-scale resolution. The UAS was equipped with a thermal camera, which in combination with the terrain data, allowed it to identify fumaroles, volcano-tectonic structures and vents and generate a catalog of 282 thermal spots. The data provide details on a nested crater complex, aligned NNE-SSW, erupting on the northern rim of the former North Crater. Our catalog of thermal spots also follows a similar alignment on the ediﬁce-scale and is also aﬀected by topography on a local scale. This paper provides rare observations at Ebeko volcano and shows details on its Vulcanian eruption style, highlighting the relevance of structural and morphologic control for the geometry of craters and tephra fallout as well as for structurally controlled geothermal activity. Keywords: Ebeko volcano; small unoccupied aircraft system (UAS), Vulcanian explosions; air fall deposits; infrared imaging; photogrammetry The 2019 Eruption Dynamics and Morphology at Ebeko Volcano Monitored by Unoccupied Aircraft Systems (UAS) and Field Stations Thomas R. Walter 1,* , Alexander Belousov 2, Marina Belousova 2, Tatiana Kotenko 2 and Andreas Auer 3 1 Department of Geophysics, GFZ Potsdam, Telegrafenberg, 14473 Potsdam, Germany 2 Institute of Volcanology and Seismology, FED RAS, 683006 Petropavlovsk, Russia; bel@kscnet.ru (A.B.); belousov@mail.ru (M.B.); sinarka2017@mail.ru (T.K.) 3 Department of Geoscience, Shimane University, Matsue 690-8504, Japan; auer@riko.shimane-u.ac.jp * Correspondence: twalter@gfz-potsdam.de Received: 20 May 2020; Accepted: 16 June 2020; Published: 18 June 2020 remote sensing remote sensing remote sensing remote sensing remote sensing 1. Introduction Eruptions, their craters and ﬁssures are known to be strongly controlled by pre-existing structures, such as fracture networks [1]. Crustal structures also play an important role during the initiation and lifespan of volcanoes, possibly aﬀecting the locations of magma pathways, hydrothermal ﬂuids and their alteration [2–5]. The eﬀects of existing crustal structures are identiﬁed at the surface, as the morphology of volcanoes evolves in close association with this structural forcing. Also the deposition and erosion of material is controlled by conduit geometry, crater morphology and other structural consequences [6,7]. As deposition of eruptive material often obscures direct views of the underlying structures, general morphological expressions can be indirectly assessed where aligned vents and craters indicate underlying zones of structural weakness [8]. Craters may form in groups and alignments [9], developing Remote Sens. 2020, 12, 1961; doi:10.3390/rs12121961 www.mdpi.com/journal/remotesensing 2 of 20 Remote Sens. 2020, 12, 1961 into crater rows linked to dikes [10] or multiple vents along active and elongated fracture zones [11]. Volcano craters that are controlled by tectonic stress ﬁelds and preexisting crustal fault structures are also aﬀected in terms of their geometry, stability and breaching direction [12], highlighting the importance of investigating these possible interactions. Pre-existing structures not only control volcanism, but also strongly aﬀect hydrothermal venting sites, as demonstrated by recent work [13–16]. The scales under consideration are important, where structural control is dominant over a large ediﬁce scale but obscured at smaller scales [15]. Variations in permeability are thought to be the main eﬀect of earlier structures, which together with lithology leads to the rather complex appearance of volcanic and geothermal ﬁelds. In addition, evolving stress ﬁelds may aﬀect sites of ﬂuid migration, volcanism and fumaroles [13,17,18]. To better understand the factors controlling the surface expression of volcanic activity, the spatial distribution of vents and thermal spots can be important parameters, which can be identiﬁed by direct and remote measurements. The spatial distribution of escaping ﬂuids and their pathways and structures can be assessed by visual and thermal aerial photography [19–21]. In addition, zones of hydrothermal alteration associated with thermal ﬂuid escape are detected by airborne surveys [22]. These studies show that the detailed spatial observations and mapping of the fumaroles may provide a detailed picture of the processes of magmatic and ﬂuid migration at active volcanoes. 1. Introduction At many volcanoes, it is still challenging to gather such information; however, frequent and powerful explosions limit access and can obscure structures. Unoccupied aircraft systems (UASs) can support observations and monitoring at hazardous volcanoes, aid in mapping volcanic structures and the quantiﬁcation of geomorphologic changes, as well as identify hydrothermal processes [23]. UASs have a number of advantages compared to standard airborne photogrammetric surveys and satellite imagery, as they are low cost, allow generating very high resolution maps and are temporally ﬂexible. Therefore, the use of UAS became a powerful tool in volcanology [23,24], as it allowed the estimation of eruption volumes [25], the detection of small topographic changes and fractures [5], sampling and gas measurements [26,27], the monitoring of deformation [28] and could even reach remote sites at distances exceeding the 100 km range [29]; moreover it could be used for many other volcanologic purposes [23–25]. Especially at explosive volcanoes, the use of a UAS is often the only aﬀordable way to obtain samples and high-resolution terrain models, as vividly demonstrated for the Agung crisis [30] and steam-driven explosion structures at Merapi volcano, Indonesia [31], topographic changes at the ﬂanks of Colima, Mexico [32] and the rapid growth and collapse at the dome of Santiaguito [28] and Fuego, Guatemala [33]. UASs are especially useful for structural assessments, allowing the study of the appearance, formation, geomorphology and dynamics of volcano craters and crater lakes at unprecedented resolution [18,23,24,31,34]. The quality of UAS-derived photogrammetric products [23] oﬀers the possibility of low cost and repeated overﬂights and monitoring at volcanic craters even during eruptive activity [35], identiﬁcation of structures, ﬁssures and faults [5,36] and the measurement of structures and fall deposits at craters [37]. Therefore, at Ebeko volcano, we gathered such photogrammetric data in an attempt to better understand eruption sites and the locations of ﬂuid escape. Ebeko is otherwise diﬃcult to access and frequent explosions have further increased since 2016 and put conventional aircrafts and operators at risk. 2. Geologic Setting The active cr chain of Ebeko (Figure 1d,f) hosts three main craters referred to as the South Crater, Middle Cr and North Crater, with temporally varying cold and hot lakes indicative of a shallow groundw level and interaction with the geothermal system [46]. Relatively strong eruptions of the Vulcan type occur episodically, with an activity that appears to migrate along the NNE-SSW Vernad Ridge, such as eruptions in 1934–35, 1965, 2011 and 2016–2017 that occurred from the Middle Cr and in 1967–1971, 1987–1991, 2009–2010, 2016–20 from the North Crater and a deep funnel on northeastern rim [41,47,48]. Sometimes eruptions have even occurred simultaneously at differ craters. Starting in April 2018, the eruptions in the active funnel in the North Crater migrated to fo a new crater located further westward, which is currently the only active New North Crater. Volca tectonic processes, degassing and an increased geothermal gradient are relevant on land and i ff h i f d f i i fl i fil [49] Figure 1. (a) Location map in northern Kuriles. (b) Southern Kamchatka and Paramushir Island, w Ebeko volcano being the northernmost volcano on the island. (c) Satellite color infrared (Sentine bands 8,4,3 from 13 September 2017) showing vegetation in red and bare soils and volcanic depos in gray. The Vernadsky Ridge (white dotted line) is defined by fissures and cone alignments (releva cones and crater vents are indicated by white circle symbology). (d) Close-up of Ebeko volcano, w a distance to the town of Severo-Kurilsk of only 7 km. (e) Eruption at Ebeko as seen from Seve Kurilsk on 11 July 2019 and (f) UAS view on 14 July 2019 showing the three craters aligned NN SSW, with the current eruptions in the New North Crater, a cold lake in the Middle Crater a fumaroles in the South Crater. Figure 1. (a) Location map in northern Kuriles. (b) Southern Kamchatka and Paramushir Island, with Ebeko volcano being the northernmost volcano on the island. (c) Satellite color infrared (Sentinel-2 bands 8,4,3 from 13 September 2017) showing vegetation in red and bare soils and volcanic deposits in gray. The Vernadsky Ridge (white dotted line) is deﬁned by ﬁssures and cone alignments (relevant cones and crater vents are indicated by white circle symbology). (d) Close-up of Ebeko volcano, with a distance to the town of Severo-Kurilsk of only 7 km. 2. Geologic Setting The active cr chain of Ebeko (Figure 1d,f) hosts three main craters referred to as the South Crater, Middle Cr and North Crater, with temporally varying cold and hot lakes indicative of a shallow groundw level and interaction with the geothermal system [46]. Relatively strong eruptions of the Vulcan type occur episodically, with an activity that appears to migrate along the NNE-SSW Vernad Ridge, such as eruptions in 1934–35, 1965, 2011 and 2016–2017 that occurred from the Middle Cr and in 1967–1971, 1987–1991, 2009–2010, 2016–20 from the North Crater and a deep funnel on northeastern rim [41,47,48]. Sometimes eruptions have even occurred simultaneously at diffe craters. Starting in April 2018, the eruptions in the active funnel in the North Crater migrated to fo a new crater located further westward, which is currently the only active New North Crater. Volca tectonic processes, degassing and an increased geothermal gradient are relevant on land and ff h f d f fl f l 2-pyroxenes. The morphology of Ebeko reaches 1142 m above sea level (as we measured by GNSS), yet the active cones are only approximately 250 m high [40]. The active crater chain of Ebeko (Figure 1d,f) hosts three main craters referred to as the South Crater, Middle Crater and North Crater, with temporally varying cold and hot lakes indicative of a shallow groundwater level and interaction with the geothermal system [46]. Relatively strong eruptions of the Vulcanian type occur episodically, with an activity that appears to migrate along the NNE-SSW Vernadsky Ridge, such as eruptions in 1934–35, 1965, 2011 and 2016–2017 that occurred from the Middle Crater and in 1967–1971, 1987–1991, 2009–2010, 2016–20 from the North Crater and a deep funnel on its northeastern rim [41,47,48]. Sometimes eruptions have even occurred simultaneously at diﬀerent craters. Starting in April 2018, the eruptions in the active funnel in the North Crater migrated to form a new crater located further westward, which is currently the only active New North Crater. Volcano-tectonic processes, degassing and an increased geothermal gradient are relevant on land and also continue to occur oﬀshore, as inferred from seismic reﬂection proﬁles [49]. The majority of erupted products are andesites and basaltic andesites [45] that appear hig porphyritic with 2-pyroxenes. The morphology of Ebeko reaches 1142 m above sea level (as measured by GNSS), yet the active cones are only approximately 250 m high [40]. 2. Geologic Setting (e) Eruption at Ebeko as seen from Severo-Kurilsk on 11 July 2019 and (f) UAS view on 14 July 2019 showing the three craters aligned NNE-SSW, with the current eruptions in the New North Crater, a cold lake in the Middle Crater and fumaroles in the South Crater. Figure 1. (a) Location map in northern Kuriles. (b) Southern Kamchatka and Paramushir Island, w Ebeko volcano being the northernmost volcano on the island. (c) Satellite color infrared (Sentin bands 8,4,3 from 13 September 2017) showing vegetation in red and bare soils and volcanic depo in gray. The Vernadsky Ridge (white dotted line) is defined by fissures and cone alignments (relev cones and crater vents are indicated by white circle symbology). (d) Close-up of Ebeko volcano, w a distance to the town of Severo-Kurilsk of only 7 km. (e) Eruption at Ebeko as seen from Seve Kurilsk on 11 July 2019 and (f) UAS view on 14 July 2019 showing the three craters aligned NN SSW, with the current eruptions in the New North Crater, a cold lake in the Middle Crater fumaroles in the South Crater Figure 1. (a) Location map in northern Kuriles. (b) Southern Kamchatka and Paramushir Island, with Ebeko volcano being the northernmost volcano on the island. (c) Satellite color infrared (Sentinel-2 bands 8,4,3 from 13 September 2017) showing vegetation in red and bare soils and volcanic deposits in gray. The Vernadsky Ridge (white dotted line) is deﬁned by ﬁssures and cone alignments (relevant cones and crater vents are indicated by white circle symbology). (d) Close-up of Ebeko volcano, with a distance to the town of Severo-Kurilsk of only 7 km. (e) Eruption at Ebeko as seen from Severo-Kurilsk on 11 July 2019 and (f) UAS view on 14 July 2019 showing the three craters aligned NNE-SSW, with the current eruptions in the New North Crater, a cold lake in the Middle Crater and fumaroles in the South Crater. Figure 1. (a) Location map in northern Kuriles. (b) Southern Kamchatka and Paramushir Island, w Ebeko volcano being the northernmost volcano on the island. (c) Satellite color infrared (Sentin bands 8,4,3 from 13 September 2017) showing vegetation in red and bare soils and volcanic depo in gray. The Vernadsky Ridge (white dotted line) is defined by fissures and cone alignments (relev cones and crater vents are indicated by white circle symbology). 2. Geologic Setting Ebeko is a composite cone located on Paramushir Island in the Kurile Islands arc, where the Paciﬁc Plate subducts under the Okhotsk Plate [38,39]. Ebeko is located only 7 km WNW from the town of Severo-Kurilsk and 320 km SW of Petropavlovsk. The volcano is a source of hazards to daily hikers and the nearby town, producing air falls, poisonous gases and lahars [40–42]. Geologic reconstruction showed that the volcano’s activity initiated in the Holocene over strongly eroded basement rocks of the Vernadsky Ridge [43], which is a tectonically active zone hosting a number of ﬁssures, NNE-SSW oriented lineaments and chains of craters [40,44] (Figure 1). The majority of erupted products are andesites and basaltic andesites [45] that appear highly porphyritic with 3 of 20 3 Remote Sens. 2020, 12, 1961 Remote Sens 2020 12 2-pyroxenes. The morphology of Ebeko reaches 1142 m above sea level (as we measured by GNSS), yet the active cones are only approximately 250 m high [40]. The active crater chain of Ebeko (Figure 1d,f) hosts three main craters referred to as the South Crater, Middle Crater and North Crater, with temporally varying cold and hot lakes indicative of a shallow groundwater level and interaction with the geothermal system [46]. Relatively strong eruptions of the Vulcanian type occur episodically, with an activity that appears to migrate along the NNE-SSW Vernadsky Ridge, such as eruptions in 1934–35, 1965, 2011 and 2016–2017 that occurred from the Middle Crater and in 1967–1971, 1987–1991, 2009–2010, 2016–20 from the North Crater and a deep funnel on its northeastern rim [41,47,48]. Sometimes eruptions have even occurred simultaneously at diﬀerent craters. Starting in April 2018, the eruptions in the active funnel in the North Crater migrated to form a new crater located further westward, which is currently the only active New North Crater. Volcano-tectonic processes, degassing and an increased geothermal gradient are relevant on land and also continue to occur oﬀshore, as inferred from seismic reﬂection proﬁles [49]. The majority of erupted products are andesites and basaltic andesites [45] that appear hig porphyritic with 2-pyroxenes. The morphology of Ebeko reaches 1142 m above sea level (as measured by GNSS), yet the active cones are only approximately 250 m high [40]. 2. Geologic Setting (d) Close-up of Ebeko volcano, w a distance to the town of Severo-Kurilsk of only 7 km. (e) Eruption at Ebeko as seen from Sev Kurilsk on 11 July 2019 and (f) UAS view on 14 July 2019 showing the three craters aligned N SSW, with the current eruptions in the New North Crater, a cold lake in the Middle Crater fumaroles in the South Crater. Figure 1. (a) Location map in northern Kuriles. (b) Southern Kamchatka and Paramushir Island, with Ebeko volcano being the northernmost volcano on the island. (c) Satellite color infrared (Sentinel-2 bands 8,4,3 from 13 September 2017) showing vegetation in red and bare soils and volcanic deposits in gray. The Vernadsky Ridge (white dotted line) is deﬁned by ﬁssures and cone alignments (relevant cones and crater vents are indicated by white circle symbology). (d) Close-up of Ebeko volcano, with a distance to the town of Severo-Kurilsk of only 7 km. (e) Eruption at Ebeko as seen from Severo-Kurilsk on 11 July 2019 and (f) UAS view on 14 July 2019 showing the three craters aligned NNE-SSW, with the current eruptions in the New North Crater, a cold lake in the Middle Crater and fumaroles in the South Crater. Ebeko volcano is renowned for its intense fumarolic activity and picturesque sulfur towers cluster in four main fields [41,46], which is also why most previous studies considered geochemistry and petrology of the volcano, its hydrothermal system and associated altera products [50,51]. Gas measurements by mobile DOAS systems reveal that 90% of SO2 emissi (approximately 100 t/d) escape from the active North Crater and only 10% or less escape from o Ebeko volcano is renowned for its intense fumarolic activity and picturesque sulfur towers that cluster in four main ﬁelds [41,46], which is also why most previous studies considered the geochemistry and petrology of the volcano, its hydrothermal system and associated alteration products [50,51]. Gas measurements by mobile DOAS systems reveal that 90% of SO2 emissions (approximately 100 t/d) escape from the active North Crater and only 10% or less escape from other fumarole ﬁelds [52], 4 of 20 Remote Sens. 2020, 12, 1961 suggesting that ﬂuid pathways are channelized by the active conduit system, although the numbers may signiﬁcantly increase during active periods to an SO2 ﬂux of over 200 t/d [53]. 2. Geologic Setting Selected fumarole ﬁelds have been intensely studied, with some showing temperatures exceeding 500 ◦C [53] and others with conical subterranean structures that connect the fumarole ﬁelds at the surface with hydrothermal reservoirs at a depth of 10 m [54] or even 20–40 m [42]. The sites of fumaroles are known to appear and disappear or migrate laterally, sometimes (as in 1983) with the activity shifting by 200 m or more [40]; however, a systematic mapping of the fumarole ﬁelds, the crater morphology and ﬂuid pathways has not been conducted yet. Instrumental and permanent monitoring is very sparse. In 2019, there was one time lapse camera and one seismic station located in western Severo-Kurilsk town at a distance of 7.2 km from the volcano. High levels of natural and artiﬁcial noise do not permit the registration of weak seismic activity and the identiﬁcation of volcanic explosions on seismograms is possible in only a few cases. Previously (in the Soviet Union era), frequent gas sampling was performed by the Institute of Volcanology [40]. Previous studies used thermal survey data and geochemical analysis for precursory and eruption activity assessments [40], which is why modern drone-based and geophysical records will also provide an important basis for robust assessment of the changes at this volcano. Sporadic ﬁeld surveys allow for the sampling of temperature, gas and water at sites of ﬂuid expulsion [55,56] before the new eruption episode started in October 2016. A modern and comprehensive geophysical characterization of this new activity has not yet been achieved. 3. Data and Methods We conducted a 10-day ﬁeld campaign in July 2019, with daily trips from a base camp (156.0605◦E, 50.6883◦N, 700 m) during which we installed a geophone seismometer, as well as time lapse cameras to record and count the duration of explosions, wind drift and the height of explosions, airfall and associated columns. We conducted repeated UAS overﬂights on two days with low wind and clear atmosphere conditions. As the UAS carried visual and infrared cameras, the data revealed detailed views of the ground and interior of the crater associated with a major explosion. 3.1. Remote Sensing by an Unoccupied Aircraft System (UAS) UAS surveys were attempted on several days but were only successful on 14 and 15 July 2019 due to clear sky and low wind conditions. The 14 July ﬂights were recorded immediately before and after one of the largest explosions. The 15 July ﬂights were achieved for general mapping purposes and acquiring a dense image overlap for referencing. UASs were Phantom 4 pro consumer drones equipped with a 20 MPixel optical camera (5472 × 3078 pixels) and a 35 mm focal of 24, set to record images at 0.5 s intervals. The launch site of all UAS ﬂights was at the highest point of Ebeko volcano at 1142 m above sea level (a.s.l.) at E156.0160◦N50.6859◦. The ﬂight height of the surveys was then 300 m above this launchpad at 1442 m a.s.l. All ﬂight paths were preplanned but controlled manually to quickly respond to volcanic explosions, ash fall and sudden wind gusts. The UAS also carried a home-built plate hosting a thermal infrared FLIR Tau2 640 × 512 radiometric camera with a 13 mm lens, recording at wavebands between 7.5 and 13.5 µm, which was linked to a Teax frame grabber and a GPS geotagging system recording at an 8 Hz interval. We deﬁned the calibrated temperature range to 0–500 ◦C because it was assumed that this range would cover the expected apparent temperature ﬁeld at Ebeko volcano. Thermal data depend on various object and environmental properties, which here are mainly the emissivity of the object, the distance, solar reﬂection, viewing angle and the presence of particles/gases in the electromagnetic radiation path [57] and these properties need to be considered to improve the results [58]. In this study, we assumed a constant emissivity of 0.95, which is typical for volcanic environments [59] and a constant ﬂight height (300 m). Other environmental eﬀects that may inﬂuence the derived temperature, such as atmospheric attenuation, were considered using the radiometric Remote Sens. 2020, 12, 1961 5 of 20 correction theme in ThermoViewer software (Teax Technology, vs. 1.3.13), a transmissivity of 0.7, an environmental and path temperature of 10 ◦C and a humidity of 50%. In total, we recorded 4800 RGB images using the optical UAS camera and 11,300 thermal infrared images using the FLIR camera, all in the nadir ﬁeld of view. The image data set was manually controlled for artifacts and cleaned. 3.1. Remote Sensing by an Unoccupied Aircraft System (UAS) The thermal image data set was preprocessed in ThermoViewer software and exported in radiometric jpeg form. We processed all the UAS photogrammetric data (both optical and thermal infrared) using the Structure-from-Motion (SfM) computer vision approach [60]. Using Agisoft Metashape (vs. 1.5.2) software, we achieved point cloud reconstruction on optical and thermal data chunks to reconstruct the three-dimensional morphology, structure and temperature ﬁeld of the Ebeko volcano summit. Geoinformatics analysis was then performed in ArcGIS (vs. 10.2.1), where we investigated the morphology, identiﬁed thermal spots and measured the dimensions and changes in the optical and infrared dataset. Due to the diﬃcult and hazardous terrain, it was impractical to implement a ground control point (GCP) network and to create reliable georeferencing. The onboard GNSS geotagging that was used was yielding SfM products assumed to be consistent in the within-model horizontal distance measurement errors (0.2–0.3 m) being approximately one order of magnitude lower than the positional error [61]. We associated all models with respect to the best and largest UAS dataset acquired on 14 July 2019 (18:00–18:30 UTC) for accurate and consistent georeferencing by point matching the models. This reference model was based on 900 selected high-quality nadir photographs, yielding 39,000 tie points and a dense cloud consisting of 81 million points used to generate a 19 cm digital elevation model and a 9.8 cm orthomosaic with a total error of 1.9 m. The UAS ﬂights conducted just before and after the explosion were shorter and covered the active crater region only but were conducted at the same heights (300 m above the launch pad), yielding 12,000 tie points, 55 million dense points, DEMs of 20 cm and orthomosaics of 10 cm, respectively. The dataset was compared against the reference dataset, which yielded relative errors that had an RMS of 0.3 m for the optical dataset and 0.5 m for the thermal datasets used in this study. 3.2. Seismic Observations As a seismic unit, we used a 4.5 Hz three-component geophone (3D Geophone PE-6/B manufactured by SENSOR Nederland). A single unit was taken with us in the ﬁeld at a location (156.023057◦E, 50.694460◦N, 1028 m) very close (600 m) to the center of the active North Crater and was used to identify explosive events. The geophone instrument was buried at 1 m depth, ﬁxed in the ground using three steel spikes and covered by a plastic lid and then by tephra material for insulation. As a data logger, we used a DSS CUBE recorder to save the data with a sampling frequency of 50 Hz and internal batteries as a power supply, as well as a built-in GPS for accurate clock synchronization. The data were analyzed using the Python package Pyrocko and the Snuﬄer toolbox [62] to show the waveforms, perform event picking and investigate the spectral density associated with the explosive activity. We bandpassed the data (highpass 0.2, lowpass 2 Hz) and ﬁrst visually compared alleged explosion events to camera data. Based on this, we deﬁne an STA/LTA picker. Short events associated with small rockfalls or earthquakes were not further considered in this study. For spectrogram generation, we considered a window length of 100 s, overlapping 75%, with the Hamming taper function, to produce daily plots, allowing us to obtain an overview of the occurrence of explosions. All timings are given in UTC (local time is UTC+11). 3.3. Time-Lapse Camera Observations We installed time lapse cameras to remotely record the occurrence of explosions, determine wind drift during the explosions and help interpret the other data, such as to distinguish between explosions and outgassing events in the seismic records. Remote Sens. 2020, 12, 1961 6 of 20 The cameras we used were Reconyx UltraFire trail cameras, which were set to record images every 15 s at a resolution of 2304 × 1296 pixels, 72 dpi. The 5 mm lens has a full frame equivalent focal length of 37 mm. Internal lithium batteries powered the cameras. The time-lapse cameras have no internal GPS clock, which we controlled by synchronization to a GNSS station before the installation and controlled again at the end of the experiment for possible time drift (which were all found to be below the sampling interval and therefore considered nonsigniﬁcant). We placed three cameras on small tripods, one camera on the South Crater rim close to the UAS launchpad (156.0137◦E, 50.6852◦N, 1130 m) and the other two other to the east of the active crater, which were frequently covered by ash, which is why we concentrate our analysis on the South Crater rim camera. The camera data were then analyzed using kymograph recording [63,64], which is a sophisticated method of photo sequence analysis. We perform this analysis by considering time lapse camera image data stacks and by choosing a vertical line through the active crater in all images (2880 images in a 12-hr interval) and plotting the RGB pixel values of this line along a time axis. The kymograph is therefore a time-space plot that allows us to quickly identify the occurrence, height and duration of explosions as seen in the camera images (Figure 2). Remote Sens. 2020, 12, x FOR PEER REVIEW 6 of 21 small tripods, one camera on the South Crater rim close to the UAS launchpad (156.0137°E, 50.6852°N, 1130 m) and the other two other to the east of the active crater, which were frequently covered by ash, which is why we concentrate our analysis on the South Crater rim camera. The camera data were then analyzed using kymograph recording [63,64], which is a sophisticated method of photo sequence analysis. 3.3. Time-Lapse Camera Observations We perform this analysis by considering time lapse camera image data stacks and by choosing a vertical line through the active crater in all images (2880 images in a 12-hr interval) and plotting the RGB pixel values of this line along a time axis. The kymograph is therefore a time-space plot that allows us to quickly identify the occurrence, height and duration of explosions as seen in the camera images (Figure 2). Figure 2. Field deployment (a) perspective map view of the active Ebeko crater, location of the camera and the geophone seismic station. The unoccupied aircraft system (UAS) launch site and flight path (indicated by blue cameras) are shown. (b) Time lapse camera looking northward to the active New North Crater (steaming in background) and (c) geophone (yellow) installed and buried at 1 m depth recorded by a DSS data cube (right). (d) The UAS launch site and explosion at the active crater on 2019-07-13 at 23:40 UTC. Figure 2. Field deployment (a) perspective map view of the active Ebeko crater, location of the camera and the geophone seismic station. The unoccupied aircraft system (UAS) launch site and ﬂight path (indicated by blue cameras) are shown. (b) Time lapse camera looking northward to the active New North Crater (steaming in background) and (c) geophone (yellow) installed and buried at 1 m depth recorded by a DSS data cube (right). (d) The UAS launch site and explosion at the active crater on 2019-07-13 at 23:40 UTC. Figure 2. Field deployment (a) perspective map view of the active Ebeko crater, location of the camera and the geophone seismic station. The unoccupied aircraft system (UAS) launch site and flight path (indicated by blue cameras) are shown. (b) Time lapse camera looking northward to the active New North Crater (steaming in background) and (c) geophone (yellow) installed and buried at 1 m depth recorded by a DSS data cube (right). (d) The UAS launch site and explosion at the active crater on 2019-07-13 at 23:40 UTC. Figure 2. Field deployment (a) perspective map view of the active Ebeko crater, location of the camera and the geophone seismic station. The unoccupied aircraft system (UAS) launch site and ﬂight path (indicated by blue cameras) are shown. 4.1. General Geomorphology and Structural Analysis The ~9.8 cm resolution orthomosaic reveals details of the structures and, in comparison to the 19 cm DEM, a detailed geomorphological database (Figure 3). We clearly distinguish the three main craters (the South Crater, Middle Crater hosting a lake and North Crater) and the small new crater in the north referred to as the New North Crater, with areas of 42, 58, 80 and 21 × 10ˆ3 m2, respectively. The main craters are aligned NNE-SSW at an azimuth of N14◦E with very regular spacing; the centers of the South and Middle Craters are 236 m apart and those of the Middle and North Crater are 239 m apart. The center of the New North Crater is 110 m away from the center of the North Crater. As the crater morphologies overlap, their assemblage is nested, deﬁning an elongated NNE-SSW-oriented crater depression (Figure 3a). The diameters of the South, Middle and North Craters in the W-E direction are 284 m, 270 m and 304 m, respectively. The new and active crater (the New North Crater) located in the north of the North Crater has a W-E diameter of 211 m. The rim of this new crater is lower on the southern side (1090 m a.s.l.) than on the west (1102 m), north (1105 m) and east (1115 m). The inner walls are steep on the north side with slopes exceeding 60◦and gentle on the southern side, generally elongated from E-W (211 m E-W versus/165 m N-S, aspect ratio 1.28). The deepest point in the New North Crater is at 1045 m, meaning that the maximum depth of the crater is approximately 70 m. Therefore, the ﬂoor of the New North Crater is lower than the elevation of the North Crater (1085 m a.s.l.), lower than the lake level of the Middle Crater (1078 m) and below the elevation of the South Crater (1124 m). A large number of bombs and bomb sags can be seen surrounding the active crater (Figure 3a and Figure 6) but are also found at distances greater than 500 m. Some small subsidence pits also formed due to ash-covered snow melting above fumaroles and sites of former lakes (sp in Figure 3), as seen on the western rim of the North Crater or 200 m to the northeast of the active crater (Figure 3b). 3.3. Time-Lapse Camera Observations (b) Time lapse camera looking northward to the active New North Crater (steaming in background) and (c) geophone (yellow) installed and buried at 1 m depth recorded by a DSS data cube (right). (d) The UAS launch site and explosion at the active crater on 2019-07-13 at 23:40 UTC. 7 of 20 Remote Sens. 2020, 12, 1961 4. Results The results are presented in two parts. First, we present the general morphology and structural analysis together with thermal spot density distribution. Second, we describe the records associated with a major explosion. The results are presented in two parts. First, we present the general morphology and structural analysis together with thermal spot density distribution. Second, we describe the records associated with a major explosion. 4.1. General Geomorphology and Structural Analysis The orthomosaic of the thermal camera images reveals the largest anomaly inside the New North Crater, with apparent temperatures reaching 220 ◦C. We note that this temperature is likely damped by steaming of the crater seen in simultaneous optical images (compare Figure 3a,c). The Middle Crater is not expressed by high temperature and represents the coldest region. Temperature anomalies are found inside the South Crater at two main zones, each with a diameter ~50–120 m, but also on the crater ﬂanks (Figure 3i). In addition, we identify a large number of moderate temperature anomalies exceeding 50 ◦C on the eastern ﬂank of Ebeko volcano, following the dominant NNE-SSW alignment (Figure 3f). Some of these temperature anomalies have dimensions exceeding 50 m in diameter, representing distinct fumarole ﬁelds. Close comparison of aerial photos, digital terrain models and infrared images allows mapping of the fumaroles, thermal springs and other hot points. We distinguish 282 thermal spots, identiﬁed as localized thermal spots surrounded by a cold region. These thermal spots are clustered in four main ﬁelds, which are 36 spots in the New North Crater (Figure 3b), 42 spots in the South Crater (Figure 3b,h) and 118 spots on the east ﬂank at the locations of fumaroles and sulfur towers (Figure 3f). The thermal spots identiﬁed in the New North Crater reveal the highest temperatures; therefore, the small number of identiﬁed spots might be misleading. In addition, we ﬁnd some isolated anomalies, such as those temporarily exposed in the thick snow cover to the east of the lake-inﬁlled Middle Crater or at the sites of large bombs surrounding the active crater. Locations outside the active craters and the E-ﬂank are rare, with less than 8% of all thermal spots identiﬁed. Remote Sens. 2020, 12, 1961 Remote Sens. 2020, 12, x F 8 of 20 8 of 21 8 of 20 8 of 21 Figure 3. Results from the UAS survey. (a) Orthophoto generated from optical UAS camera data using the Structure from Motion (SfM) approach. (b) Thermal spots associated with hot springs and fumaroles identified from thermal cameras and optical data (shown by red dots) and cluster analysis (red shades indicate high clustering density) overlapped on a shaded relief digital terrain model generated from the optical UAS data. sp - subsidence pits. (c) Thermal infrared orthophoto. White boxes show close-ups as illustrated in the center and lowest row of the figure. 4.1. General Geomorphology and Structural Analysis Note that thermal spots cluster in fields, with a local alignment associated with ravine and ridge topography (panel e). Thermal spots are also located at sites of former eruption vents, as in the South Crater (panel i). The orthomosaic of the thermal camera images reveals the largest anomaly inside the New North Figure 3. Results from the UAS survey. (a) Orthophoto generated from optical UAS camera data using the Structure from Motion (SfM) approach. (b) Thermal spots associated with hot springs and fumaroles identiﬁed from thermal cameras and optical data (shown by red dots) and cluster analysis (red shades indicate high clustering density) overlapped on a shaded relief digital terrain model generated from the optical UAS data. sp—subsidence pits. (c) Thermal infrared orthophoto. White boxes show close-ups as illustrated in the center and lowest row of the ﬁgure. Note that thermal spots cluster in ﬁelds, with a local alignment associated with ravine and ridge topography (panel e). Thermal spots are also located at sites of former eruption vents, as in the South Crater (panel i). 4.2. Explosions Observed by Instrumental Network, Geomorphology and Structural Analysis Figure 3. Results from the UAS survey. (a) Orthophoto generated from optical UAS camera data using the Structure from Motion (SfM) approach. (b) Thermal spots associated with hot springs and fumaroles identified from thermal cameras and optical data (shown by red dots) and cluster analysis (red shades indicate high clustering density) overlapped on a shaded relief digital terrain model generated from the optical UAS data. sp - subsidence pits. (c) Thermal infrared orthophoto. White boxes show close-ups as illustrated in the center and lowest row of the figure. Note that thermal spots cluster in fields, with a local alignment associated with ravine and ridge topography (panel e). Thermal spots are also located at sites of former eruption vents, as in the South Crater (panel i). Figure 3. Results from the UAS survey. (a) Orthophoto generated from optical UAS camera data using the Structure from Motion (SfM) approach. (b) Thermal spots associated with hot springs and fumaroles identiﬁed from thermal cameras and optical data (shown by red dots) and cluster analysis (red shades indicate high clustering density) overlapped on a shaded relief digital terrain model generated from the optical UAS data. sp—subsidence pits. (c) Thermal infrared orthophoto. White boxes show close-ups as illustrated in the center and lowest row of the ﬁgure. 4.1. General Geomorphology and Structural Analysis Note that thermal spots cluster in ﬁelds, with a local alignment associated with ravine and ridge topography (panel e). Thermal spots are also located at sites of former eruption vents, as in the South Crater (panel i). The orthomosaic of the thermal camera images reveals the largest anomaly inside the 4.2. Explosions Observed by Instrumental Network, Geomorphology and Structural Analysis The orthomosaic of the thermal camera images reveals the largest anomaly inside the Explosions Observed by Instrumental Network, Geomorphology and Structural Analysis Crater, with apparent temperatures reaching 220 °C. We note that this temperature is likely damped by steaming of the crater seen in simultaneous optical images (compare Figure 3a,c). The Middle Crater is not expressed by high temperature and represents the coldest region. Temperature The instrumental network provided important data for contextualization of the remote sensing observations from UAS data. Explosions exclusively occurred in the New North Crater and no activity was observed in the other craters. Our camera allowed for the visualization of the occurrence, wind drift and scale of the explosions. Remote Sens. 2020, 12, 1961 9 of 20 The comparison of the camera kymograph with the seismic spectrogram reveals a very good agreement of the retrieved event information during clear sky conditions (Figure 4), with 34 events during daytime (black arrow symbology in Figure 4b). The seismometer allowed the counting of explosion events, which represented 192 events with durations over 5 s, with an average of 34 min intervals between the eruptions for the entire measurement period of 10–14 July 2019. However, a closer look at the records documents very uneven activity, as revealed on the clear sky day of 13 July 2019, when the camera kymograph allows for the conﬁrmation of the occurrence of dark ash-laden clouds. In the ﬁrst 6 h of daylight, only minor activity was associated with steaming and mild explosions, such as at 20:05 UTC (8:05 local time), drifting westwards. Then, eruptive activity strongly increased, with 11 major events in the second half of the day, the strongest identiﬁed by camera at 23:40, 1:15 and 8:29 UTC. The seismic data generally conﬁrm this but also reveal a number of events in the < 10 Hz spectral frequency (although with smaller amplitudes and durations), associated with degassing pulses and white plumes, as observed by the cameras. The between-explosion activity is mainly related to degassing, as observed by the white steam plume escaping from the New North Crater. The strongest steaming occurs immediately after explosions, gradually decreasing until almost virtual absence before the next explosion is initiated. The maximum eruption heights cannot be deduced from the close camera location, but the seismic strength and duration of the signal concurs with the visual strength of the eruption, exceeding the image frame in height (which was set to 500 m, Figure 3c). Remote Sens. 2020, 12, x FOR PEER REVIEW 10 of 21 Figure 4. Instrumental records. The orthomosaic of the thermal camera images reveals the largest anomaly inside the Explosions Observed by Instrumental Network, Geomorphology and Structural Analysis (a) Camera kymograph generated by the vertical RGB line (y-axis) versus time (x-axis) shows 12 main eruptions on one day (dusk to dawn on 2018-07-13 to 2018-07-14 UTC). The first large event after > 3-h of quiescence occurred at 23:40. (b) Spectrogram generated for seismic station show clear explosion occurrences (dark vertical lines). The events picked using the LTA-STA trigger are indicated by small black arrows. Eruption at 23:40 monitored closely by the UAS is highlighted by white timing. Note that the occurrence of eruption multiplets (at 20:06 and 8:29) was preceded and followed by pronounced lag times. Figure 4. Instrumental records. (a) Camera kymograph generated by the vertical RGB line (y-axis) versus time (x-axis) shows 12 main eruptions on one day (dusk to dawn on 2018-07-13 to 2018-07-14 UTC). The ﬁrst large event after > 3-h of quiescence occurred at 23:40. (b) Spectrogram generated for seismic station show clear explosion occurrences (dark vertical lines). The events picked using the LTA-STA trigger are indicated by small black arrows. Eruption at 23:40 monitored closely by the UAS is highlighted by white timing. Note that the occurrence of eruption multiplets (at 20:06 and 8:29) was preceded and followed by pronounced lag times. Figure 4. Instrumental records. (a) Camera kymograph generated by the vertical RGB line (y-axis) versus time (x-axis) shows 12 main eruptions on one day (dusk to dawn on 2018-07-13 to 2018-07-14 Figure 4. Instrumental records. (a) Camera kymograph generated by the vertical RGB line (y-axis) versus time (x-axis) shows 12 main eruptions on one day (dusk to dawn on 2018-07-13 to 2018-07-14 Figure 4. Instrumental records. (a) Camera kymograph generated by the vertical RGB line (y-axis) versus time (x-axis) shows 12 main eruptions on one day (dusk to dawn on 2018-07-13 to 2018-07-14 UTC). The first large event after > 3-h of quiescence occurred at 23:40. (b) Spectrogram generated for seismic station show clear explosion occurrences (dark vertical lines). The events picked using the LTA-STA trigger are indicated by small black arrows. Eruption at 23:40 monitored closely by the UAS is highlighted by white timing. Note that the occurrence of eruption multiplets (at 20:06 and 8:29) was preceded and followed by pronounced lag times. Figure 4. Instrumental records. (a) Camera kymograph generated by the vertical RGB line (y-axis) versus time (x-axis) shows 12 main eruptions on one day (dusk to dawn on 2018-07-13 to 2018-07-14 UTC). The orthomosaic of the thermal camera images reveals the largest anomaly inside the Explosions Observed by Instrumental Network, Geomorphology and Structural Analysis The ﬁrst large event after > 3-h of quiescence occurred at 23:40. (b) Spectrogram generated for seismic station show clear explosion occurrences (dark vertical lines). The events picked using the LTA-STA trigger are indicated by small black arrows. Eruption at 23:40 monitored closely by the UAS is highlighted by white timing. Note that the occurrence of eruption multiplets (at 20:06 and 8:29) was preceded and followed by pronounced lag times. We investigate the 23:40 eruption in further detail (Figure 5). The camera data show an eruption cloud initiating at 23:39:45, exceeding 200 m 15 s later, drifting westward and reaching a height of 400 m at 23:40:15 and above 500 m (which is the image limit) at 23:40:45 UTC. The eruption lasted at least 2 min, which was also confirmed in the seismic data (Figure 5). In fact, the seismogram suggests We investigate the 23:40 eruption in further detail (Figure 5). The camera data show an eruption cloud initiating at 23:39:45, exceeding 200 m 15 s later, drifting westward and reaching a height of 400 m at 23:40:15 and above 500 m (which is the image limit) at 23:40:45 UTC. The eruption lasted at least 2 min, which was also conﬁrmed in the seismic data (Figure 5). In fact, the seismogram suggests 10 of 20 Remote Sens. 2020, 12, 1961 that the eruption initiates rapidly, possibly in two pulses, with a power spectral density peak at 4 Hz. The spectrogram reveals a > 10 Hz frequency lasting 160 s and a much longer and lower frequency of < 10 Hz lasting approximately 420 s. The comparison of optical and seismic data suggests that the higher frequency is dominated during explosions, followed by a < 10 Hz frequency signal tail associated with gas jets and steaming activity. Remote Sens. 2020, 12, x FOR PEER REVIEW 11 of 21 Figure 5. Geophone seismic records (radial component, E-W) and camera documentation of the 23:40 eruption event on 13 July 2019. (a) Seismogram of the 23:40 event, revealing a rapid emergence, two main amplitude peaks and a long coda. (b) The Power Spectral Density (PSD) indicates a peak at 3–4 Hz. (c) The spectrogram reveals a dominant lower frequency but also a 2-min lasting 10–40 Hz frequency episode associated with bomb and rock falls. (d) Photographs of the 23:40 event. Eruption height exceeded 400 m, airfall towards the west (left in north-looking camera view). The orthomosaic of the thermal camera images reveals the largest anomaly inside the Explosions Observed by Instrumental Network, Geomorphology and Structural Analysis Figure 5. Geophone seismic records (radial component, E-W) and camera documentation of the 23:40 eruption event on 13 July 2019. (a) Seismogram of the 23:40 event, revealing a rapid emergence, two main amplitude peaks and a long coda. (b) The Power Spectral Density (PSD) indicates a peak at 3–4 Hz. (c) The spectrogram reveals a dominant lower frequency but also a 2-min lasting 10–40 Hz frequency episode associated with bomb and rock falls. (d) Photographs of the 23:40 event. Eruption height exceeded 400 m, airfall towards the west (left in north-looking camera view). Figure 5. Geophone seismic records (radial component, E-W) and camera documentation of the 23:40 eruption event on 13 July 2019. (a) Seismogram of the 23:40 event, revealing a rapid emergence, two main amplitude peaks and a long coda. (b) The Power Spectral Density (PSD) indicates a peak at 3–4 Hz. (c) The spectrogram reveals a dominant lower frequency but also a 2-min lasting 10–40 Hz frequency episode associated with bomb and rock falls. (d) Photographs of the 23:40 event. Eruption height exceeded 400 m, airfall towards the west (left in north-looking camera view). Figure 5. Geophone seismic records (radial component, E-W) and camera documentation of the 23:40 eruption event on 13 July 2019. (a) Seismogram of the 23:40 event, revealing a rapid emergence, two main amplitude peaks and a long coda. (b) The Power Spectral Density (PSD) indicates a peak at 3–4 Hz. (c) The spectrogram reveals a dominant lower frequency but also a 2-min lasting 10–40 Hz frequency episode associated with bomb and rock falls. (d) Photographs of the 23:40 event. Eruption height exceeded 400 m, airfall towards the west (left in north-looking camera view). UAS flights conducted in the 20 min immediately before and after the 23:40 eruption indicate mportant changes (Figure 6) inside and surrounding the active crater. A comparison of apparent emperature fields reveals that before the explosion, the outer flanks are cold and the inner crater is expressed by apparent temperatures mainly between 120 and 160 °C (Figure 6a,d). The flight onducted immediately after the explosions shows a pronounced deposition field 81,200 m2 in size, marked by elevated temperature on the outer NE flank of the active crater (Figure 6b), which is in agreement with the prevailing wind direction of N70°W. The orthomosaic of the thermal camera images reveals the largest anomaly inside the Explosions Observed by Instrumental Network, Geomorphology and Structural Analysis The temperature-derived deposition zone s composed of a large number of small and distinguishable particles, which were interpreted to be bombs and fine materials (Figure 6b). The temperature decreases with distance to the New North Crater, but the isolines of equal temperature show some pronounced complexities (Figure 6c). Temperature isolines show a strong morphological dependency, such as the inner part of the large North Crater being elevated in temperature (Figure 6c). The temperature field inside the active crater UAS ﬂights conducted in the 20 min immediately before and after the 23:40 eruption indicate important changes (Figure 6) inside and surrounding the active crater. A comparison of apparent temperature ﬁelds reveals that before the explosion, the outer ﬂanks are cold and the inner crater is expressed by apparent temperatures mainly between 120 and 160 ◦C (Figure 6a,d). The ﬂight conducted immediately after the explosions shows a pronounced deposition ﬁeld 81,200 m2 in size, marked by elevated temperature on the outer NE ﬂank of the active crater (Figure 6b), which is in agreement with the prevailing wind direction of N70◦W. The temperature-derived deposition zone is composed of a large number of small and distinguishable particles, which were interpreted to be bombs and ﬁne materials (Figure 6b). The temperature decreases with distance to the New North Crater, but the isolines of equal temperature show some pronounced complexities (Figure 6c). Temperature isolines show a strong morphological dependency, such as the inner part of the large North Crater being elevated in temperature (Figure 6c). The temperature ﬁeld inside the active crater at this time 11 of 20 Remote Sens. 2020, 12, 1961 is between 160 and 220 ◦C for most of the pixels and therefore strongly increased compared to the pre-eruption survey. Remote Sens. 2020, 12, x FOR PEER REVIEW 12 of 21 Figure 6. New North Crater before and after the 23:40 event on 13 July 2019 recorded by a UAS thermal camera. (a) Before the eruption, the thermal infrared orthomosaic shows main temperature zones. The enlarged crater area (panel d) is indicated by a white rectangle. Note that the data are the same as in Figure 3c but with different color scales to allow comparison to UAS data after the explosion. (b) After the eruption, the temperature increased inside the active crater but also on the western flank, indicating the deposition zone. Enlarged area (panel e). The orthomosaic of the thermal camera images reveals the largest anomaly inside the Explosions Observed by Instrumental Network, Geomorphology and Structural Analysis (c) Temperature isolines (interval 20 ◦C) derived from data shown in panel b. (d–f) Enlarged inner crater. (f) Slope map calculated by the gradient of the digital terrain model, revealing structures aligned along the previous crater rim. y g g g g g p crater rim. Structural analysis reveals that a clear WNW-ESE temperature gradient is seen in the northern inner slope of the crater. This gradient can be better understood by considering the morphological scars that are oriented WNW-ESE, as shown in the slope map (Figure 6f), implying that the temperature field (and deposition of hot fresh material) is affected by morphology and/or by the fluid pathways changing at the rim of the previous North Crater where this New North Crater has evolved. An interesting finding is the zonation of the temperature fields inside the crater, revealing a generally elevated crater field, a main field with increased temperature and an inner field. The inner field is l t d i th NNE SSW di ti d th f i il t th t f th i t Structural analysis reveals that a clear WNW-ESE temperature gradient is seen in the northern inner slope of the crater. This gradient can be better understood by considering the morphological scars that are oriented WNW-ESE, as shown in the slope map (Figure 6f), implying that the temperature ﬁeld (and deposition of hot fresh material) is aﬀected by morphology and/or by the ﬂuid pathways changing at the rim of the previous North Crater where this New North Crater has evolved. An interesting ﬁnding is the zonation of the temperature ﬁelds inside the crater, revealing a generally elevated crater ﬁeld, a main ﬁeld with increased temperature and an inner ﬁeld. The inner ﬁeld is elongated in the NNE-SSW direction and therefore similar to the arrangements of the main craters and the alignment of temperature anomalies and thermal spots on the ﬂank of Ebeko volcano. The orthomosaic of the thermal camera images reveals the largest anomaly inside the Explosions Observed by Instrumental Network, Geomorphology and Structural Analysis (c) Temperature isolines (interval 20 °C) derived from data shown in panel b. (d–f) Enlarged inner crater. (f) Slope map calculated by the gradient of the digital terrain model revealing structures aligned along the previous Figure 6. New North Crater before and after the 23:40 event on 13 July 2019 recorded by a UAS thermal camera. (a) Before the eruption, the thermal infrared orthomosaic shows main temperature zones. The enlarged crater area (panel d) is indicated by a white rectangle. Note that the data are the same as in Figure 3c but with diﬀerent color scales to allow comparison to UAS data after the explosion. (b) After the eruption, the temperature increased inside the active crater but also on the western ﬂank, indicating the deposition zone. Enlarged area (panel e). (c) Temperature isolines (interval 20 ◦C) derived from data shown in panel b. (d–f) Enlarged inner crater. (f) Slope map calculated by the gradient of the digital terrain model, revealing structures aligned along the previous crater rim. Figure 6. New North Crater before and after the 23:40 event on 13 July 2019 recorded by a UAS thermal camera. (a) Before the eruption, the thermal infrared orthomosaic shows main temperature zones. The enlarged crater area (panel d) is indicated by a white rectangle. Note that the data are the same as in Figure 3c but with different color scales to allow comparison to UAS data after the explosion. (b) After the eruption, the temperature increased inside the active crater but also on the western flank, indicating the deposition zone. Enlarged area (panel e). (c) Temperature isolines (interval 20 °C) derived from data shown in panel b. (d–f) Enlarged inner crater. (f) Slope map l l d b h di f h di i l i d l li li d l h i Figure 6. New North Crater before and after the 23:40 event on 13 July 2019 recorded by a UAS thermal camera. (a) Before the eruption, the thermal infrared orthomosaic shows main temperature zones. The enlarged crater area (panel d) is indicated by a white rectangle. Note that the data are the same as in Figure 3c but with diﬀerent color scales to allow comparison to UAS data after the explosion. (b) After the eruption, the temperature increased inside the active crater but also on the western ﬂank, indicating the deposition zone. Enlarged area (panel e). 5.1. Limitations and Performance 5.1. Limitations and Performance f While the instruments and analysis methods used provide the first detailed accounts of the new eruption episode at Ebeko volcano, a number of limitations are relevant to mention. Most of our analysis is based on UAS data. We used a simple consumer drone with a 30-min flight time, which, when carrying the FLIR Tau2 thermal infrared camera, was reduced to only 18–20 min. Therefore, even though multiple UAS flights were conducted, we could not monitor the initiation of an eruption inside the crater by UAS. Observation of the initiation of an eruption by UAS may shed some light on the surface expression of the conduit geometry. Our UAS flights were conducted before and after eruptions so that we could only approximately interpret the location and dimension of the surface expression of the conduit While the instruments and analysis methods used provide the ﬁrst detailed accounts of the new eruption episode at Ebeko volcano, a number of limitations are relevant to mention. Most of our analysis is based on UAS data. We used a simple consumer drone with a 30-min ﬂight time, which, when carrying the FLIR Tau2 thermal infrared camera, was reduced to only 18–20 min. Therefore, even though multiple UAS ﬂights were conducted, we could not monitor the initiation of an eruption inside the crater by UAS. Observation of the initiation of an eruption by UAS may shed some light on the surface expression of the conduit geometry. Our UAS ﬂights were conducted before and after eruptions so that we could only approximately interpret the location and dimension of the surface expression of the conduit. expression of the conduit. As the UAS flights were conducted during a very high activity phase of the volcano, with tephra and even decimeter-sized bombs found at distances exceeding 500 m, we were not able to record ground control points (GCPs). This limitation comes with increased uncertainties of the absolute position and possible error and artifact propagation. We attempted to control this uncertainty by comparing the SfM point clouds against each other, but cannot rule out any systematic positioning errors. g and the alignm 5. Discussion 5. Discussion Ebeko volcano displays Vulcanian explosive activity at the northern edge of the North Crater, where a new 70-m deep explosive funnel has evolved. Using the UAS data, details of the geomorphology and structures of this New North Crater and its environment were mapped. The digital terrain model together with orthophotos and infrared imagery reveals that the crater formed on the northern continuation of a crater row aligned NNE-SSW and was strongly affected by other pre-existing structures as sketched in Figure 7. We compared the results to in situ geophysical observations and were able to determine (i) the general eruption frequency and NNE-SSW alignment of partially overlapping craters, (ii) the NNE-SSW alignment of temperature anomaly fields, (iii) the expression of ~300 thermal spots, (iv) the geometry of the newly formed crater and its inner l d h d f l d h l l Ebeko volcano displays Vulcanian explosive activity at the northern edge of the North Crater, where a new 70-m deep explosive funnel has evolved. Using the UAS data, details of the geomorphology and structures of this New North Crater and its environment were mapped. The digital terrain model together with orthophotos and infrared imagery reveals that the crater formed on the northern continuation of a crater row aligned NNE-SSW and was strongly aﬀected by other pre-existing structures as sketched in Figure 7. We compared the results to in situ geophysical observations and were able to determine (i) the general eruption frequency and NNE-SSW alignment of partially overlapping craters, (ii) the NNE-SSW alignment of temperature anomaly ﬁelds, (iii) the expression of ~300 thermal spots, (iv) the geometry of the newly formed crater and its inner temperature anomaly and (v) the deposition of new materials associated with a single explosion. These results are novel for the Ebeko site but may also help to better understand other volcanoes where access and structures are 12 of 20 Remote Sens. 2020, 12, 1961 diﬃcult to reach, highlighting the eﬃcient use of UAS together with time lapse and geophone records for temporal monitoring. In the following, we ﬁrst discuss the limitations and performance of our approaches and then elaborate on the structural control as well as the wider implications of this work. g and the alignm 5. Discussion emote Sens 0 0, , O EE E IE 3 o limitations and performance of our approaches and then elaborate on the structural control as well as the wider implications of this work. Figure 7. Synthesis of the structural architecture and analysis of this work highlighting the fluid pathways affected by inferred volcano-tectonic structures. Labels 1 to 5 describe (1) the general eruption frequency and NNE-SSW alignment of partially overlapping craters, (2) the NNE-SSW alignment of fumarole thermal fields obscured on smaller scales by morphology, (3) the expression of ~300 thermal spots, (4) the elongation of the newly formed crater and its inner elliptical temperature anomaly possibly associated with an elliptical conduit and (5) the deposition of new materials. The presence of shallow reservoirs or an aquifer is based on earlier studies [42,54]. Figure 7. Synthesis of the structural architecture and analysis of this work highlighting the ﬂuid pathways aﬀected by inferred volcano-tectonic structures. Labels 1 to 5 describe (1) the general eruption frequency and NNE-SSW alignment of partially overlapping craters, (2) the NNE-SSW alignment of fumarole thermal ﬁelds obscured on smaller scales by morphology, (3) the expression of ~300 thermal spots, (4) the elongation of the newly formed crater and its inner elliptical temperature anomaly possibly associated with an elliptical conduit and (5) the deposition of new materials. The presence of shallow reservoirs or an aquifer is based on earlier studies [42,54]. Figure 7. Synthesis of the structural architecture and analysis of this work highlighting the fluid pathways affected by inferred volcano-tectonic structures. Labels 1 to 5 describe (1) the general eruption frequency and NNE-SSW alignment of partially overlapping craters, (2) the NNE-SSW alignment of fumarole thermal fields obscured on smaller scales by morphology, (3) the expression of ~300 thermal spots, (4) the elongation of the newly formed crater and its inner elliptical temperature anomaly possibly associated with an elliptical conduit and (5) the deposition of new materials. The presence of shallow reservoirs or an aquifer is based on earlier studies [42,54]. Figure 7. Synthesis of the structural architecture and analysis of this work highlighting the ﬂuid pathways aﬀected by inferred volcano-tectonic structures. g and the alignm 5. Discussion Labels 1 to 5 describe (1) the general eruption frequency and NNE-SSW alignment of partially overlapping craters, (2) the NNE-SSW alignment of fumarole thermal ﬁelds obscured on smaller scales by morphology, (3) the expression of ~300 thermal spots, (4) the elongation of the newly formed crater and its inner elliptical temperature anomaly possibly associated with an elliptical conduit and (5) the deposition of new materials. The presence of shallow reservoirs or an aquifer is based on earlier studies [42,54]. 5.1. Limitations and Performance 5.1. Limitations and Performance Moreover, the coverage by the UAS, although flying over the most relevant parts of the volcano was not ideal as the western flank and the surroundings could not be included in the As the UAS ﬂights were conducted during a very high activity phase of the volcano, with tephra and even decimeter-sized bombs found at distances exceeding 500 m, we were not able to record ground control points (GCPs). This limitation comes with increased uncertainties of the absolute position and possible error and artifact propagation. We attempted to control this uncertainty by comparing the SfM point clouds against each other, but cannot rule out any systematic positioning errors. Moreover, the coverage by the UAS, although ﬂying over the most relevant parts of the volcano, was not ideal, as the western ﬂank and the surroundings could not be included in the analysis. This Remote Sens. 2020, 12, 1961 13 of 20 limitation was due to the rapidly changing wind conditions and fog/cloud formation, especially on the western ﬂanks. The thermal infrared UAS survey was conducted from a ﬂight height of 300 m above the launch pad (1142 m a.s.l) so that the base of the New North Crater was seen in the UAS camera images from a distance of approximately 400 m. Precise measurements of the apparent temperature strongly depend on distance to the target [65], so the true temperature may be underestimated by our measurements. Independent measurements at fumaroles suggest temperatures up to 490 ◦C [52] or even exceeding 500 ◦C in the new explosive funnel in 2015 [53]. In addition, steaming activity may dampen the infrared signal and reduce the apparent temperature, while the strength of the method is that the structural analysis is less aﬀected by such parameters [18,28]. We therefore note that the true temperature might be higher than the measured temperature, although the temperatures of only 160–220 ◦C in the inner crater as measured immediately (within ~20 min) after an explosion were indeed surprising. 5.1. Limitations and Performance 5.1. Limitations and Performance While we cannot explain these low temperatures, we may speculate that (i) the eruptive products are indeed relatively low temperatures due to cooling by surface water (the crater ﬂoor is lower than the nearby cold water level in the maar-like Middle Crater), (ii) ﬁne-grained (possibly remobilized) ash quickly covers the materials and dampens the temperature or (iii) our measurements are biased by measurement artifacts arising from small-scale thermal structures, large measurement distances and steaming eﬀects. The measurements performed using time lapse cameras and a single geophone instrument gave simple but highly useful information on eruption occurrence, wind drift and eruption strength. The instruments were properly working and were hence used for a ﬁrst-order characterization of the eruption activity and intervals. The comparison of camera and seismic records indicated that none of the methods alone would have been complete, as we observed strong seismicity during periods of degassing (which by mistake could be interpreted as an explosion), as well as strong nearly horizontal westward drift of eruption plumes (which by mistake might have been missed by the cameras placed further east on the volcano slopes). As no other systematic measurements are accessible for the earlier activity episodes at Ebeko, we cannot compare our results to previous records. 5.2. Structural Inﬂuence Using the UAS data, we identiﬁed approximately 300 thermal spots. Earlier volcanologic mapping was conducted based on aerial photography but dates back over 30 years (the most comprehensive photogrammetric survey was in 1988) and could distinguish between thermal areas and main fumaroles and pots of thermal waters, counting only 25 such sites [41]. As no thermal infrared camera was available during this time, the work relied on a combination of remote sensing and ﬁeld validation. However, as previous researchers noted the migration of fumarole sites by as much as 200 m (as in 1983; [40]), our detailed and accessible maps provide a unique basis for further studies on the dynamics of thermal spots and fumaroles. Previous studies reveal that both magmatic and hydrothermal activity is migrating, appearing at one site while ceasing at another site, with possible systematic trends following the NNE-SSW structures. The eruptive activity at Ebeko migrated back and forth along the NNE-SSW crater row, with explosions from the Middle Crater (1934-35, 1965, 2011 and 2016-2017) and from the North Crater (1967-1971, 1987-1991, 2009-2010, 2016-20) and some of the cases exhibited simultaneous explosions at both craters. In addition to the changing vent positions, a migration of individual fumaroles and thermal ﬁelds was also identiﬁed. One may therefore conjecture that ﬂuid pathways seal and reopen, with systematics that on a large scale are strongly controlled by the hypothesized NNE-SSW fracture system. Furthermore, we found it puzzling why maar-like lakes occasionally developed along this system, either cold (as currently in the Middle Crater) or hot (as it was in 2005–2007 in the North Crater). Our ﬁndings suggest that both magmatic pathways and geothermal pathways are strongly structurally controlled by either fracture sets (e.g., the E-ﬂank thermal ﬁeld) and/or pre-existing vents (e.g., thermal ﬁeld in the South Crater) as summarized in Figure 7. A cold water inﬁll in the maar-like 14 of 20 14 of 20 Remote Sens. 2020, 12, 1961 Middle Crater may indicate that thermal ﬂuids are not rising there, which could be associated with a currently impermeable system. We may conjecture that the diﬀerent magmatic and geothermal pathways are either competing with each other, where activity at one venting site causes a decrease in the other venting site or are temporally connected. 5.3. Eruption Dynamics The eruption dynamics at Ebeko are characterized by Vulcanian eruptions [47] forming bread-crust bombs exceeding 5 m in diameter. Another recent study tested the performance of the atmospheric electric potential gradient, allowing monitoring of the passage of ash plumes over Severo-Kurilsk [67]. Using our seismic and camera records, we could not measure the far ﬁeld drift but the occurrence of the explosions, as well as the associated near-ﬁeld ash plume and eﬀects. For the four-day period of 10–14 July 2019, we counted 192 events, yielding an average interval of 34 min between the onset of eruptions. We also note (see Section 4.2), however, a strong irregular activity. On 13 July 2019, in the ﬁrst 6 h of daylight, only minor activity was associated with steaming and mild explosions, such as at 20:05 UTC (8:05 local time), strongly increasing later that day with 11 major events in the second half of the day (strongest 23:40, 1:15 and 8:29 UTC, respectively). We further ﬁnd that eruptions often occur in clusters, with double or triple eruptions occurring within a few minutes only, followed by pronounced periods of quiescence. While our short data set and limited number of events do not warrant a more rigorous statistical testing of this idea, the repose gap and clustered explosions might be comparable to the behavior of other (Strombolian type) eruptions at volcanoes [68,69] and geysers [70]. The comparison to Strombolian and geyser explosions is particularly interesting, as the dynamics of Vulcanian-type eruptions also shows periodic behavior and recharge-time-dependent eruption size [71]. A similarity further exists in the presence of double or triple eruptions, i.e., eruptions occurring in very close succession followed by a long repose period (approximately 3 h in our dataset, see also Figure 3c). Therefore, one may speculate that the eruption dynamics at Ebeko are an example of Vulcanian style explosions that show an apparent irregularity modulated by explosions that occur in close succession. Whether this mechanism is controlled from a deep depth by rising ﬂuids, at shallow depths or even at aquifer levels is diﬃcult to ascertain from our available data. 5.2. Structural Inﬂuence It is possible that hydrothermal activity changes the permeability, as has been proposed at other locations [66], which may help us to understand the sealing of one vent and the reopening of the other vent. Eye-witness accounts during earlier explosions suggest that the activity occurred simultaneously from two opposing vents at the active funnel of the North Crater, which may support this hypothesis of temporally connected pathways. Most of the identiﬁed thermal spots cluster in ﬁelds described earlier (e.g., [46,51,52,54,56]). These ﬁelds elongate along E-W ravines but also cluster in a NNE-SSW direction, similar to the arrangements of the nested craters (South, Middle, North Crater and New North crater). We identify a weakly expressed but clearly distinguished zone of thermal ﬁelds aligned on the eastern ﬂank of Ebeko. This large-scale (ediﬁce-scale) feature is not similarly seen on smaller scales. Considering any singular thermal ﬁeld, the distribution of thermal spots appears to be aﬀected by morphology and other local eﬀects and are often aligned subparallel to erosional ravines and ridges, which are east-west on the eastern slope of Ebeko. This observation, a large-scale structural control obscured by morphology and ravines on a smaller scale, is in line with ﬁndings made elsewhere [15], highlighting the importance of the scales under consideration. In this view, structural control is dominant on a large ediﬁce scale but obscured on smaller scales by morphology and other eﬀects. 5.5. Final Remark on Hazard Aspects This study is based on one of the ﬁrst comprehensive measurements realized for decades at this site and may motivate forthcoming researchers to conduct more dedicated surveys or even implement continuous monitoring. Ebeko is a unique laboratory case for studying volcano-tectonic structures and adds a further destination for the study of Vulcanian style behavior. Ebeko volcano poses a signiﬁcant hazard to the local population. A 2500-year reconstruction of the eruptive history of Ebeko [78] showed that all kinds of hazards may be expected; the most relevant hazards for the local population are the eﬀusion of lava ﬂows, tephra falls, passing eruptive plumes and lahars, which is further complemented by signiﬁcant hazards to the aviation industry [79]. In recent years, and during our stay, the volcano sends gases and tephra dominantly to easterly directions, resulting in gases and tephra falls aﬀecting the town of Severo-Kurilsk. Following a catastrophic tsunami in 1952, the town was relocated, unfortunately now being built on alluvial fans of the main rivers Kuz’minka and Matrosskaya originating near Ebeko volcano, with high lahar risk that can reach the town within 20–30 min without any warning [78]. Therefore, close monitoring of the volcano using a combination of in-situ and remote methods is recommended for early warning. 5.4. Eruption Deposition and Isopach Analysis Tephra deposition, in particular, may obscure the structural architecture of volcanoes, but is diﬃcult to monitor and quantify in the ﬁeld. Analysis of tephra allows us to derive important information on explosive eruptions and intensity and allows indirect assessment of eruption height and dynamics [72]; therefore, parameters on tephra depositions and thickness are critical for the assessment of related volcano hazards [73,74]. In particular, information on the dispersion and distribution of 15 of 20 15 of 20 Remote Sens. 2020, 12, 1961 tephra surrounding explosive vents is essential for understanding the volcano [75]; however, data are commonly limited due to poor preservation and lack of access to the deposition zones. As pointed out earlier [76], both the proximal and distal locations are challenging to elaborate in the ﬁeld, despite the relevance for understanding those deposits. In particular, the proximal region of deposition is complex due to local plume and topographic eﬀects. Characterization of the gradient of deposit thickness and clast size is aﬀected by subjectivity and has remained challenging and rarely preserved due to post-eruption processes, which is why traditional isopach reconstructions are associated with major uncertainties and subjective decisions [77]. Accurate ﬁeld assessments are highly relevant for developing improved modeling and forecasting techniques [74,76]. As we show, the UAS data may be used for tephra deposit isopach mapping and together with thermal sensors, even allows us to record the associated temperature eﬀects. We demonstrate that the deposition area can be deduced by a temperature anomaly, indicating complex isolines associated with local-scale morphology. We did not attempt to compute the volume of the deposition, which in future works can be regularly inferred from DEM diﬀerences under consideration of ground control points. 6. Conclusions We achieved a geophysical, geomorphological and structural analysis at Ebeko volcano, a site of intense Vulcanian explosions in 2019. We conducted repeat UAS campaigns and investigated seismic and camera records. The data reveal the occurrence of ~200 explosive events during the recording period, with an eruption interval of 34 min on average, although we noted that the explosions and lag times were not regular. We could generate cm-scale digital terrain models and orthomosaics of the optical and thermal infrared cameras carried by the UAS over the erupting crater. We also found that this new crater follows the general NNE-SSW alignment of the chain of craters that developed since the beginning of the Holocene [80]. The new crater, which initiated as a small explosive funnel on the northern rim of the former North Crater, has now evolved dimensions that are comparable to those of the other craters. The depth of the new crater is even below the water level of the maar-like Middle Crater and is aﬀected by other pre-existing structures. In summary, our analysis (Figure 7) revealed (i) the general eruption frequency and NNE-SSW alignment of partially overlapping craters, (ii) the NNE-SSW alignment of fumarole thermal ﬁelds, which are often obscured at smaller scales by morphology, (iii) the expression of ~300 thermal spots, (iv) the elongation of the newly formed crater and its inner temperature anomaly and (v) the deposition of new materials associated with a single explosion. These results are novel for the Ebeko site, but may also help to better understand other volcanoes where access and structures are 16 of 20 Remote Sens. 2020, 12, 1961 16 of 20 diﬃcult to reach, highlighting the eﬃcient use of UAS together with time lapse and geophone stations for temporal monitoring. diﬃcult to reach, highlighting the eﬃcient use of UAS together with time lapse and geophone stations for temporal monitoring. Author Contributions: Drone data, seismic data and time-lapse camera data were collected by T.R.W., A.B. and M.B. Data processing, SfM, seismic data analysis and kymography by T.R.W. Volcanologic observations and historical analysis by A.B., M.B. and T.K. Dynamic interpretation and discussion contribution by A.A. T.R.W. wrote the manuscript with contributions of all authors. All authors have read and agreed to the published version of the manuscript. Funding: This is a contribution to VOLCAPSE, a research project funded by the European Research Council under the European Union’s H2020 Programme/ERC consolidator grant n. 6. Conclusions [ERC-CoG 646858]. Acknowledgments: The manuscript beneﬁted from thoughtful reviews by Tom Pering and an anonymous reviewer. We thank Stefan Mikulla for assisting the instrument preparation and Sebastian Heimann for help with his Pyrocko and snuﬄer python scripts (https://pyrocko.org/). We appreciate the hospitality and organization help by Oleg Stuchinsky and the staﬀof the seismic observatory of Russian Geophysical Survey in Severo-Kurilsk. We additionally thank to ESA for providing Sentinel-2 data. All data presented is available on request from the corresponding author of this study. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Gudmundsson, A. 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In Proceedings of the Volcanic Ash and Aviation Safety: First International Symposium, Seattle, DC, USA, 8–12 July 1991; pp. 57–64. © 2020 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/). © 2020 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/W3186472623	https://www.mdpi.com/2223-7747/10/7/1465/pdf?version=1626769002	English	null	Identification and Expression Analysis of the Genes Involved in the Raffinose Family Oligosaccharides Pathway of Phaseolus vulgaris and Glycine max	Plants	2,021	cc-by	16,390	Citation: de Koning, R.; Kiekens, R.; Toili, M.E.M.; Angenon, G. Identiﬁcation and Expression Analysis of the Genes Involved in the Rafﬁnose Family Oligosaccharides Pathway of Phaseolus vulgaris and Glycine max. Plants 2021, 10, 1465. https://doi.org/10.3390/ plants10071465 Keywords: common bean; Fabaceae; legumes; qRT-PCR; rafﬁnose family oligosaccharides (RFOs); RNA-seq; soybean Keywords: common bean; Fabaceae; legumes; qRT-PCR; rafﬁnose family oligosaccharides (RFOs); RNA-seq; soybean Received: 11 June 2021 Accepted: 13 July 2021 Published: 16 July 2021 1 Research Group Plant Genetics, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Ramon.De.Koning@vub.be (R.d.K.); Raphael.Kiekens@vub.be (R.K.); Mary.Esther.Muyoka.Toili@vub.be (M.E.M.T.) 2 Department of Horticulture, Jomo Kenyatta University of Agriculture and Technology, Juja 01001, Kiambu County, Kenya * Correspondence: Geert.Angenon@vub.be; Tel.: +32-2-629-1935 plants plants plants plants Identiﬁcation and Expression Analysis of the Genes Involved in the Rafﬁnose Family Oligosaccharides Pathway of Phaseolus vulgaris and Glycine max e Koning 1 , Raphaël Kiekens 1 , Mary Esther Muyoka Toili 1,2 and Geert Angenon 1,* 1 Research Group Plant Genetics, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Ramon.De.Koning@vub.be (R.d.K.); Raphael.Kiekens@vub.be (R.K.); Mary.Esther.Muyoka.Toili@vub.be (M.E.M.T.) 2 Department of Horticulture, Jomo Kenyatta University of Agriculture and Technology, Juja 01001, Kiambu County, Kenya * Correspondence: Geert.Angenon@vub.be; Tel.: +32-2-629-1935 1 Research Group Plant Genetics, Vrije Universiteit Brussel, 1050 Brussels, Belgium; Ramon.De.Koning@vub.be (R.d.K.); Raphael.Kiekens@vub.be (R.K.); Mary.Esther.Muyoka.Toili@vub.be (M.E.M.T.) 2 Department of Horticulture, Jomo Kenyatta University of Agriculture and Technology, Juja 01001, Kiambu County, Kenya * Correspondence: Geert.Angenon@vub.be; Tel.: +32-2-629-1935 Abstract: Rafﬁnose family oligosaccharides (RFO) play an important role in plants but are also considered to be antinutritional factors. A profound understanding of the galactinol and RFO biosynthetic gene families and the expression patterns of the individual genes is a prerequisite for the sustainable reduction of the RFO content in the seeds, without compromising normal plant development and functioning. In this paper, an overview of the annotation and genetic structure of all galactinol- and RFO biosynthesis genes is given for soybean and common bean. In common bean, three galactinol synthase genes, two rafﬁnose synthase genes and one stachyose synthase gene were identiﬁed for the ﬁrst time. To discover the expression patterns of these genes in different tissues, two expression atlases have been created through re-analysis of publicly available RNA-seq data. De novo expression analysis through an RNA-seq study during seed development of three varieties of common bean gave more insight into the expression patterns of these genes during the seed development. The results of the expression analysis suggest that different classes of galactinol- and RFO synthase genes have tissue-speciﬁc expression patterns in soybean and common bean. With the obtained knowledge, important galactinol- and RFO synthase genes that speciﬁcally play a key role in the accumulation of RFOs in the seeds are identiﬁed. These candidate genes may play a pivotal role in reducing the RFO content in the seeds of important legumes which could improve the nutritional quality of these beans and would solve the discomforts associated with their consumption. 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. The common bean (Phaseolus vulgaris) and soybean (Glycine max) are of outstanding importance for human and animal nutrition. They have a high nutritional quality, contain- ing large amounts of proteins, carbohydrates, vitamins, minerals and dietary ﬁbers. Beans are used as a major source of calories and proteins in developing countries, and they are the main legumes in vegetarian diets [1–4]. Furthermore, diets consisting mainly of cereals can be complemented with legumes to increase the essential nutrients acquired, making them ideal components to include in our diets [5]. 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/). A major drawback of the consumption of beans in large quantities is the presence of several anti-nutritional factors. Beans contain high amounts of rafﬁnose family oligosaccha- rides (RFOs) [1,6–9]. These sugars contain one or more α-1,6 galactosyl bonds which make them indigestible for humans and monogastric animals because they lack α-galactosidase, the enzyme required to break down these sugars. Instead, these sugars undergo fermenta- tion by gut microbiota in the large intestine resulting in the production of gases such as https://www.mdpi.com/journal/plants Plants 2021, 10, 1465. https://doi.org/10.3390/plants10071465 Plants 2021, 10, 1465 2 of 21 2 of 21 methane and carbon dioxide [1,10]. The production of gases does not pose any problems for humans or animals when small quantities of beans are consumed. On the contrary, the breakdown of small amounts of RFOs might stimulate the growth of beneﬁcial bacteria in the large intestine providing RFOs a potential small probiotic effect [11,12]. However, the consumption of large amounts of beans results in the production of gases which leads to unwanted ﬂatulence and digestive disturbances [13]. The presence of RFOs in the feed also results in shorter transit times through the digestive tract, reducing the absorption of nutrients and causing poor weight gain [14,15]. Lowering the amount of RFOs in the seeds could improve the nutritional quality of these beans and would solve the discomforts associated with their consumption [14]. 1. Introduction Since RFOs have many functions in the plant, a thorough understanding of the galactinol and RFO biosynthetic gene families and the expression patterns of the individual genes is needed to be able to reduce the RFO content in the seeds, without compromising normal plant development and functioning. p g p p g RFOs play an important role in the transport and storage of carbon within the plant [16]. Sucrose produced as a result of photosynthesis in mesophyll cells (source), can move via the bundle sheath cells located at the minor veins, into specialized compan- ion cells which are also known as intermediary cells. Here RFO biosynthetic enzymes are present which convert sucrose into RFOs. The RFOs are then loaded into the phloem according to a polymer trapping model [17]. This model speciﬁes that, due to the large size of the RFOs, the diffusion back into the mesophyll cells is prevented. The osmotic pressure facilitates the migration towards the sieve elements where the RFOs are then transported to other parts of the plant (sinks) where they can be broken down by alkaline α-galactosidase [16–20]. The production of RFOs in the intermediary cells allows plants to maintain a high sugar concentration in the phloem [19]. It is also suggested that RFOs play a role in the abiotic stress responses of plants. This is mainly based on the observations that the RFO content increases in plants as a response to various stresses such as drought, heat, cold and salt stress [21–26]. Several different mechanisms in which RFOs provide protection against abiotic stress have been proposed. During abiotic stress, RFOs could stabilize membranes and proteins with hydrophilic interactions, act as reactive oxygen species scavengers or serve as osmolytes to reduce water loss [25,27–30]. In leguminous crops, RFOs are mainly stored in the seeds under normal growth conditions. They are produced de novo during the maturation of the seed and protect the seed against seed desiccation and provide seed longevity for storage [31–36]. During germination, they are rapidly broken down by acidic α-galactosidases providing energy and carbon to the young seedling [37,38]. However, it needs to be noted that they are not essential for germination in soybean [39]. Four different members of the RFO family are found in plants: rafﬁnose, stachyose, verbascose and ajugose, which are respectively, tri-, tetra-, penta- and hexasaccharides [37]. 1. Introduction Soybean and common bean primarily produce, rafﬁnose and stachyose, and to a lesser extent, verbascose in their seeds [10,14]. The production of RFOs is mainly dependent on the inﬂux of galactinol which functions as a galactosyl donor for the RFO synthesis. Galactinol is produced by galactinol synthase (GolS) which catalyzes the galactosyl transfer from uridine diphosphate galactose to myo-inositol forming galactinol [37]. Rafﬁnose synthase (RS) catalyzes the formation of rafﬁnose, by transferring a galactosyl moiety from galactinol to sucrose. This reversible reaction produces the ﬁrst RFO in the RFO biosynthesis pathway [40]. The larger RFOs, stachyose and verbascose, can both be formed by stachyose synthase (SS) which has, contrary to RS, a broader substrate speciﬁcity [41]. For the formation of stachyose and subsequently verbascose, SS uses respectively, rafﬁnose or stachyose as a galactosyl acceptor. As a galactosyl donor, either galactinol, stachyose or galactosyl cyclitols can be used. Furthermore, SS also has the ability to synthesize these galactosyl cyclitols by facilitating the galactosyl transfer from galactinol to a cyclitol [37]. Galactosyl cyclitols contain, like RFOs, an α-1,6 bond. It is important to point out that galactosyl cyclitols potentially have a similar function as RFOs during seed development due to their high similarity in structure and contribute to the digestive problems humans Plants 2021, 10, 1465 3 of 21 and monogastric animals face after consumption of beans [37,42]. An alternative route to produce higher RFOs (stachyose, verbascose and ajugose), independent of galactinol, has only been reported in two species of the Lamiaceae family (Ajuga reptans and Coleus blumei) where the enzyme galactan:galactan galactosyltransferase (GGT) catalyzes the transfer of a galactosyl moiety from one RFO to another [43,44]. The GGT enzyme has only been found in the vacuoles of leaves, which makes the synthesis of higher RFOs in seeds unlikely to be dependent on this pathway [41]. p p y [ ] Previous attempts to lower the amount of RFOs in the seeds have shown to be success- ful in G. max. Valentine et al. (2017) reduced the rafﬁnose and stachyose content in seeds from respectively, 0.63% and 3.79% in the wild type to 0.11% and 1.21% in a transgenic line using a silencing construct targeting a RS isoform [14]. In a recent study by Le et al. (2020) two GolS genes were knocked out, reducing the total RFO content by 35.2% [45]. 1. Introduction However, more progress can be made to lower the RFO content in the seeds more efﬁciently without compromising normal plant development and functioning. An in-depth study of all genes involved can elucidate which strategies can be best perused to achieve this. In contrast to G. max, the RFO biosynthesis pathway is still understudied in P. vulgaris. In this paper, an overview of the annotation and genetic structure of all galactinol- and RFO biosynthesis genes is given for soybean and common bean. In P. vulgaris, three GolS genes, two RS genes and one SS gene were identiﬁed for the ﬁrst time. To discover the expression patterns of these genes in different tissues, two expression atlases have been created through a re-analysis of publicly available RNA-seq studies. Furthermore, little is known about the expression patterns of these galactinol- and RFO biosynthesis genes during seed develop- ment in common bean. De novo expression analysis through an RNA-seq study during seed development in three varieties of common bean gave more insight into the expression patterns of these genes and seed speciﬁc genes involved in the RFO production have been identiﬁed. The results of the expression analysis indicate tissue-speciﬁc expression patterns of different classes of galactinol- and RFO synthase genes in soybean and common bean. With the obtained knowledge, suitable candidate genes to alter the expression levels of the galactinol- and RFO synthase genes are proposed to lower the amount of RFOs in the seeds. 2. Results Analysis of the phylogenetic relationships and gene structures of galactinol synthase (GolS). (a) Evolutionary relationship of GolS enzymes of G. max, P. vulgaris and other well-characterized taxa (A. thaliana and C. arietinum). The GolS enzymes of P. vulgaris and G. max are subdivided into three different classes based on their clustering (GolS1, GolS2 and GolS3. The phylogenetic tree was made in MEGA X (v10.2.4) using the neighbor-joining algorithm combined with a bootstrap test of 1000 replicates (next to the branches, the percentage of replicate trees in which the associated taxa clustered together is shown) [46–48]. The Poisson correction method was used to compute the evolutionary distances with the number of amino acid substitutions per site as a unit [49]. The phylogenetic tree was drawn to scale using the calculated evolutionary distances as branch lengths. This analysis involved 23 amino acid sequences. For each sequence pair, all ambiguous positions were removed using the pairwise deletion option and the ﬁnal dataset consisted of 355 positions. The multiple sequence alignment was created using the MUSCLE algorithm; (b) structures of genes encoding the GolS enzymes were visualized using GSDS software [50]. Nine possible RFO biosynthesis genes have been predicted in P. vulgaris and fourteen in G. max using our bioinformatics pipeline. Most proteins encoded by these genes are annotated as potential RFO biosynthesis enzymes but could also be wrongly annotated. To distinguish RS from alkaline α-galactosidase two conserved motifs (FMxLGTEAxxLG and SGDPxGTxWLQGCHMVHC) were used (Figure A1 in Appendix A). These motifs are present in the amino acid sequence of RS but absent in alkaline α-galactosidase [51]. RS can be distinguished from SS because of an amino acid insert present only in SS (Figure A2 in Appendix A) [40,52]. A phylogenetic tree of the amino acid sequences has been made to evaluate the evolutionary relationship of the different RFO biosynthetic and hydrolytic enzymes of P. vulgaris, G. max and other well-characterized taxa: A. thaliana, Pisum sativum, Vigna angularis, Solanum lycopersicum, Oryza sativa and Zea mays (Figure 2a). The phyloge- netic tree clusters 4 different groups of enzymes with high certainty (bootstrap score > 88): RS, SS, alkaline α-galactosidase and α-galactosidase. In addition, gene features of these enzymes were visualized (Figure 2b). The RS genes in G. max and P. vulgaris all consist of 5 exons and 4 introns. The genes encoding SS consist of 4 exons and 3 introns. 2. Results 2.1. In Silico Identiﬁcation of the Galactinol- and RFO Biosynthetic Enzymes in Phaseolus vulgaris and Glycine max Using a customized bioinformatics pipeline, three GolS genes were identiﬁed in P. vulgaris, (Phvul.001G215300, Phvul.001G223700 and Phvul.007G203400) and six GolS genes in G. max (Glyma.03G229800, Glyma.19G227800, Glyma.20G094500, Glyma.03G222000, Glyma.19G219100 and Glyma.10G145300). Some genes encode multiple predicted isoforms. In P. vulgaris, only for Phvul.001G215300 two isoforms are predicted. In G. max, two iso- forms for Glyma.19G227800 and three isoforms for Glyma.10G145300 are predicted. A phylogenetic tree has been made using the amino acid sequences of the predicted proteins of the GolS enzymes of P. vulgaris, G. max and well-characterized GolS enzymes of Ara- bidopsis thaliana and Cicer arietinum (Figure 1a). Furthermore, the gene structures of the GolS enzymes are presented in Figure 1b. Based on the clustering of the P. vulgaris and G. max enzymes in the phylogenetic tree, the GolS enzymes of G. max and P. vulgaris were categorized into three classes: galactinol synthase 1 (GolS1), galactinol synthase 2 (GolS2) and galactinol synthase 3 (GolS3). The predicted primary transcript isoforms of the GolS1 genes consist of three exons and two introns. The GolS2 and GolS3 genes consist of four exons and three introns. 4 of 21 Plants 2021, 10, 1465 Figure 1. Analysis of the phylogenetic relationships and gene structures of galactinol synthase (GolS). (a) Evolutionary relationship of GolS enzymes of G. max, P. vulgaris and other well-characterized taxa (A. thaliana and C. arietinum). The GolS enzymes of P. vulgaris and G. max are subdivided into three different classes based on their clustering (GolS1, GolS2 and GolS3. The phylogenetic tree was made in MEGA X (v10.2.4) using the neighbor-joining algorithm combined with a bootstrap test of 1000 replicates (next to the branches, the percentage of replicate trees in which the associated taxa clustered together is shown) [46–48]. The Poisson correction method was used to compute the evolutionary distances with the number of amino acid substitutions per site as a unit [49]. The phylogenetic tree was drawn to scale using the calculated evolutionary distances as branch lengths. This analysis involved 23 amino acid sequences. For each sequence pair, all ambiguous positions were removed using the pairwise deletion option and the ﬁnal dataset consisted of 355 positions. The multiple sequence alignment was created using the MUSCLE algorithm; (b) structures of genes encoding the GolS enzymes were visualized using GSDS software [50]. Figure 1. 2. Results This is in contrast with the alkaline α-galactosidase genes which mostly consist of 13 exons although quite a large variation can be seen in this group. Some of the alkaline α-galactosidase genes consist of 1, 7, 12 or 14 predicted exons. From these results, it can be concluded that P. vul- garis most likely contains at least two RS genes (Phvul.009G175400 and Phvul.004G007100) and only one SS gene (Phvul.001G214300). G. max has three RS genes (Glyma.05G003900, Plants 2021, 10, 1465 5 of 21 Glyma.06G179200 and Glyma.05G040300) and one SS gene (Glyma.19G217700). Based on the phylogenetic tree, the RS enzymes of G. max and P. vulgaris have consequently been categorized into two classes: rafﬁnose synthase 1 (RS1) and rafﬁnose synthase 2 (RS2). An overview of the genes involved in the RFO biosynthesis pathway in G. max and P. vulgaris can be found in Table 1. The corresponding names of the genes found in Table 1 will be used in the rest of the text. Figure 2. Analysis of the phylogenetic relationships and gene structures of the rafﬁnose family oligosaccharides’ biosynthetic and catabolic enzymes. (a) Evolutionary relationship of the rafﬁnose synthase (RS), stachyose synthase (SS) and (alkaline) α-galactosidase (AGA) enzymes of G. max, P. vulgaris and other well-characterized enzymes of different taxa (A. thaliana, P. sativum, V. angularis, S. lycopersicum, O. sativa and Z. mays). The RS enzymes of P. vulgaris and G. max are subdivided into two different classes (RS1 and RS2) based on their clustering. The phylogenetic tree was made in MEGA X (v10.2.4) using the neighbor-joining algorithm combined with a bootstrap test of 1000 replicates (next to the branches are the percentage of replicate trees in which the associated taxa clustered together is shown) [46–48]. The Poisson correction method was used to compute the evolutionary distances with the number of amino acid substitutions per site as a unit [49]. The phylogenetic tree was drawn to scale using the calculated evolutionary distances as branch lengths. This analysis involved 43 amino acid sequences. For each sequence pair, all ambiguous positions were removed using the pairwise deletion option and the ﬁnal dataset consisted of 1030 positions. The multiple sequence alignment was created using the MUSCLE algorithm. (b) Structures of genes encoding the RFO synthase enzymes were visualized using GSDS software [50]. Figure 2. Analysis of the phylogenetic relationships and gene structures of the rafﬁnose family oligosaccharides’ biosynthetic and catabolic enzymes. 2. Results (a) Evolutionary relationship of the rafﬁnose synthase (RS), stachyose synthase (SS) and (alkaline) α-galactosidase (AGA) enzymes of G. max, P. vulgaris and other well-characterized enzymes of different taxa (A. thaliana, P. sativum, V. angularis, S. lycopersicum, O. sativa and Z. mays). The RS enzymes of P. vulgaris and G. max are subdivided into two different classes (RS1 and RS2) based on their clustering. The phylogenetic tree was made in MEGA X (v10.2.4) using the neighbor-joining algorithm combined with a bootstrap test of 1000 replicates (next to the branches are the percentage of replicate trees in which the associated taxa clustered together is shown) [46–48]. The Poisson correction method was used to compute the evolutionary distances with the number of amino acid substitutions per site as a unit [49]. The phylogenetic tree was drawn to scale using the calculated evolutionary distances as branch lengths. This analysis involved 43 amino acid sequences. For each sequence pair, all ambiguous positions were removed using the pairwise deletion option and the ﬁnal dataset consisted of 1030 positions. The multiple sequence alignment was created using the MUSCLE algorithm. (b) Structures of genes encoding the RFO synthase enzymes were visualized using GSDS software [50]. Plants 2021, 10, 1465 6 of 21 Table 1. Overview of the enzymes involved in the RFO biosynthesis pathway and corresponding genes. Species Gene Name Accession Number * Function Chromo- Some Start (bp) End (bp) Protein Length (AA) # Exons # Transcripts P. vulgaris PvGolS1 Phvul.001G215300 Galactinol synthase Chr1 47,166,933 47,168,674 339 3 2 G. max GmGolS1_A Glyma.03G222000 Chr3 42,494,623 42,497,111 339 3 1 G. max GmGolS1_B Glyma.19G219100 Chr19 47,148,225 47,150,373 335 3 1 P. vulgaris PvGolS2 Phvul.001G223700 Chr1 47,870,097 47,872,698 327 4 1 G. max GmGolS2_A Glyma.03G229800 Chr3 43,172,457 43,175,687 331 4 1 G. max GmGolS2_B Glyma.19G227800 Chr19 47,911,130 47,914,214 330 4 2 P. vulgaris PvGolS3 Phvul.007G203400 Chr7 32,610,928 32,612,577 326 4 1 G. max GmGolS3_A Glyma.10G145300 Chr10 38,014,453 38,016,396 328 4 3 G. max GmGolS3_B Glyma.20G094500 Chr20 33,759,417 33,761,555 324 4 1 P. vulgaris PvRS1 Phvul.004G007100 Rafﬁnose synthase Chr4 519,197 523,594 763 5 1 G. max GmRS1 Glyma.05G003900 Chr5 307,461 312,091 758 5 1 P. vulgaris PvRS2 Phvul.009G175400 Chr9 26,053,801 26,057,657 777 5 1 G. max GmRS2_A Glyma.06G179200 Chr6 15,217,419 15,223,877 810 5 2 G. max GmRS2_B Glyma.05G040300 Chr5 3,593,378 3,598,821 782 5 1 P. vulgaris PvSS Phvul.001G214300 Stachyose synthase Chr1 47,049,258 47,052,441 857 4 2 G. 2. Results max GmSS Glyma.19G217700 Chr19 47,033,812 47,037,286 860 4 1 * Accession numbers were adopted from Phytozome v12.1 (P. vulgaris v2.1; G. max Wm82.a2.v1). Overview of the enzymes involved in the RFO biosynthesis pathway and corresponding genes. 2.2. Transcriptomic Analysis of the RFO Biosynthetic Pathway 2.2. Transcriptomic Analysis of the RFO Biosynthetic Pathway 2.2. Transcriptomic Analysis of the RFO Biosynthetic Pathway 2.2.1. Expression Analysis of Galactinol- and RFO Biosynthesis Genes in Glycine max and Phaseolus vulgaris by RNA-seq Re-Analysis 2.2.1. Expression Analysis of Galactinol- and RFO Biosynthesis Genes in Glycine max and Phaseolus vulgaris by RNA-seq Re-Analysis An expression atlas has been made from the RNA-seq data of studies SRP038111 (G. max cv. Wm82) and SRP030614 (P. vulgaris cv. BAT93) [53,54]. The normalized expres- sion data, represented as transcripts per million (TPM), of the different GolS, RS and SS genes is shown in Figure 3 for G. max cv. Wm82 and Figure 4 for P. vulgaris cv. BAT93 during ﬁve different growth stages. The different classes of GolS (GolS1, GolS2 and GolS3) and RS (RS1 and RS2), as deﬁned by the phylogenetic tree analysis, show tissue-speciﬁc expression patterns for both G. max cv. Wm82 and P. vulgaris cv. BAT93. Figure 3. Heatmap of the normalized expression values (TPM) of the galactinol synthase (GmGolS1-3), rafﬁnose synthase (GmRS1-2) and stachyose synthase (GmSS) genes in various plant tissues during different developmental stages in G. max cv. Wm82 (SRP038111). The rows represent plant tissues in ﬁve different developmental stages: emergence stage, early vegetative stage, late vegetative stage, ﬂowering stage and seed developmental stage. More detailed information of the exact growth stage of the tissues can be found after the tissues names between brackets, with growth stage abbreviations (VE: emergence stage; V1: ﬁrst-node stage; V7: seventh-node stage; EM: early maturation stage of the seed; MM: mid maturation stage of the seed; DAF: days after ﬂowering) adapted from Fehr et al. [55]. The columns represent the different genes and their expression levels, represented with a color gradient in which white indicates no expression (0 TPM) and red the highest expression (200+ TPM). Hierarchical clustering was performed between the columns to obtain gene clusters. Figure 3. Heatmap of the normalized expression values (TPM) of the galactinol synthase (GmGolS1-3), rafﬁnose synthase (GmRS1-2) and stachyose synthase (GmSS) genes in various plant tissues during different developmental stages in G. max cv. Wm82 (SRP038111). * Accession numbers were adopted from Phytozome v12.1 (P. vulgaris v2.1; G. max Wm82.a2.v 2. Results The rows represent plant tissues in ﬁve different developmental stages: emergence stage, early vegetative stage, late vegetative stage, ﬂowering stage and seed developmental stage. More detailed information of the exact growth stage of the tissues can be found after the tissues names between brackets, with growth stage abbreviations (VE: emergence stage; V1: ﬁrst-node stage; V7: seventh-node stage; EM: early maturation stage of the seed; MM: mid maturation stage of the seed; DAF: days after ﬂowering) adapted from Fehr et al. [55]. The columns represent the different genes and their expression levels, represented with a color gradient in which white indicates no expression (0 TPM) and red the highest expression (200+ TPM). Hierarchical clustering was performed between the columns to obtain gene clusters. 7 of 21 Plants 2021, 10, 1465 Figure 4. Heatmap of the normalized expression values, presented in transcripts per million (TPM), of the galactinol synthase (PvGolS1-3), rafﬁnose synthase (PvRS1-2) and stachyose synthase (PvSS) genes in various plant tissues during different developmental stages in P. vulgaris cv. BAT93 (SRP030614). The rows represent plant tissues in ﬁve different developmental stages: emergence stage, early vegetative stage, late vegetative stage, ﬂowering stage and seed developmental stage. More detailed information of the exact growth stage of the tissues can be found after the tissues name between brackets, indicated in days after sowing (DAS) and with growth stage abbreviations (V1: emergence stage; V2: primary leaves stage; V3: ﬁrst trifoliate leaf stage; V4: third trifoliate leaf stage; R5: preﬂowering stage; R6: ﬂowering stage; R9: maturity stage; MM: mid maturation stage of the seed) adapted from Vlasova et al. [53]. The columns represent the different genes and their expression levels, represented with a color gradient in which white indicated no expression (0 TPM) and red the highest expression (200+ TPM). Hierarchical clustering was performed between the columns to obtain gene clusters. The normalized expression data of RNA-seq study SRP038111 in G. max cv. Wm82 shows that the GolS1 genes (GmGolS1_A and GmGolS1_B) are expressed highly in the hypocotyl during the emergence stage (resp. 59.4 and 128.2 TPM) and early vegetative stage (resp. 38.0 and 113.7 TPM). During the late vegetative stage, both genes are expressed in different types of tissues; GmGolS1_B, however, is mainly expressed in the stem node (122.4 TPM), ﬂower bud (80.5 TPM), leaf bud (51.6 TPM) and trifoliate leaf (39.4 TPM) during the late vegetative stage. 2. Results In soybean, the SS gene, GmSS, is expressed in the hypocotyl during the emergence stage and early vegetative stage (resp. 34.8 and 20.6 TPM). During the late vegetative stage, it is expressed in the trifoliate leaf (23.4 TPM) and ﬂower bud (21.5 TPM). In the seed developmental stage, it is expressed in the seed during the mid-maturation stage of the seed (214.0 TPM). The hierarchical clustering of the genes in the heatmap in Figure 3 also shows that the different GolS, RS and SS genes within one class have tissue-speciﬁc expression patterns and generally cluster close together. However, the GS2 genes do not cluster together mainly because of the expression difference in the seeds. l i A h l d d f h A d g g y p In P. vulgaris cv. BAT93, the normalized expression data of the RNA-seq study SRP030614 (Figure 4) shows that PvGolS1 is expressed mainly in the primary leaf (334.7 TPM), epicotyl (214.6 TPM), hypocotyl (208.1 TPM) and to a lesser extent in the primary root (48.0 TPM) and cotyledons (47.5 TPM) during the emergence stage. During the early vegetative stage, it is mainly expressed in the hypocotyl (105.8 TPM) and to a lesser extent in the primary leaf (22.3 TPM), ﬁrst trifoliate leaf (19.7 TPM) and the neck of the root (14.2 TPM). During the late vegetative stage, it is mainly expressed in the stem (107.3 TPM). During this stage, expression can also be seen in the hypocotyl (27.4 TPM) and trifoliate leaf (13.4 TPM) and the axial meristem (10.6 TPM). In the ﬂowering stage, it is expressed in the stem node (56.7 TPM), the root (37.1 TPM), the trifoliate leaf (28.3 TPM) and axial meris- tem (8.9 TPM). During the seed developmental stage, it is the only gene of this pathway that is expressed in the seed during the mid-maturation stage (196.8 TPM). In addition, PvGolS1 shows the most TPM variation throughout the tissues in comparison with the other genes analyzed. Furthermore, in the primary root, a contrasting expression is seen, with low PvGolS1 expression compared to higher expression for all other genes except PvRS2. The common bean GolS2 gene, PvGOLS2, is almost exclusively expressed in the primary root (438.26 TPM) during the emergence stage. 2. Results Furthermore, GmGolS1_B shows a higher expression in all stages of ﬂower development. These two genes are highly expressed during the mid-maturation stage of the seed, with GmGolS1_A being expressed two times higher than GmGolS1_B in the seeds with expression levels of 721.7 TPM and 328.2 TPM, respectively. At the mid-maturation stage of the seed, both genes are expressed more in comparison with any other galactinol or RFO synthase gene. When observing the GolS2 genes (GmGolS2_A and GmGolS2_B) it can be seen that GmGolS2_A is highly expressed in the ﬂower during the ﬂowering stage, with expression levels of 176.2 TPM during the opening and 183.4 TPM ﬁve days after the opening of the ﬂower. During the other developmental stages, no signiﬁcant expression is observed. GmGolS2_B is mainly expressed in the seeds during the mid-maturation stage of the seeds (280.9 TPM) and less pronounced in the ﬂower during the opening (30.0 TPM) and ﬁve days after the opening (25.1 TPM). The GolS3 genes (GmGolS3_A and GmGolS3_B) are both expressed in the roots (resp. 190.8 and 41.0 TPM) and hypocotyl (resp. 73.9 and 125.7 TPM) during the emergence stage. GmGolS3_B is Plants 2021, 10, 1465 8 of 21 mainly expressed in the hypocotyl (109.6 TPM) and to a lower extent in the primary leaf (30.8 TPM) during the early vegetative stage. In the late vegetative stage, GmGolS3_B is mainly expressed in the ﬂower bud (103.6 TPM), stem node (54.3 TPM) and trifoliate leaf (53.5 TPM). During the ﬂowering stage, both genes are expressed in the ﬂower bud (resp. 20.3 and 49.4 TPM) and in the ﬂower during the opening (resp. 26.7 and 87.0 TPM), 5 days after the opening (resp. 37.8 and 64.1 TPM) and during the senescence (resp. 29.2 and 80.3 TPM). During the mid-maturation stage of the seed, GmGolS3_A is primarily expressed (95.2 TPM). The soybean RS1 gene, GmRS1, is highly expressed in the primary leaf (355.9 TPM) during the early vegetative stage and in the trifoliate leaf (46.6 TPM) in the late vegetative stage. In the early vegetative stage, it is also expressed in the leaf bud (42.6 TPM) and hypocotyl (39.7 TPM). During the seed developmental stage, it is almost not expressed in the seed (13.6 TPM). This is in contrast with the RS2 genes (GmRS2_A and GmRS2_B) that are only expressed in the seed during the mid-maturation stage of the seed (resp. 123.0 and 109.4 TPM). 2. Results The GolS3 gene, PvGolS3, is also primarily expressed in the primary root (512.2 TPM) and to a lesser extent in the hypocotyl (140.0 TPM), the cotyledons (118.0 TPM), epicotyl (74.4 TPM) and primary leaf (66.0 TPM) during the emergence stage. In the early vegetative stage, it is expressed in the neck of the root (11.9 TPM) and during the late vegetative stage, expression is observed in the axial meristem (24.6 TPM) and hypocotyl (21.9 TPM). During the ﬂowering stage, PvGolS3 is also mainly expressed in the roots (40.6 TPM) and to a lesser extent in the stem node (12.3 TPM). In common bean, the RS1 gene, PvRS1, is primarily expressed in the primary root (298.2 TPM) during the emergence stage and to a lesser extent in the primary leaf (47.0 TPM), the cotyledons (42.8 TPM), the epicotyl (37.2 TPM) and hypocotyl (35.9 TPM). During the early vegetative stage, it is expressed in the primary leaf (10.5 TPM) and during the late vegetative stage in the trifoliate leaf (5.3 TPM). PvRS2 is only expressed during the emergence stage, where it shows expression in the primary leaf (224.2 TPM) and to a lesser extent in the epicotyl (36.9 TPM), cotyledons (36.9 TPM) and hypocotyl (32.2 TPM). Finally, the common bean SS gene, PvSS, is primarily expressed in the primary root (153.3 TPM) and hypocotyl (147.8 TPM) and to a lesser extent in the primary leaf (58.5 TPM), cotyledons (42.1 TPM) and epicotyl (36.5 TPM) during the emergence stage. Plants 2021, 10, 1465 9 of 21 2.2.2. De Novo Expression Analysis of Galactinol- and RFO Biosynthesis Genes during Seed Development in P. vulgaris 2.2.2. De Novo Expression Analysis of Galactinol- and RFO Biosynthesis Genes during Seed Development in P. vulgaris Only a limited amount of information is available on the seed development in common bean. The RNA-seq study SRP030614 only contains one datapoint of the expression in the seeds (79 DAS, R9). To get a better understanding of the expression of the galactinol- and RFO biosynthesis genes during the seed development, an expression atlas was made by RNA-seq analysis for the common bean cultivars Canadian wonder, Pinto and Rosecoco. The expression levels were measured during early (15 DAF), mid (20 DAF) and late (30 and 35 DAF) maturation stages of seed development using four biological repeats. 2. Results In all cultivars, PvGolS1 is expressed at a much higher level in comparison with the other galactinol- and RFO biosynthesis genes for all developmental stages (Figure 5). During the early stage of the seed development (15 DAF), PvGolS1 is expressed in all three cultivars with expression levels of 8.5 TPM (SE = 1.6) in Canadian wonder, 132.6 TPM (SE = 54.5) in Pinto and 11.1 TPM (SE = 2.7) in Rosecoco. From here, the expression level increases over time, with expression levels during the mid-maturation stage of the seed (20 DAF) of 58.3 TPM (SE = 30.1) in Canadian wonder, 445.6 TPM (SE = 77.9) in Pinto and 128.1 TPM (SE = 31.7) in Rosecoco. The highest expression of PvGolS1 was measured in the late stage of seed development (30 and 35 DAF). Here the expression level increased up to 4684.2 TPM (SE = 212.0) in Canadian wonder and 3351.0 TPM (SE = 1107.2) in Rosecoco at 35 DAF. In Pinto, the highest expression was measured at 30 DAF, with an expression level of 1305.9 TPM (SE = 190.4). In all cultivars, expression of the other galactinol- and RFO biosynthesis genes was low to undetectable in the early and mid-maturation stages. Only a slight expression of PvSS, and to a minor extent PvRS2 and PvGolS2, was measured during the mid-maturation stage of seed development in Pinto with expression levels of respectively, 16.8 TPM (SE = 2.1), 4.6 TPM (SE = 1.2) and 3.2 TPM (SE = 0.7). The expression of PvGolS2 increased over time during the late stage of the seed development with the highest expression level measured at 35 DAF in Canadian wonder (154.6 TPM; SE = 11.3), Pinto (188.7 TPM; SE = 9.4) and Rosecoco (156.9 TPM; SE = 21.3). PvRS2 was also mainly expressed during the late stage of the seed development with the highest expression level measured at 35 DAF in Canadian wonder (89.4 TPM; SE = 7.0) and Rosecoco (61.7 TPM; SE = 13.7). In Pinto, the highest expression was measured at 30 DAF (29.9 TPM; SE = 8.0). PvGolS3 and PvRS1 were not expressed in the seeds. Figure 5. RNA-seq expression analysis of the galactinol- and RFO biosynthesis genes during seed development in P. vulgaris cvs. Canadian wonder, Pinto and Rosecoco. 2.2.3. Validation of RNA-Sequencing Results 2.2.3. Validation of RNA-Sequencing Results The RNA-seq results of the galactinol- and RFO biosynthesis genes during the seed development were validated by measuring the expression values of the six galactinol and RFO synthase genes in the different developmental stages of the seeds of P. vulgaris cv. Rosecoco using qRT-PCR. When the differential gene expression levels of these genes were compared for the different developmental stages of the seed, a similar expression pattern was observed in both the RNA-seq and qRT-PCR data (Figure 6). Because most of these genes exhibit very low expression levels during the early and mid-maturation stages of seed development (15 DAF vs. 20 DAF), it is more difﬁcult to compare the differential gene expression patterns for these stages. However, the overall correlation between the RNA-seq and qPCR results was high, indicated by a Spearman’s rank correlation coefﬁcient (rs) of 0.77. Especially when comparing the differential gene expressions during the mid and late maturity stages of the seeds, a high correlation could be seen (rs = 0.93). In addition, the qRT-PCR results conﬁrmed the absence of the expression of PvGolS3 and PvRS1 in the seeds during the seed development. Figure 6. Validation of the RNA-seq data using qRT-PCR. The gene expression ratios of GolS1 (PvGolS1), GolS2 (PvGolS2), RS2 (PvRS2) and SS (PvSS) are represented as a result of the relative comparison of the different developmental stages of the seeds of P. vulgaris cv. Rosecoco: 15 vs. 20 days after ﬂowering (DAF), 20 vs. 30 DAF and 30 vs. 35 DAF. These ratios are expressed as log2fold changes (Log2 FC). The housekeeping gene β-tubulin was used for the normalization of the qRT-PCR data [56]. Error bars represent the standard error. Figure 6. Validation of the RNA-seq data using qRT-PCR. The gene expression ratios of GolS1 (PvGolS1), GolS2 (PvGolS2), RS2 (PvRS2) and SS (PvSS) are represented as a result of the relative comparison of the different developmental stages of the seeds of P. vulgaris cv. Rosecoco: 15 vs. 20 days after ﬂowering (DAF), 20 vs. 30 DAF and 30 vs. 35 DAF. These ratios are expressed as log2fold changes (Log2 FC). The housekeeping gene β-tubulin was used for the normalization of the qRT-PCR data [56]. Error bars represent the standard error. 2. Results The normalized expression values of GolS1 (PvGolS1), GolS2 (PvGolS2), GolS3 (PvGolS3), RS1 (PvRS1), RS2 (PvRS2) and SS (PvSS) in the seed are shown during different developmental stages: 15, 20, 30 and 35 days after ﬂowering (DAF). The expression values are log2 transformed and expressed in transcripts per million (TPM). Error bars represent the standard error. Figure 5. RNA-seq expression analysis of the galactinol- and RFO biosynthesis genes during seed development in P. vulgaris cvs. Canadian wonder, Pinto and Rosecoco. The normalized expression values of GolS1 (PvGolS1), GolS2 (PvGolS2), GolS3 (PvGolS3), RS1 (PvRS1), RS2 (PvRS2) and SS (PvSS) in the seed are shown during different developmental stages: 15, 20, 30 and 35 days after ﬂowering (DAF). The expression values are log2 transformed and expressed in transcripts per million (TPM). Error bars represent the standard error. Plants 2021, 10, 1465 10 of 21 3. Discussion However, the RS1 and SS genes appear to have only one copy in G. max as in P. vulgaris. After the WGD, chromosomes were subject to rearrangements and deletions resulting in gene loss [65]. It is possible that duplicates of the RS1 and SS genes were lost from the G. max genome during these rearrangements. We subdivided the different galactinol and RFO biosynthesis genes into different classes based on the sequence and structure similarity of the genes between P. vulgaris and G. max. The chromosomal location of the genes in the different classes corresponds with syntenic relationship between these two species (Figure S1 of the Supplementary Materials) [63,64]. Indeed, duplication and speciation events can lead to the formation of genes with novel functionalities or different expression patterns. However, the high homology between the newly identiﬁed genes indicates a high probability that these genes still have the same function which is supported by the research done in G. max [14,45,57]. When the expression patterns are compared in G. max cv. Wm82 it is interesting to see that in general the different classes of galactinol- and RFO biosynthesis genes cluster together in the expression heatmap generated from the re-analysis data of the RNA-seq study. This indicates that the galactinol- and RFO biosynthesis genes within one class have a conserved expression pattern. This is further supported when the expression patterns of the different classes were compared between G. max and P. vulgaris. However, not all plant tissues are equally represented in the RNA-seq re-analysis studies of P. vulgaris cv. BAT93 and G. max cv. Wm82, which needs to be taken into account. Our novel RNA-seq study measured the expression levels of the galactinol- and RFO synthase genes in the seeds of P. vulgaris cv. Canadian wonder, cv. Rosecoco and cv. Pinto during the early, mid and late maturation stage of the seed. A similar expression pattern can be seen for all the galactinol- and RFO biosynthesis genes in these three cultivars and hence the expression patterns were only validated in one variety of P. vulgaris, namely, cv. Rosecoco. g y In multiple plant species such as Coffea arabica and Zea mays, tissue-speciﬁc expression patterns for GolS genes have been observed [58,66,67]. In C. arabica, for example, higher expression levels of CaGolS1 were measured in the leaves in comparison with CaGolS2 and CaGolS3. 3. Discussion A lot of progress has already been made for G. max in the identiﬁcation of the genes involved in the RFO biosynthesis pathway. Our study conﬁrms the results of the earlier identiﬁed genes involved in the RFO pathway. Le et al. (2020) identiﬁed the same six GolS genes and showed that GmGolS1_A and GmGolS2_B both contribute to the production of RFOs through two CRISPR/Cas9-mediated knockout lines [45]. Dierking and Bilyeu (2008) identiﬁed the same three RS genes and Valentine et al. (2017) proved that GmRS2 contributes to the production of rafﬁnose with the use of a soybean line in which GmRS2 was silenced [7,14]. Qiu et al. (2015) identiﬁed and characterized the same SS gene as in our study [57]. This validates the performance of our bioinformatics pipeline and no new additional galactinol and RFO biosynthesis genes were found in G. max. In contrast, the galactinol- and RFO synthase genes have not yet been identiﬁed in P. vulgaris until now. We identiﬁed three GolS genes in P. vulgaris, named PvGolS1, PvGolS2 and PvGolS3. PvGolS1 consists of 3 exons, while both PvGolS2 and PvGolS3 contain 4 exons. This is in accordance with the genetic structures of other well-characterized GolS genes [23,51,57–60]. Previous research showed that AtSIP2 in A. thaliana was incorrectly annotated as an RS gene and encodes an alkaline α-galactosidase with substrate speciﬁcity for rafﬁnose [61]. We found that in P. vulgaris, many genes annotated as potential RS genes in the databases were incorrectly annotated and had similar gene sequences and structures as those of alkaline α-galactosidases. We identiﬁed two RS genes, PvRS1 and PvRS2, and one SS gene, Plants 2021, 10, 1465 11 of 21 11 of 21 PvSS, in P. vulgaris. Their genetic structure corresponds with well-characterized RS and SS genes of species other than P. vulgaris [40,51,52,57,62]. The presence of PvSS and PvGolS1 in P. vulgaris was also reported by Moghaddam et al. (2018) in a Genome-Wide Association Study (GWAS), where they showed their involvement in the RFO biosynthesis pathway [9]. y ( ), y y p y [ ] G. max and P. vulgaris are two closely related species within the Fabaceae family. Around 11 million years ago, G. max underwent a whole-genome duplication (WGD) and consistently we found, for almost all galactinol- and RFO synthase genes in P. vulgaris, a duplicate in G. max [63,64]. 3. Discussion This indicates that the general expression pattern of the GolS1 class genes is conserved between G. max and P. vulgaris. However, in G. max, GmGolS1_A is mostly active during the seed development, whereas GmGolS1_B is mostly active in the other vegetative tissues. The GolS2 genes in G. max are the only genes that do not cluster together in the expression heatmap of G. max cv. Wm82 and show a distinct expression pattern compared to each other. GmGolS2_A is mainly expressed in the ﬂower, whereas GmGolS2_B shows its highest expression in the seeds during the seed mid-maturation stage. Both genes are to a lesser extent expressed in the hypocotyls. In P. vulgaris, PvGolS2 shows a high expression in the primary root during the emergence stage and to a lower extent in the hypocotyl, epicotyl and stem. During seed development, PvGolS2 is expressed mainly in the late stage of seed development in all three P. vulgaris cultivars. These results suggest that PvGolS2 of P. vulgaris and GmGolS2_B of G. max play a role during the seed development, whereas GmGolS2_A seems to have a speciﬁc role in the ﬂower in G. max. It is interesting to note that the GolS3 genes in P. vulgaris and G. max do not play a major role during the seed development in comparison with the other GolS genes. In G. max, only a low expression can be seen in the seeds whereas in P. vulgaris no expression of PvGolS3 was detected in the RNA-seq re-analysis data of cv. BAT93 and the novel RNA-seq analysis data of cvs. Rosecoco, Pinto and Canadian wonder. The GolS3 genes are primarily expressed in the roots, cotyledon and hypocotyl in both G. max and P. vulgaris. In G. max, GmGolS3_A and GmGolS3_B are also expressed in the ﬂower and leaves. The GolS3 genes show, in comparison with the other galactinol and RFO synthase genes, the highest expression in the roots, indicating a possible function for this class in the roots. Tissue-speciﬁc expression patterns can also be seen for the RS genes. The RS1 gene in G. max and P. vulgaris is mainly expressed in the leaves and hypocotyl. In P. vulgaris, PvRS1 is additionally expressed in the primary root during the emergence stage. Expression of the RS1 gene cannot be seen in either species during seed development. 3. Discussion max, GmGolS1_A is mostly active during the seed development, whereas GmGolS1_B is mostly active in the other vegetative tissues. The GolS2 genes in G. max are the only genes that do not cluster together in the expression heatmap of G. max cv. Wm82 and show a distinct expression pattern compared to each other. GmGolS2_A is mainly expressed in the ﬂower, whereas GmGolS2_B shows its highest expression in the seeds during the seed mid-maturation stage. Both genes are to a lesser extent expressed in the hypocotyls. In P. vulgaris, PvGolS2 shows a high expression in the primary root during the emergence stage and to a lower extent in the hypocotyl, epicotyl and stem. During seed development, PvGolS2 is expressed mainly in the late stage of seed development in all three P. vulgaris cultivars. These results suggest that PvGolS2 of P. vulgaris and GmGolS2_B of G. max play a role during the seed development, whereas GmGolS2_A seems to have a speciﬁc role in the ﬂower in G. max. It is interesting to note that the GolS3 genes in P. vulgaris and G. max do not play a major role during the seed development in comparison with the other GolS genes. In G. max, only a low expression can be seen in the seeds whereas in P. vulgaris no expression of PvGolS3 was detected in the RNA-seq re-analysis data of cv. BAT93 and the novel RNA-seq analysis data of cvs. Rosecoco, Pinto and Canadian wonder. The GolS3 genes are primarily expressed in the roots, cotyledon and hypocotyl in both G. max and P. vulgaris. In G. max, GmGolS3_A and GmGolS3_B are also expressed in the ﬂower and leaves. The GolS3 genes show, in comparison with the other galactinol and RFO synthase genes, the highest expression in the roots, indicating a possible function for this class in the roots. Rosecoco. The transcript levels of PvGolS1 were higher than other galactinol- and RFO synthase genes. This result is uniform between the RNA-seq re-analysis data of P. vulgaris cv. BAT93 and our RNA-seq data. Besides the expression in the seeds, PvGolS1 also shows expression in all vegetative tissues of P. vulgaris during the different developmental stages. The expression of PvGolS1 is especially high in the hypocotyl, epicotyl, stem, leaves and roots which corresponds with the expression pattern of the GolS1 genes in G. max. 3. Discussion Little to no expression was measured for CaGolS2 in all tissues and CaGolS3 was mainly expressed in the roots and ﬂowers [66]. In this paper, we show this is also the case for G. max and P. vulgaris. In G. max, GmGolS1_A and GmGolS1_B are both highly expressed during the mid-maturation stage of the seeds, with GmGolS1_A being expressed two times higher than GmGolS1_B. At this stage, both genes are expressed more in soybean in comparison with the other galactinol- and RFO synthase genes indicating that they have a possible signiﬁcant role during seed development. Indeed, their involvement in the RFO biosynthesis pathway during seed development in soybean is conﬁrmed by Le et al. (2020) [45]. Knocking out GmGolS1_A in G. max resulted in a signiﬁcant reduction of the total RFO content in the seed, whereas the double knockout line of both GmGolS1_A and GmGolS1_B further reduced the total RFO content but to a lesser extent. This indicates that especially GmGolS1_A is important during seed development. The function of the GolS1 genes is not limited to seed development. Both GolS1 genes in G. max show expression in other organs, including the roots, hypocotyl, stem node, leaves and ﬂower during the different developmental stages, however, GmGolS1_B shows a higher expression level in these tissues in comparison with GmGolS1_A. In P. vulgaris, PvGolS1 also shows a relatively high expression level during seed development, especially in the mid and late stages of seed maturation. This was observed in our novel RNA-seq study in P. vulgaris cvs. Rosecoco, Pinto and Canadian wonder and was validated with qRT-PCR in P. vulgaris cv. Plants 2021, 10, 1465 12 of 21 12 of 21 Rosecoco. The transcript levels of PvGolS1 were higher than other galactinol- and RFO synthase genes. This result is uniform between the RNA-seq re-analysis data of P. vulgaris cv. BAT93 and our RNA-seq data. Besides the expression in the seeds, PvGolS1 also shows expression in all vegetative tissues of P. vulgaris during the different developmental stages. The expression of PvGolS1 is especially high in the hypocotyl, epicotyl, stem, leaves and roots which corresponds with the expression pattern of the GolS1 genes in G. max. This indicates that the general expression pattern of the GolS1 class genes is conserved between G. max and P. vulgaris. However, in G. 3. Discussion In contrast, the RS2 genes are mainly active in the seeds during the mid and late maturation stage in P. vulgaris and G. max. In soybean and chickpea seeds, rafﬁnose mainly accumulates in the late stage of seed development which corresponds with the expression pattern seen for the RS2 genes in common bean and soybean [34,68,69]. Furthermore, research done by Valentine et al. (2017) showed that by silencing the GmRS2_A gene in G. max, the rafﬁnose and stachyose content in the seeds signiﬁcantly decreased [14]. In P. vulgaris, expression of PvRS2 can also be seen during the emergence stage of the plant, mainly in the primary leaves. However, no expression of PvRS2 can be seen in the later developmental stages. This suggests that the RS1 genes in P. vulgaris and G. max are mainly active in the vegetative tissue, whereas the RS2 genes are primarily active in the seeds. These results are partly in contrast with the qRT-PCR results of Dierking and Bilyeu (2008) that showed comparable expression levels for GmRS1 and GmRS2_A genes in the seeds and leaves of G. max [7]. These authors further showed that GmRS1 was not associated with the seed rafﬁnose and stachyose content, which corresponds with our observations. They suggested that that GmRS2_B is probably not associated with the seeds’ rafﬁnose and stachyose content because GmRS1 and GmRS2_B are located on the same chromosome. However, our observations suggest that GmRS2_B is also responsible for the production of rafﬁnose in the seeds. The SS single-copy gene in G. max and P. vulgaris mainly shows expression in the primary and trifoliate leaves, the hypocotyl and cotyledon after emergence. It also shows expression in the seeds, primarily during the seeds’ mid and late maturation stage. In P. vulgaris, PvSS is also expressed in the primary root during the emergence stage. The elevated expression level of SS in the seeds during the mid and late maturation stage of the seed development Plants 2021, 10, 1465 13 of 21 13 of 21 in G. max and P. vulgaris corresponds with the observations made in the seeds of soybean and chickpea where the stachyose content also increased during this period [34,68,69]. The combined results of the gene expression atlases demonstrate that the different classes of galactinol- and RFO synthase genes show tissue-speciﬁc expression patterns in soybean and common bean grown under standard conditions (Figure 7). 3. Discussion The different classes of galactinol- and RFO synthase genes most likely also show a speciﬁc expression pattern during abiotic stress conditions [23,26,31,32]. For example, in C. arabica, the GolS gene CaGolS1 showed an increased expression level during drought, heat and salt stress. The GolS gene CaGolS1, which was almost not expressed during standard growth conditions, had elevated transcript levels during drought and salt stress and CaGolS3 was primarily expressed during drought stress [66]. Further research in P. vulgaris and G. max could give a better understanding of the role of the different galactinol- and RFO synthase classes during abiotic stress. Figure 7. Overview of the RFO metabolic pathway and corresponding genes in P. vulgaris and G. max. The galactinol- and RFO synthase genes for P. vulgaris and G. max are shown to the right of the metabolic pathway. The main expression of these genes, either in the seed or the vegetative tissue, is indicated behind the gene names and the expression level of these genes in the seed are indicated in color, ranging from red (high), orange (average) to blue (low or no expression). Figure 7. Overview of the RFO metabolic pathway and corresponding genes in P. vulgaris and G. max. The galactinol- and RFO synthase genes for P. vulgaris and G. max are shown to the right of the metabolic pathway. The main expression of these genes, either in the seed or the vegetative tissue, is indicated behind the gene names and the expression level of these Figure 7. Overview of the RFO metabolic pathway and corresponding genes in P. vulgaris and G. max. The galactinol- and RFO synthase genes for P. vulgaris and G. max are shown to the right of the metabolic pathway. The main expression of these genes, either in the seed or the vegetative tissue, is indicated behind the gene names and the expression level of these genes in the seed are indicated in color, ranging from red (high), orange (average) to blue (low or no expression). Plants 2021, 10, 1465 14 of 21 14 of 21 Lowering the amount of RFOs in the seeds could improve the nutritional quality of these beans and would solve the discomforts associated with their consumption [14]. However, RFOs also play an important role in the seed, protecting it against desiccation, providing longevity during storage and as an energy source during germination [31–38]. In A. 3. Discussion thaliana, it was shown that seed vigor is not correlated to the absolute amount of a speciﬁc RFO molecule but rather to the total amount of RFOs and the ratio of RFOs to sucrose [51]. Contrastingly, when a wild-type soybean was compared with a low RFO variety, no signiﬁcant difference was observed in terms of germination rate [39]. When choosing target genes this should be taken into account and further research should determine the optimal balance between seed health and the beneﬁts for the consumer. Furthermore, RFOs are also needed for sugar transport through the phloem and protect the plant against abiotic stress. Gene targets that would not compromise normal plant development and functioning should be selected. One possible strategy is the targeting of seed-speciﬁc galactinol- and RFO synthase genes responsible for the production of RFOs in the seeds. With the current insights of the expression patterns, suitable candidate genes to alter the expression levels of the galactinol- and RFO synthase genes are proposed to lower the amount of RFOs in the seeds. In soybean, the GolS gene, GmGolS1_A, and the RS genes, GmRS2_A and GmRS2_B, form interesting genes to target because of their expression patterns, indicating that their main activity is during seed development (Figure 7). In common bean, PvRS2 is the only gene with a seed-speciﬁc expression pattern, making it an interesting candidate to knock out. The GolS1 gene, PvGolS1, shows a very high expression level in the seeds of the common bean, indicating its importance in the accumulation of galactinol in the seeds. However, this gene is also expressed in many other plant tissues. Targeting this gene could potentially lead to unwanted phenotypic side effects. However, this does not necessarily have to be the case as was observed in a soybean double mutant where both the GolS1 genes were knocked out, with no adverse effects on the plants’ growth [45]. Besides the formation of stachyose, SS also facilitates the formation of galactinol cyclitols which additionally contribute to the digestive problems associated with RFOs [37,42]. Stachyose is also the main RFO compound present in common bean and soybean seeds. This makes the SS gene also an interesting candidate gene [1,69]. Targeting a combination of these proposed GolS, RS and SS genes will most likely lead to the best result to lower the quantities of RFOs in the seeds. 3. Discussion This should increase the nutritional value and decrease the ﬂatulence and stomach discomforts associated with the consumption of common bean and soybean. 4.1. Plant Material Seeds from P. vulgaris cvs. Pinto, Rosecoco and Canadian wonder, obtained from the Kenya Agricultural and Livestock Research Organization (Nairobi, Kenya) were grown in a greenhouse (Brussels, Belgium) under a 16/8 h light/dark regime. Seeds were harvested at 15, 20, 30 and 35 days after ﬂowering using the ﬂash freezing method with liquid nitrogen and stored at −80 ◦C. 4.2. In Silico Identiﬁcation of the Galactinol and RFO Biosynthetic Genes in Phaseolus vulgaris and Glycine max The amino acid sequences of well-characterized galactinol- and RFO synthase en- zymes of Arabidopsis thaliana (AT2G47180.1, AT1G56600.1, AT1G09350.1, AT1G60470.1, AT5G23790.1, AT4G26250.1, AT1G60450.1, AT5G30500.1, NP_198855.1, NP_192106.3), Cicer arietinum (AMP59727.1, AMP59729.1), Pisum sativum (CAD20127.2), Oryza sativa (XP_015621501.1), Zea mays (NP_001354805.1) and Vigna angularis (CAB64363.1) were ini- tially used as queries against the protein and gene databases of the National Center for Biotechnology Information (NCBI) and Phytozome databases to search for the orthologs in P. vulgaris and G. max using BLASTP and TBLASTN (Table S1) [23,40,51,52,60–62,70–78]. The resulting gene/protein sequences were further used to perform a combination of Plants 2021, 10, 1465 15 of 21 15 of 21 BLASTP, TBLASTN and BLASTN to ﬁnd all potential galactinol and RFO biosynthetic genes in P. vulgaris and G. max. A multiple sequence alignment of the amino acid se- quences was made using the MUSCLE algorithm in MEGA X (v10.2.4) and screened for conserved motifs to distinguish the different RFO synthase enzymes [48]. Two conserved motifs (FMxLGTEAxxLG and SGDPxGTxWLQGCHMVHC) were used by the motif search method of MEGA X(v10.2.4) to distinguish RS from alkaline α-galactosidase [51]. To further distinguish SS from RS the presence of a speciﬁc amino acid insert, only present in SS as described in Peterbauer et al. (1999), has been evaluated through multiple sequence alignment [52]. Sequence similarity and identity were evaluated and compared with other well-characterized galactinol- and RFO synthase enzymes of A. thaliana (AT2G47180.1, AT1G56600.1, AT1G09350.1, AT1G60470.1, AT5G23790.1, AT4G26250.1, AT1G60450.1, AT5G30500.1, NP_198855.1, NP_192106.3), C. arietinum (AMP59727.1, AMP59729.1), P. sativum (CAD20127.2), O. sativa (XP_015621501.1), Z. mays (NP_001354805.1) and V. angu- laris (CAB64363.1). Isoforms where predicted based on the annotation of the Phytozome database. To evaluate the evolutionary relationship, a phylogenetic tree was made in MEGA X (v10.2.4) using the amino acid sequences of the different galactinol and RFO biosyn- thetic and hydrolytic enzymes of P. vulgaris, G. max and well-annotated species A. thaliana (AT2G47180.1, AT1G56600.1, AT1G09350.1, AT1G60470.1, AT5G23790.1, AT4G26250.1, AT1G60450.1, AT5G30500.1, NP_198855.1, NP_192106.3, OAP05273.1, NP_191190.2, NP_00 1031855.1), C. arietinum (AMP59727.1, AMP59729.1), P. sativum (CAD20127.2), O. sativa (XP_015621501.1), textls[-5]emphZ. mays (NP_001354805.1, AAQ07251.2, NP_001105794.2, NP_001105775.2) and V. angularis (CAB64363.1), Cucumis melo (AAM75139.1, AAM75140.1) and Solanum lycopersicum (AAF04591.1) using the neighbor-joining algorithm combined with a bootstrap test of 1000 replicates (Table S1) [46,48,51,58,79]. The Poisson correction method was used to compute the evolutionary distances with the number of amino acid substitutions per site as a unit [49]. 4.2. In Silico Identiﬁcation of the Galactinol and RFO Biosynthetic Genes in Phaseolus vulgaris and Glycine max The phylogenetic tree was drawn to scale using the calculated evolutionary distances as branch lengths. The features of the galactinol and RFO biosynthesis genes were visualized using Gene Structure Display Server 2.0 (GSDS) software [50]. BLASTP, TBLASTN and BLASTN to ﬁnd all potential galactinol and RFO biosynthetic genes in P. vulgaris and G. max. A multiple sequence alignment of the amino acid se- quences was made using the MUSCLE algorithm in MEGA X (v10.2.4) and screened for conserved motifs to distinguish the different RFO synthase enzymes [48]. Two conserved motifs (FMxLGTEAxxLG and SGDPxGTxWLQGCHMVHC) were used by the motif search method of MEGA X(v10.2.4) to distinguish RS from alkaline α-galactosidase [51]. To further distinguish SS from RS the presence of a speciﬁc amino acid insert, only present in SS as described in Peterbauer et al. (1999), has been evaluated through multiple sequence alignment [52]. Sequence similarity and identity were evaluated and compared with other well-characterized galactinol- and RFO synthase enzymes of A. thaliana (AT2G47180.1, AT1G56600.1, AT1G09350.1, AT1G60470.1, AT5G23790.1, AT4G26250.1, AT1G60450.1, AT5G30500.1, NP_198855.1, NP_192106.3), C. arietinum (AMP59727.1, AMP59729.1), P. sativum (CAD20127.2), O. sativa (XP_015621501.1), Z. mays (NP_001354805.1) and V. angu- laris (CAB64363.1). Isoforms where predicted based on the annotation of the Phytozome database. To evaluate the evolutionary relationship, a phylogenetic tree was made in MEGA X (v10.2.4) using the amino acid sequences of the different galactinol and RFO biosyn- thetic and hydrolytic enzymes of P. vulgaris, G. max and well-annotated species A. thaliana (AT2G47180.1, AT1G56600.1, AT1G09350.1, AT1G60470.1, AT5G23790.1, AT4G26250.1, AT1G60450.1, AT5G30500.1, NP_198855.1, NP_192106.3, OAP05273.1, NP_191190.2, NP_00 1031855.1), C. arietinum (AMP59727.1, AMP59729.1), P. sativum (CAD20127.2), O. sativa (XP_015621501.1), textls[-5]emphZ. mays (NP_001354805.1, AAQ07251.2, NP_001105794.2, NP_001105775.2) and V. angularis (CAB64363.1), Cucumis melo (AAM75139.1, AAM75140.1) and Solanum lycopersicum (AAF04591.1) using the neighbor-joining algorithm combined with a bootstrap test of 1000 replicates (Table S1) [46,48,51,58,79]. The Poisson correction method was used to compute the evolutionary distances with the number of amino acid substitutions per site as a unit [49]. The phylogenetic tree was drawn to scale using the calculated evolutionary distances as branch lengths. The features of the galactinol and RFO biosynthesis genes were visualized using Gene Structure Display Server 2.0 (GSDS) software [50]. 4.3. Transcriptome Analysis of the RFO Biosynthesis Pathway 4.3.1. Expression Atlas of Galactinol- and RFO Biosynthesis Genes in G. max and P. vulgaris by RNA-seq Re-Analysis 4.3.1. Expression Atlas of Galactinol- and RFO Biosynthesis Genes in G. max and P. 4.2. In Silico Identiﬁcation of the Galactinol and RFO Biosynthetic Genes in Phaseolus vulgaris and Glycine max vulgaris by RNA-seq Re-Analysis The sequence read archive (SRA) of the International Nucleotide Sequence Database Collaboration (INSDC) was screened for publicly available RNA-seq studies that could be used for the creation of an expression atlas of G. max and P. vulgaris under normal conditions. For P. vulgaris, study SRP030614 containing RNA-seq data of genotype BAT93, as described in Vlasova et al. (2016), was used for the creation of an expression atlas [53]. The common bean plants of this study were grown under a 16 h light and 8 h dark photoperiod at ±25 ◦C and 80% humidity. The study consists of 61 run accession ﬁles (SRR) containing raw read ﬁles (FASTQ) comprising 453 247 Mbases. The RNA-seq data of study SRP038111, as described in Shen et al. (2014), was used for the creation of an expression atlas for G. max cv. Wm82 [54]. The soybean plants in this study were grown during the growing season at the experimental station of the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences (38◦06′56′′ N 114◦32′00′′ E). This study consists of 28 SRR ﬁles containing raw read ﬁles (FASTQ) comprising 181 065 Mbases. FASTQC (v0.11.8) was used for the quality control of the FASTQ ﬁles. The raw read ﬁles were converted into gene count ﬁles using a customized Bash script adapted from Kiekens et al. (unpublished; manuscript in preparation) [80]. In short, raw read ﬁles were trimmed using Sickle (v1.33) with the threshold value for the fragment length set at 35 bp and the threshold for the quality score set at 30 [81]. STAR (v2.6.0) was used to map the trimmed reads on the Wm82.a2.v1 genome assembly for G. max and the Pvulgaris_442_v2.1 genome assembly for P. vulgaris (http://phytozome.jgi.doe.gov/; accessed on 15 May 2020) [74,82]. The number of sequence reads per gene was calculated using HTSeq (v0.9.1) [83]. The resulting count ﬁles were normalized for both the gene length and sequencing depth and were expressed Plants 2021, 10, 1465 16 of 21 16 of 21 in transcripts per million (TPM) [84]. Heatmaps were created using Pheatmap (v1.0.12) within R studio (v1.4.1106) [85]. in transcripts per million (TPM) [84]. Heatmaps were created using Pheatmap (v1.0.12) within R studio (v1.4.1106) [85]. 4.3.2. Expression Atlas of Galactinol- and RFO Biosynthesis Genes during the Seed Development in P. 4.2. In Silico Identiﬁcation of the Galactinol and RFO Biosynthetic Genes in Phaseolus vulgaris and Glycine max vulgaris by De Novo RNA-seq Analysis At four different developmental stages (15, 20, 30 and 35 days after ﬂowering (DAF)) seed samples of P. vulgaris cvs. Pinto, Rosecoco and Canadian wonder were used to extract RNA from using the RNeasy PowerPlant Kit (Qiagen, Hilden, Germany, CAT #13500-50). The quality and quantity of the RNA samples were measured using a Thermo Scientiﬁc (Waltham, MA, USA) NanoDrop 1000 Spectrophotometer and the RNA integrity was determined using the bleach gel electrophoresis protocol described by Aranda et al. [86]. Four biological repeats for each developmental stage were sent for sequencing to the Ge- nomics Core facility (Leuven, Belgium). The library was prepared using the QuantSeq 3′ mRNA-Seq Library Prep Kit (FWD) (Lexogen, Wien, Austria, CAT #015.96) and Illumina’s HiSeq4000 was used for sequencing. The resulting raw read ﬁles were converted into gene count ﬁles using a customized Bash script based on the optimized QuantSeq FWD/REV Data Analysis Pipeline (Available at https://www.lexogen.com/wp-content/uploads/20 21/01/015UG108V0310_QuantSeq-Data-Analysis-Pipeline-on-BlueBee-Platform_2021-01- 20.pdf; accessed on 15 Aug 2020). The quality of the raw read ﬁles was checked us- ing FASTQC (v0.11.8) and trimmed using BBDuk from the BBmap suite (v38.50b, set- tings: -k13 -ktrim r -useshortkmers t -mink 5 -qtrim r -trimq 10 -minlength 20). FastQC (v0.11.8) was also used to check the quality of the trimmed ﬁles. Mapping of the reads on the Pvulgaris_442_v2.1 genome assembly (P. vulgaris v2.1, DOE-JGI and USDA-NIFA, http://phytozome.jgi.doe.gov/; accessed on 15 May 2020) was performed using STAR (v2.6.0) after which Qualimap 2 (v2.2.1) was used to control the quality of the map- ping [74,82,87]. The number of sequence reads per gene was calculated using HTSeq (v0.9.1) [83]. The resulting count ﬁles were normalized for both the gene length and se- quencing depth and were expressed in transcripts per million (TPM) [84]. A more detailed analysis of the RNA-seq data will be published elsewhere by Toili et al. (unpublished; manuscript in preparation) [88]. 4.4. Validation of RNA-Sequencing Results in P. vulgaris cv. Rosecoco 4.4. Validation of RNA-Sequencing Results in P. vulgaris cv. Rosecoco The results of the novel RNA-seq study were validated in one variety of P. vulgaris, namely, cv. Rosecoco. In this variety, the expression levels of the galactinol- and RFO biosynthesis genes were measured during different stages of the seed development using qRT-PCR. The primers used for qRT-PCR were designed using primer-BLAST [89]. Stan- dard settings were used for the product size ranging from 70 to 200 bases and melting temperature ranging from 58.0 to 62.0 ◦C with an optimum of 60.0 ◦C and a maximum temperature difference of 2 ◦C. The concentration of dNTPs was set to 0.4 and the concen- tration of divalent cations was set to 3.0 in the advanced settings. To ensure no genomic DNA could be ampliﬁed, only primers that spanned an exon-exon junction were chosen. Standard curves were made to verify the speciﬁcity and ampliﬁcation efﬁciency of each primer pair. β-tubulin was used as a reference gene [56]. An overview of the primers used for qRT-PCR can be found in Table S2 of the Supplementary Materials. q pp y RNA samples to be used for the qRT-PCR were extracted from 15, 20, 30 and 35 DAF seeds of P. vulgaris cv. Rosecoco (see Section 4.3.2) and treated with RQ1 RNase-Free DNase (Promega, Madison, WI, USA, CAT #M6101) to remove genomic DNA. cDNA was synthesized using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientiﬁc, CAT #K1631). GoTaq qPCR Master Mix (Promega, CAT # A6001) was used to load the samples on the CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) to perform qRT-PCR. The following settings were used: 95 ◦C for 3 min followed by 40 cycles of 15 s at 95 ◦C and 1 min at 60 ◦C each. The ﬂuorescence was measured after each cycle. The dissociation was analyzed starting at 65.0 ◦C and increasing till 95.0 ◦C with increments of 0.5 ◦C every 5 s. After every increment, the ﬂuorescence signal was measured. For each developmental stage, the gene expression was Plants 2021, 10, 1465 17 of 21 17 of 21 measured of three biological repeats. The comparative ∆CT method was used to normalize the cDNAs threshold cycle (Ct) values observed by qRT-PCR using β-tubulin as a reference gene [56,90,91]. The differential expression levels of the galactinol- and RFO synthase genes between the different developmental stages (15 vs. 4.4. Validation of RNA-Sequencing Results in P. vulgaris cv. Rosecoco 20 DAF, 20 vs. 30 DAF and 30 vs. 35 DAF) were calculated using the 2−∆∆Ct method [91]. g Within R studio (v1.4.1106), DESeq2 (v1.30.1) was used to calculate the differential expression levels of the galactinol- and RFO synthase genes between the different devel- opmental stages (15 vs. 20 DAF, 20 vs. 30 DAF and 30 vs. 35 DAF) using the RNA-seq count ﬁles of P. vulgaris cv. Rosecoco [92]. To compare the results of the RNA-seq and qRT-PCR data, the log2 fold changes were represented in a bar chart and the correla- tion between the RNA-seq and qRT-PCR results were calculated using Spearman’s rank correlation coefﬁcient. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/plants10071465/s1, Figure S1: Visualization of the syntenic relationship between P. vulgaris and G. max; Table S1: Overview of the enzymes used for the identiﬁcation of the galactinol- and RFO synthase genes in P. vulgaris and G. max; Table S2: Primers used for qRT-PCR. Author Contributions: Conceptualization, R.d.K., R.K. and G.A.; methodology, R.d.K., R.K. and G.A.; software, R.d.K. and R.K.; validation, R.d.K., G.A., R.d.K. and M.E.M.T.; formal analysis, R.d.K., R.K. and M.E.M.T.; investigation, R.d.K. and M.E.M.T.; resources, G.A.; data curation, R.d.K. and R.K.; writing—original draft preparation, R.d.K.; writing—review and editing, R.d.K., G.A., M.E.M.T. and R.K.; visualization, R.d.K. and R.K.; supervision, R.d.K. and G.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was partially funded by VLI-UOS, grant number KE2017IUC037A101. Data Availability Statement: The data generated and analyzed in this study is available in this paper and the Supplementary Materials. The raw reads have been uploaded to the European Nucleotide Archive database (study PRJEB45523). Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Appendix A A multiple sequence alignment was performed using the MUSCLE algorithm in MEGA X of the protein sequences of potential RFO synthase enzymes of G. max and P. vulgaris together with well-characterized RFO synthase and alkaline α-galactosidase enzymes of O. sativa, C. melo, Z. mays, A. thaliana, P. sativum, V. angularis, S. afﬁnis and A. meridionalis. RS can be distinguished from SS because of an insert present only in SS [52]. Appendix A Figure A1. Visualization of the amino acid motifs present in rafﬁnose synthase (RS) but absent in alkaline α-galactosidase. A multiple sequence alignment was performed using the MUSCLE algorithm in MEGA X of the protein sequences of potential RFO synthase enzymes of G. max and P. vulgaris together with well-characterized RFO synthase and alkaline α-galactosidase enzymes of O. sativa, C. melo, Z. mays, A. thaliana, P. sativum, V. angularis, Stachys afﬁnis and Alonsoa meridionalis. To distinguish RS from alkaline α-galactosidase, two conserved motifs, FMxLGTEAxxLG and SGDPxGTxWLQGCHMVHC, were used. These motifs are present in the amino acid sequence of RS but absent in alkaline α-galactosidase [51]. Figure A1. Visualization of the amino acid motifs present in rafﬁnose synthase (RS) but absent in alkaline α-galactosidase. A multiple sequence alignment was performed using the MUSCLE algorithm in MEGA X of the protein sequences of potential RFO synthase enzymes of G. max and P. vulgaris together with well-characterized RFO synthase and alkaline α-galactosidase enzymes of O. sativa, C. melo, Z. mays, A. thaliana, P. sativum, V. angularis, Stachys afﬁnis and Alonsoa meridionalis. To distinguish RS from alkaline α-galactosidase, two conserved motifs, FMxLGTEAxxLG and SGDPxGTxWLQGCHMVHC, were used. These motifs are present in the amino acid sequence of RS but absent in alkaline α-galactosidase [51]. Plants 2021, 10, 1465 18 of 21 Figure A2. Visualization of the amino acid insert only present in stachyose synthase (SS). A multiple sequence alignment was performed using the MUSCLE algorithm in MEGA X of the protein sequences of potential RFO synthase enzymes of G. max and P. vulgaris together with well-characterized RFO synthase and alkaline α-galactosidase enzymes of O. sativa, C. melo, Z. mays, A. thaliana, P. sativum, V. angularis, S. afﬁnis and A. meridionalis. RS can be distinguished from SS because of an insert present only in SS [52]. Figure A2. Visualization of the amino acid insert only present in stachyose synthase (SS). A multiple sequence alignment was performed using the MUSCLE algorithm in MEGA X of the protein sequences of potential RFO synthase enzymes of G. max and P. vulgaris together with well-characterized RFO synthase and alkaline α-galactosidase enzymes of O. sativa, C. melo, Z. mays, A. thaliana, P. sativum, V. angularis, S. afﬁnis and A. meridionalis. RS can be distinguished from SS because of an insert present only in SS [52]. Figure A2. 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https://openalex.org/W2921474396	https://www.mdpi.com/1996-1944/16/4/1479/pdf?version=1675962664	English	null	Effects of Finish Line Design and Fatigue Cyclic Loading on Phase Transformation of Zirconia Dental Ceramics: A Qualitative Micro-Raman Spectroscopic Analysis	Materials	2,019	cc-by	7,788	Citation: Bodzon-Kulakowska, A.; Młodawska, W.; Mielczarek, P.; Lachowicz, D.; Suder, P.; Smoluch, M. Mammalian Oocyte Analysis by MALDI MSI with Wet-Interface Matrix Deposition Technique. Materials 2023, 16, 1479. https:// doi.org/10.3390/ma16041479 Keywords: oocytes; MALDI; imaging; single cell; lipidomics; wet-interface Keywords: oocytes; MALDI; imaging; single cell; lipidomics; wet-interface Anna Bodzon-Kulakowska 1 , Wiesława Młodawska 2, Przemyslaw Mielczarek 3 , Dorota Lachowicz 4 , Piotr Suder 1 and Marek Smoluch 1,* Anna Bodzon-Kulakowska 1 , Wiesława Młodawska 2, Przemyslaw Mielczarek 3 , Dorota Lachowicz 4 , Piotr Suder 1 and Marek Smoluch 1,* 1 Department of Analytical Chemistry and Biochemistry, Faculty of Materials Science and Ceramics, AGH University of Science and Technology, A. Mickiewicza 30, 30-059 Krakow, Poland 1 Department of Analytical Chemistry and Biochemistry, Faculty of Materials Science and Ceramics, Department of Analytical Chemistry and Biochemistry, Faculty of Materials Science and Ceramics, AGH University of Science and Technology, A. Mickiewicza 30, 30-059 Krakow, Poland 2 Department of Animal Reproduction, Anatomy and Genomics, University of Agriculture in Krakow, Al. Mickiewicza 24/28, 30-059 Krakow, Poland 3 Laboratory of Proteomics and Mass Spectrometry, Maj Institute of Pharmacology, Polish Academy of Sciences Smetna 12, 31-343 Krakow, Poland 3 Laboratory of Proteomics and Mass Spectrometry, Maj Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, 31-343 Krakow, Poland 3 Laboratory of Proteomics and Mass Spectrometry, Maj Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, 31-343 Krakow, Poland 4 Academic Centre for Materials and Nanotechnology, AGH University of Science and Technology, 4 Academic Centre for Materials and Nanotechnology, AGH University of Science and Technology, A. Mickiewicza 30, 30-059 Krakow, Poland * Correspondence: smoluch@agh.edu.pl Abstract: Oocytes are a special kind of biological material. Here, the individual variability of a single cell is important. It means that the opportunity to obtain information about the lipid content from the analysis of a single cell is signiﬁcant. In our study, we present a method for lipid analysis based on the MALDI-based mass spectrometry imaging (MSI) approach. Our attention was paid to the sample preparation optimization with the aid of a wet-interface matrix deposition system (matrix spraying). Technical considerations of the sample preparation process, such as the number of matrix layers and the position of the spraying nozzle during the matrix deposition, are presented in the article. Additionally, we checked if changing the 2,5-dihydroxybenzoic acid (DHB) and 9-Aminoacridine (9AA) matrix concentration and their solvent composition may improve the analysis. Moreover, the comparison of paraformaldehyde-ﬁxed versus nonﬁxed cell analysis was performed. We hope that our approach will be helpful for those working on lipid analyses in extraordinary material such as a single oocyte. Our study may also offer clues for anybody interested in single-cell analysis with the aid of MALDI mass spectrometry imaging and the wet-interface matrix deposition method. materials materials 1. Introduction Academic Editor: Francesco Inchingolo The process of oocyte maturation and the acquisition of developmental competence is complex, regulated by many factors linked to oocyte metabolism, and is still not fully understood. Research suggests that not only carbohydrates but also lipids are used as fun- damental energy donors for oocytes during development and maturation [1–3]. In general, in mammalian oocytes lipids are mainly stored in the form of lipid droplets (LDs) con- taining mostly phospholipids (PLs), neutral triacylglycerols (TAGs), and sterol esters [4–7]. Lipid content and composition undergo dynamic changes during folliculogenesis and oocyte ﬁnal maturation [1,8]. Total lipid composition, type of lipids, and fatty acid (FAs) content of oocytes have been shown to differ signiﬁcantly among mammalian species [9,10]. It is generally accepted that lipid-rich oocytes of pigs and cats have visually darker ap- pearance in bright-ﬁeld microscopy than those with smaller intracellular lipid stores, for example, in mice and rats [6,10,11]. Even oocytes derived from the same ovary may visually differ with respect to ooplasm appearance/darkness level. Studies have shown that bovine oocytes classiﬁed into different “darkness categories” differ in the proportion of certain unsaturated (oleic, linoleic, and arachidonic) and saturated (stearic) FAs content [12] and Received: 1 December 2022 Revised: 4 January 2023 Accepted: 1 February 2023 Published: 9 February 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/materials Materials 2023, 16, 1479. https://doi.org/10.3390/ma16041479 Materials 2023, 16, 1479 2 of 15 after in vitro fertilization (IVF) displayed different capacity for cleavage and blastocyst formation in vitro [13]. after in vitro fertilization (IVF) displayed different capacity for cleavage and blastocyst formation in vitro [13]. Unfortunately, “darkness level” is an extremely inaccurate type of measurement, even if somehow connected to the lipid composition in the cells. Therefore, getting to know the composition of intracytoplasmic lipids is essential for a deeper understanding of the role of lipids and their metabolism in controlling the mechanisms of oocyte development. Such knowledge creates the possibility to improve oocyte quality by modulating lipid metabolism (for example, by the addition of speciﬁc lipid compounds to culture media) and may ﬁnd application in increasing the efficiency of in vitro embryo production in mammals. y pp g y y p For lipid study and visualization in mammalian oocytes various techniques are used, each having its advantages and limitations, enabling lipid analysis in different aspects. Exemplary, gas chromatography allows for the evaluation of the total lipid content, the ratio of FAs content, and composition. However, this technique is expensive and, for suc- cessful analysis, requires a large number (usually several hundred) of oocytes. Fluorescent staining, using such dyes as Nile red or BODIPY 493/503 in association with confocal laser microscopy analysis, is commonly used for LDs imaging and semi-quantiﬁcation in a single oocyte. However, this approach failed to provide detailed information about the different types and content of accumulated lipids. Electron microscopy allows the visualization and assessment of the position, size, and form of LDs within a single cell but does not provide any information about lipid species or their content (for review, see [14]). Proposed here, MALDI (matrix-assisted laser desorption ionization) mass spectrome- try imaging originates from the mass spectrometry-based analytical techniques, allowing for the simultaneous analysis of the majority of lipids building the cell membrane of a single oocyte, as well as the lipid composition of the cytoplasm. MALDI-MSI is a very sensitive method, detecting molecules at the pico- or even femtomolar level, which makes it the method of choice for lipid analysis from a single cell (for concise review, see: [15]). Furthermore, as it is possible to measure, almost simultaneously, various cells placed on the glass target plate one after another, it is possible to easily compare a constituent of different kinds of oocytes. Finally, it allows us to understand the biological background of the differences detected between them. The MALDI-based analysis uses the so-called matrix, being the weak organic acid (like dihydroxybenzoic, cinnamic, or others), which must be applied onto the sample to be imaged. Matrix spraying and matrix sublimation are currently the two most popular matrix deposition techniques. During matrix sublimation, no liquid phase is used, which should minimize the problem of molecules delocalization in the tissue. For lipids, it usually offers a signiﬁcant enhancement of the signal acquired [16]. Due to the characteristics of sublimation methods (the ease of preparation, the puriﬁcation of matrix during sublimation, and no need for expensive devices), they are still under development. This means that the new devices that are invented offer better control of the parameters of matrix deposition [17,18] Spraying techniques offer more efﬁcient analyte extraction and crystallization, essen- tial when protein analysis is considered. Moreover, the possibility of manipulating spraying parameters offers the option of matrix deposition similar to the sublimation process. De- vices for matrix spraying are expensive but, in the meantime, they are more versatile. The efﬁcient methodology of oocyte measurements using MALDI-MSI was proposed and used by our team previously [7,19]. This approach was based on the older system for matrix deposition, ImagePrepTM, which uses ultrasonic spraying from the metallic surface. The aim of the study presented here is to optimize the lipid analysis from the single oocyte with the aid of SunCollect system. It is recognized as the “wet-interface matrix deposition method” and is based on an optimized and highly efﬁcient spray generator that produces extremely small matrix droplets. The efﬁcient coverage of the cells by the matrix prior to MALDI-MSI analysis is one of the most important optimization steps, so, in our opinion, it deserves to be described in detail. Our remarks could be useful for those who would like to use MSI technology in cellular lipids’ qualitative and semi- quantitative measurements. Materials 2023, 16, 1479 3 of 15 3 of 15 During covering the oocytes with the SunCollect set-up, the number of matrix layers and the distance from the spraying tip to the cells could be adjusted. 2.2. MALDI Matrices During the experiments, the following chemicals were used. Solvents: acetonitrile (ACN) and methanol (J.T. Baker, Amsterdam, The Netherlands), both at the HPLC gra- dient grade, ethanol (Avantor, Poland), ultrapure water (Sigma-Aldrich/Merck, Darm- stadt, Germany). Matrices: 2,5-dihydroxybenzoic acid (DHB), triﬂuoroacetic acid (TFA), 9-aminoacridine (9AA), all from Sigma-Aldrich/Merck. Z = 1 mm is the highest position above the cells, and Z = 49 mm is the lowest position possible. Spraying the matrix from the topmost position results in a very dry spray, while the lowest position results in a wetter vapor and better solution penetration into the sample. It should also be remembered that the cells must be deposited onto a special surface before the matrix deposition: indium- tin-oxide covered glass (ITO glass). A conductive surface just under the cells is necessary for the ionization of the molecules during the measurement, while a transparent plate makes it easier to ﬁnd the cells on the surface during the ionization process. g p To optimize MS-based imaging results, we tested two MALDI matrices, selected by us previously as perfect for investigations on oocytes: 2,5-dihydroxybenzoic acid (DHB) for positive ionization mode and 9-aminoacridine (9AA) for negative ionization mode. Different ionization modes make a wider identiﬁcation range of various kinds of lipids. In the positive ionization mode, mainly glycerophosphocholines and sphingomyelins are detected. Meanwhile, the negative ionization mode is suitable mainly for glycerophospho- ethanolamines, glycerophosphoserines, and glycerophosphoinositols. The other lipids can also be detected depending on the sample properties. In the case of both modes, four different positions of the spraying nozzle above the sample surface and the inﬂuence of the number of the matrix layers on the quality of obtained MS spectra were examined. Additionally, we have tested how the matrix concentration and the type of matrix solvent inﬂuence the results. Moreover, oocytes brieﬂy washed with water were compared in those ﬁxed with 4% paraformaldehyde (PFA) using MALDI-MSI and AFM (Atomic Force Microscope). 2.1. Oocyte Collection and Preparation The oocytes were collected from female domestic cat (Felis catus) ovaries after a routine sterilization procedure performed at private local veterinary surgery and animal shelter (Krakow, Poland). The ovaries were transported to the laboratory in phosphate-buffered saline (PBS; Biomed, Lublin, Poland) supplemented with penicillin (100 IU/mL) and streptomycin (100 µg/mL) at 4 ◦C [20]. Cumulus-oocyte complexes (COCs) were recovered by slicing the ovaries in OPU-medium (Bioscience, Bickland Industrial Park, Falmouth, Cornwall, UK), washed in PBS and denuded of surrounding cumulus cells by mechanical pipetting, and kept in PBS at 4 ◦C up to 12 h. The oocytes were then: (i) washed three times with deionized water to remove the salts and buffer compounds (PBS-group) or (ii) ﬁxed by incubating in 4% paraformaldehyde (PFA-group) dissolved in PBS for 15 min, followed by washing thrice in ultrapure water. Subsequently, the oocytes were deposited on the ITO glass (Bruker-Daltonics, Bremen, Germany), near the white mark from the nail polish, facilitating the cell localization on the glass slide during MALDI analysis. The glass surface was covered with the appropriate MALDI matrix, which allowed for the desorption and ionization of the molecules present in the analyzed sample. To be able to properly localize the oocytes on the ITO glass, a picture of each oocyte under the binocular magniﬁer or the microscope was taken and included in some of the Figures. 2. Materials and Methods 2.1. Oocyte Collection and Preparation 2.4. MALDI Measurements Matrix-coated area containing deposited oocytes were subjected to imaging experi- ments using the MALDI–TOF/TOF UltraﬂeXtreme MS (Bruker-Daltonics, Bremen, Ger- many) with a Smartbeam II™laser (Bruker-Daltonics) operating at 2 kHz. All following MS parameters underwent initial, multistep optimization. Ions were accelerated at 25 kV with a pulsed ion extraction of 120 ns and ion suppression up to 100 Da. Spectra were recorded in positive and negative ionization modes with reﬂectron within a 200–3000 m/z. They were externally calibrated with Peptide Calibration Standard II (Bruker Daltonics, Bremen, Germany) and known matrix ions, e.g., 273.03990 m/z for DHB matrix ([2DHB-2H2O+H]+ ion) and 229.05330 m/z for 9AA matrix ([9AA+Cl]−ion). A raster width of 50 µm was applied to all samples. In total, 400 shots were collected from each ablation point with 20 shots at the raster spot, and the laser focus diameter was set to “3_medium”. FlexCon- trol version 3.4 (Bruker Daltonics) was employed for spectra acquisition, and FlexImaging version 4.0 was used for data processing and the creation of molecular images. Mmass software (version 5.5.0, Open-Source software developed by Martin Strohalm, Academy of Sciences, Prague, Czech Republic) was used for the spectra analysis [21]. 2.3. Matrix Application Before the MALDI analysis, an optical image of a glass slide with oocytes and marked ﬁducials—to be able to localize the sample—was taken. Then, the matrices were applied Materials 2023, 16, 1479 4 of 15 by a SunCollect device (SunChrom GmbH, Friedrichsdorf, Germany). For each matrix, a different number of layers were applied. The Z value—the position of the spraying nozzle above the tissue surface was also tested for different matrices. Z = 1 means the highest possible (about 50 mm) position above the tissue, whereas Z = 25 means the position about 25 mm above the tissue and is determined as the “extraction mode”. The ﬂow rate of the matrix solution was variable among layers, according to the manufacturer recommendations: 10 µL/min for the ﬁrst layer, 20 µL/min for the second layer, 30 µL/min for the third layer, 40 µL/min for the fourth layer, and 60 µL/min for all other layers. The nozzle applied the matrix solution with a line distance of 2 mm, and speed equals 600 mm/min. At the beginning, for each matrix, four different numbers of layers and four different nozzle heights were tested. Then, the best parameters were used for different solvents of the same matrix in different concentrations. 2.5. Assessment of the Results For the purpose of our analysis, peaks that represent the main groups of molecules that could be detected in the different ion modes and are widely recognized in the literature were chosen. For the negative ionization mode single fatty acids (255 (16:0), 279 (18:2), 303 (20:4)), lysophosphatidylinositols (LPI) (571 (LPI 16:0), 597 (LPI 18:1), 619 (LPI 20:4), phosphatidic acid 701.5 (PA 36:1), phosphatidylethanolamines (PE) (744.5 (PE 36:1), 762.5 (PE 38:6)) and phosphoinositides (PI) (835 (PI 34:1), 857 (PI 36:4), 885 (PI 38:4)) were selected (see Figure 1). 5 of 16 Figure 1. Peaks chosen for the analysis in the negative ionization mode (marked by their m/z ratios). Figure 1. Peaks chosen for the analysis in the negative ionization mode (marked by their m/z ratios). Figure 1. Peaks chosen for the analysis in the negative ionization mode (marked by their m/z ratios). Figure 1. Peaks chosen for the analysis in the negative ionization mode (marked by their m/z ratios). Materials 2023, 16, 1479 5 of 15 heir m/z For the positive ionization mode, lysophosphatidylcholine 577.5 (LPC 22:1), and phosphatidylcholines (756.4 (PC 32:0), 758.6 (PC 34:2), 760.5 (PC 34:1), 782.5 (PC 36:4), 786.6 (PC 36:2), 808.6 (PC 36:2 Na), 810.7 (PC 36:1 Na), m/z 1546.8 were selected (see: Figure 2). p y p p y ( ) phosphatidylcholines (756.4 (PC 32:0), 758.6 (PC 34:2), 760.5 (PC 34:1), 782.5 (PC 36:4), 786.6 (PC 36:2), 808.6 (PC 36:2 Na), 810.7 (PC 36:1 Na), m/z 1546.8 were selected (see: Figure 2). For the positive ionization mode, lysophosphatidylcholine 577.5 (LPC 22:1), and phosphatidylcholines (756.4 (PC 32:0), 758.6 (PC 34:2), 760.5 (PC 34:1), 782.5 (PC 36:4), 786.6 (PC 36:2), 808.6 (PC 36:2 Na), 810.7 (PC 36:1 Na), m/z 1546.8 were selected (see: Figure 2). p y p p y phosphatidylcholines (756.4 (PC 32:0), 758.6 (PC 34:2), 760.5 (PC 34:1), 782.5 (PC 36:4), 786.6 (PC 36:2), 808.6 (PC 36:2 Na), 810.7 (PC 36:1 Na), m/z 1546.8 were selected (see: Figure 2). Figure 2. Peaks chosen for the analysis in the positive ionization mode (marked by their m/z ratios). Figure 2. Peaks chosen for the analysis in the positive ionization mode (marked by their m/z ratios). 2.6. AFM Analysis Figure 2. Peaks chosen for the analysis in the positive ionization mode (marked by their m/z ratios). Figure 2. Peaks chosen for the analysis in the positive ionization mode (marked by their m/z ratios). 2.6. AFM Analysis ratios). 2.6. AFM Analysis 2.6. AFM Analysis The morphology of the prepared oocyte samples was studied using atomic force microscopy (AFM). AFM topography images were obtained with Dimension Icon XR microscope (Bruker, Santa Barbara, CA, USA) working in the air in the PeakForce Tapping (PFT) mode using soft silicon cantilevers of nominal spring constant of 0.4 N/m and triangular geometry tip with a nominal tip radius of 2 nm. AFM data have been The morphology of the prepared oocyte samples was studied using atomic force microscopy (AFM). AFM topography images were obtained with Dimension Icon XR microscope (Bruker, Santa Barbara, CA, USA) working in the air in the PeakForce Tapping (PFT) mode using soft silicon cantilevers of nominal spring constant of 0.4 N/m and triangular geometry tip with a nominal tip radius of 2 nm. AFM data have been processed using NanoScope Analysis 1.9 and Gwyddion 2.6 software. processed u 3. Results 3. Results 3.1. Nozzle Height and the Number of Matrix Layers 3. Results 3 1 Nozzle Height and the Number 3.1.1. Positive Ionization Mode 3.1. Nozzle Height and the Number of Matrix Layers 3.1.1. Positive Ionization Mode In the positive ionization mode, four different positions of the spraying nozzle above the sample surface were tested. The intensity of the peaks increases with the h i ht f th l ( Fi 3) W l b d th t l l h i ht ld b In the positive ionization mode, four different positions of the spraying nozzle above the sample surface were tested. The intensity of the peaks increases with the height of the nozzle (see Figure 3). We also observed that lower nozzle height could be responsible for the unwanted “splashing” of the cell (see Figure 4). height of the nozzle (see Figure 3). We also observed that lower nozzle height could be responsible for the unwanted “splashing” of the cell (see Figure 4). Figure 3. The relationship between the chosen peaks’ intensities and signal-to-noise ratio (s/n) in terms of the spraying nozzle height for the positive ionization mode, matrix: 25 mg/mL DHB (50% MeOH, 0.2% TFA), number of matrix layers: ten (L = 10). ) g splashing” of the cell (see Figure 4). responsible for the unwanted “s Figure 3. The relationship between the chosen peaks’ intensities and signal-to-noise ratio (s/n) in terms of the spraying nozzle height for the positive ionization mode, matrix: 25 mg/mL DHB (50% MeOH, 0.2% TFA), number of matrix layers: ten (L = 10). 6 of 15 Materials 2023, 16, 1479 Figure 4. The picture of the oocyte obtained after matrix application at a lower nozzle position (Z = 15) and higher nozzle position (Z = 1), positive ionization mode, number of matrix layers: ten (L = 10), size bar: 200 µm). At lower position the content of the cell is going out of it (“splashing”). Below it is presented the microscopic image of oocytes. The inﬂuence of the number of the matrix layers on the quality of obtained MS spectra l i d Th i l i t it i d ith th b f l f th k Fi 4 Th i f h b i d f i li i l l i i Figure 4. processed u 3. Results The picture of the oocyte obtained after matrix application at a lower nozzle position (Z = 15) and higher nozzle position (Z = 1), positive ionization mode, number of matrix layers: ten (L = 10), size bar: 200 µm). At lower position the content of the cell is going out of it (“splashing”). Below it is presented the microscopic image of oocytes. Figure 4. The picture of the oocyte obtained after matrix application at a lower nozzle position (Z = 15) and higher nozzle position (Z = 1), positive ionization mode, number of matrix layers: ten (L = 10), size bar: 200 µm). At lower position the content of the cell is going out of it (“splashing”). Below it is presented the microscopic image of oocytes. The inﬂuence of the number of the matrix layers on the quality of obtained MS spectra was also examined. The signal intensity increased with the number of layers for the peaks identiﬁed in the literature as derived from lipids (see: Figures 5 and 6). 7 of 15 Materials 2023, 16, 1479 Figure 5. The relationship between the chosen peaks’ intensities and signal-to-noise ratio (s/n) in terms of the number of matrix layers for the positive ionization mode, matrix: 25 mg/mL DHB (50% MeOH, 0.2% TFA), nozzle position: Z = 25. Figure 5. The relationship between the chosen peaks’ intensities and signal-to-noise ratio (s/n) in terms of the number of matrix layers for the positive ionization mode, matrix: 25 mg/mL DHB (50% MeOH, 0.2% TFA), nozzle position: Z = 25. Figure 6. The MALDI-MSI picture of the oocyte obtained after covering oocytes with different numbers of the matrix layers, positive ionization mode, (m/z 810, nozzle position: Z = 25), size bar: 200 µm. Below it is presented the microscopic image of oocytes. Figure 6. The MALDI-MSI picture of the oocyte obtained after covering oocytes with different numbers of the matrix layers, positive ionization mode, (m/z 810, nozzle position: Z = 25), size bar: 200 µm. Below it is presented the microscopic image of oocytes. 3.1.2. Negative Ionization Mode For the negative ionization mode, the number of applied matrix layers was also important, but not so obvious as for the positive ionization mode. For the oocytes washed with PBS, the most intense signal was with 14 matrix layers, and the signal-to-noise ratio was also quite high (see: Figures 7 and 8). 8 of 15 Materials 2023, 16, 1479 Figure 7. The relationship between the chosen peaks’ intensities and signal-to-noise ratio (s/n) in terms of the number of matrix layers for the negative ionization mode, matrix: 9AA 7 mg/mL 70% EtOH, nozzle position: Z = 25. Figure 7. The relationship between the chosen peaks’ intensities and signal-to-noise ratio (s/n) in terms of the number of matrix layers for the negative ionization mode, matrix: 9AA 7 mg/mL 70% EtOH, nozzle position: Z = 25. EtOH, nozzle position: Z = 25. Figure 8. The picture of the oocytes obtained after covering them with different number of the matrix layers—negative ionization mode (m/z 744 and 857), size bar: 200 µm. Below it is presented the microscopic image of oocytes. Figure 8. The picture of the oocytes obtained after covering them with different number of the matrix layers—negative ionization mode (m/z 744 and 857), size bar: 200 µm. Below it is presented the microscopic image of oocytes. Considering the spraying nozzle height for the negative mode, it seems that the lowest and the highest nozzle position allow to observe the peaks of high intensity (see: Figures 9 and 10). 9 of 15 Materials 2023, 16, 1479 Figure 9. The relationship between the chosen peaks’ intensities and signal-to-noise ratio (s/n) in terms of the spraying nozzle height for the negative ionization mode, matrix: 9AA 7 mg/mL 70% EtOH, number of matrix layers: L = 14. Figure 9. The relationship between the chosen peaks’ intensities and signal-to-noise ratio (s/n) in terms of the spraying nozzle height for the negative ionization mode, matrix: 9AA 7 mg/mL 70% EtOH, number of matrix layers: L = 14. Figure 10. The picture of the oocyte obtained after matrix application at a lower nozzle position (Z = 25 mm) and higher nozzle position (Z = 5 mm), for the negative ionization mode, size bar: 200 µm. Below it is presented the microscopic image of oocytes. Figure 10. Figure 11. The picture of the oocytes obtained after covering them with matrix of different concentra- tions and solvent compositions, size bar: 100 µm. Figure 11. The picture of the oocytes obtained after covering them with matrix of different concentra tions and solvent compositions, size bar: 100 µm. We have also tested acetonitrile as a solvent, since it is sometimes used with DHB matrix, but in this case it does not work well. There were no reasonable peaks from lipids on the mass spectrum. For some of the m/z values the picture could be created, but it had very low quality. y q y For the negative ionization mode, we used two 9AA matrix concentrations (5 mg/mL and 7 mg/mL), and two ethanol concentrations (50% and 70%, both v/v). The oocytes were covered with 14 layers of matrix, and the spraying nozzle height was established at Z = 1 mm. Here the classical choice of 7 mg/mL 9AA, 70% EtOH was also the best choice (see: Figure 12). Figure 12. The picture of the oocyte obtained after covering them with the matrix of different concentrations and solvent compositions—negative ionization mode, size bar: 200 µm. Figure 12. The picture of the oocyte obtained after covering them with the matrix of different concentrations and solvent compositions—negative ionization mode, size bar: 200 µm. Methanol solution does not work for this kind of analysis with a 9AA matrix. It is impossible to observe lipids when such a solution is used. 3.1.2. Negative Ionization Mode The picture of the oocyte obtained after matrix application at a lower nozzle position (Z = 25 mm) and higher nozzle position (Z = 5 mm), for the negative ionization mode, size bar: 200 µm. Below it is presented the microscopic image of oocytes. 3.2. Different Matrix and Solvent Concentration During the next step, we tried to assess the inﬂuence of matrix concentration and the kind of matrix solvent for the analysis results. In the positive ionization mode, we have tested 25 mg/mL DHB in 50% and 70% methanol, and 15 mg/mL DHB in the same solutions. In this experiment classical composition (25 mg DHB, MeOH 50%) worked the best (see Figure 11). 10 of 15 Materials 2023, 16, 1479 Figure 11. The picture of the oocytes obtained after covering them with matrix of different concentra- tions and solvent compositions, size bar: 100 µm. 3.3. Sample Preparation: PBS and PFA Fixing For the measurements, oocytes were prepared in two ways. In the ﬁrst approach, they were washed from the PBS (storage buffer) by immersing them for a few seconds in three subsequent drops of ultrapure water before deposition on ITO glass. In the second approach, oocytes were ﬁxed by immersing them for 15 min in 4% PFA before washing in ultrapure water (3 times as well). MS-imaging reveals the difference between these two ways of sample preparation. In Figure 13 (see also Supplementary Materials for step-by-step preparation sequence of the Materials 2023, 16, 1479 11 of 15 Figure 13), the oocyte washed with PBS is ﬂattened on the ITO glass, and its content seems to be released, whereas the oocyte ﬁxed with PFA retains its shape. The conclusions drawn from the MSI analysis were conﬁrmed by AFM (Atomic Force Microscope) measurements. AFM analysis of oocyte washed with PBS showed severely damaged cell morphology that has lost its spherical shapes (Figure 13 left). On the other hand, for PFA, we observed unaltered oocytes with a spherical shape and smooth surface. Figure 13), the oocyte washed with PBS is ﬂattened on the ITO glass, and its content seems to be released, whereas the oocyte ﬁxed with PFA retains its shape. The conclusions drawn from the MSI analysis were conﬁrmed by AFM (Atomic Force Microscope) measurements. AFM analysis of oocyte washed with PBS showed severely damaged cell morphology that has lost its spherical shapes (Figure 13 left). On the other hand, for PFA, we observed unaltered oocytes with a spherical shape and smooth surface. Figure 13), the oocyte washed with PBS is ﬂattened on the ITO glass, and its content seems to be released, whereas the oocyte ﬁxed with PFA retains its shape. The conclusions drawn from the MSI analysis were conﬁrmed by AFM (Atomic Force Microscope) measurements. AFM analysis of oocyte washed with PBS showed severely damaged cell morphology that has lost its spherical shapes (Figure 13 left). On the other hand, for PFA, we observed unaltered oocytes with a spherical shape and smooth surface. Figure 13. Oocyte washed with PBS (left) and the oocyte ﬁxed with 4% PFA (right), negative and positive ion mode, with microscopic images of oocytes in the negative ionization mode. Additionally, AFM (Atomic Force Microscope) measurement of both types of the sample, to check of the morphology differences between cells, also visible by the MALDI-IMS. 4. Discussion Measuring and imaging a single cell, even as huge as a single oocyte, is an analytical challenge, especially using a technique with many variables that may inﬂuence the result. In our approach, we decided that the overall high peak intensity on the spectra and good quality of obtained picture of the lipid distribution will be the main indicator of the quality of the proposed settings for the analysis. Additionally, we have tried to reveal some general rules inﬂuencing the quality of the MALDI-MSI measurements. During our analysis, we tested two matrices that are used in positive and negative ionization modes for the lipid measurements. p In both matrices, four different positions of the spraying nozzle above the sample surface and four different numbers of the matrix layers on the quality of obtained MS spectra were tested. In the positive ionization mode, the intensity of the peaks increases with the height of the nozzle. We also observed that lower nozzle height could be responsible for the unwanted “splashing” of the cell. Thus, the highest nozzle height (Z = 1) and ten (l = 10) matrix layers were chosen for the positive ionization mode. For the negative ionization mode, the peak intensity increased with the number of the matrix layers as for the positive ionization mode. Thus, fourteen layers (l = 14) were chosen to be optimal. In the negative ionization mode higher nozzle position (Z = 1) could be used when oocytes are only stored in PBS and washed with water. However, to obtain a signal from PFA-ﬁxed oocytes, lower nozzle height (Z = 25) seems to be necessary, especially in negative ionization mode. We may speculate that the two phenomena inﬂuence the signal intensity when we consider the nozzle height over the sprayed surface. According to the thermal desorption mechanism proposed by Dreisenwerd [22,23], higher temperature increase desorption yield during the MALDI process. Smaller matrix crystals that are produced while spraying with the higher nozzle position achieve higher temperatures during the laser desorption process. It is due to the difﬁculty in the dissipation of laser energy connected simply with the smaller number of atoms in the crystal lattice and inefﬁcient energy transfer between nearby crystals. However, proper extraction and cocrystallization are essential for some molecules and may be responsible for higher signal intensity. 3.3. Sample Preparation: PBS and PFA Fixing Size bar for images: 500 µm. Figure 13. Oocyte washed with PBS (left) and the oocyte ﬁxed with 4% PFA (right), negative and positive ion mode, with microscopic images of oocytes in the negative ionization mode. Additionally, AFM (Atomic Force Microscope) measurement of both types of the sample, to check of the morphology differences between cells, also visible by the MALDI-IMS. Size bar for images: 500 µm. For PFA-ﬁxed oocytes, it should be easier to mark the region of measurement and, thus, the measurements should be more reliable. On the other hand, the content of the cell on the glass seems to produce a higher, more reliable signal and it is easier to obtain the spectrum, especially in the negative ionization mode. To prevent the content of the cell from splashing, the highest (Z = 1) nozzle height should be used while measuring the Materials 2023, 16, 1479 12 of 15 12 of 15 oocytes just washed in water from PBS. On the other hand, to be able to obtain the signal from ﬁxed oocytes with PFA, the lowest (Z = 25) nozzle height should be used. oocytes just washed in water from PBS. On the other hand, to be able to obtain the signal from ﬁxed oocytes with PFA, the lowest (Z = 25) nozzle height should be used. 4. Discussion hand, the signal from oocytes stored in PBS and washed with water is stronger and easier to obtain (see Figure 15). However, care must be taken to mark a bigger area around the cell. A higher nozzle height (Z = 1) should be used in this case to prevent the cell content from splashing over the glass. Thus, in this case, the measurement method may depend on the sample provided and the sensitivity of the mass spectrometer. Measuring the oocytes only washed with water might be advisable when we have an instrument of a lower sensitivity. Figure 14. The comparison of the spectra obtained from different parts of the image of the oocyte washed with PBS (A)—spectrum from the splashed part of the cell (the intensity of the signal is increased), (B)—the signal directly from the cell (the inner part), (C)—the signal from the nail polish—the indicator of oocyte position, (D)—the picture of the oocyte with the marked region from which the spectra were created, (E)—overall view of the MSI image, (F)—the photo of the oocyte on the ITO glass, (G)—the photo of the same oocyte covered with matrix and overlapped by the MS image, size bar: 200 µm. Figure 14. The comparison of the spectra obtained from different parts of the image of the oocyte washed with PBS (A)—spectrum from the splashed part of the cell (the intensity of the signal is increased), (B)—the signal directly from the cell (the inner part), (C)—the signal from the nail polish—the indicator of oocyte position, (D)—the picture of the oocyte with the marked region from which the spectra were created, (E)—overall view of the MSI image, (F)—the photo of the oocyte on the ITO glass, (G)—the photo of the same oocyte covered with matrix and overlapped by the MS image, size bar: 200 µm. Figure 15. The comparison of the spectra of the oocytes washed with PBS and washed in PBA and ﬁxed with PFA in the positive and negative ionization modes. Figure 15. The comparison of the spectra of the oocytes washed with PBS and washed in PBA and ﬁxed with PFA in the positive and negative ionization modes. 4. Discussion Here, the lower nozzle position is preferred, since it allows the production of wetter vapor that better penetrates the sample and allows for better cocrystallization. We may observe it, especially in the case of paraformaldehyde-ﬁxed cells where paraformaldehyde produces cross-links between different chemical groups. It seems that the molecules need stronger extraction from the ﬁxed cell, and that is why the lower nozzle position is able to produce the interpretable signal. Considering the number of matrix layers, it seems that the higher layer number is applied, the higher the signal intensity is obtained, to some point. Unfortunately, such optimization may be limited by the availability of precious material. It is essential to mention that since in our samples we have plenty of molecules that may have different properties, the optimization procedures may indicate the trends characteristic for the particular sample source and may not be perfect for all samples. Considering the matrix concentration and the organic solvent for the matrix in the pos- itive ionization mode, we concluded that the classical matrix concentration and methanol as a solvent (25 mg/mL DHB, MeOH 50%) works the best. Changing the matrix solvent for ACN in the case of DHB does not allow for lipid measurement. The same is with the negative ionization mode, where the classic composition 7mg/mL 9AA and 70% EtOH works best. In the case of lipid analysis, ﬁxing oocytes with PFA allows them to retain its shape. The content of the cell does not “splash” on the glass and makes the marking of the region of measurement easier and more reliable (see Figure 14 and also Supplementary Materials for co-registered images of the cell with a few ion maps for the different m/z). On the other Materials 2023, 16, 1479 13 of 15 hand, the signal from oocytes stored in PBS and washed with water is stronger and easier to obtain (see Figure 15). However, care must be taken to mark a bigger area around the cell. A higher nozzle height (Z = 1) should be used in this case to prevent the cell content from splashing over the glass. Thus, in this case, the measurement method may depend on the sample provided and the sensitivity of the mass spectrometer. Measuring the oocytes only washed with water might be advisable when we have an instrument of a lower sensitivity. 5. Conclusions Due to the improvement of scientiﬁc techniques, especially because of their increased sensitivity, we can go deeper into the complicated biology of the single cell. This is important especially in the case of oocytes, where individual variability may be responsible Materials 2023, 16, 1479 14 of 15 14 of 15 for the fate of the oocyte and embryo development. The MALDI-MSI approach may analyze various kinds of lipids from a single oocyte. It is worth mentioning that this technique may be used to analyze embryos, which are more precious and harder to obtain as well. y y p To conclude, while using wet-interface matrix deposition techniques in the positive ionization mode with 25 mg/mL DHB, MeOH 50% matrix, the highest nozzle height (Z = 1) and ten (l = 10) matrix layers should be used. In the negative ionization mode, the 7 mg/mL 9AA 70% EtOH matrix works best. A high nozzle position (Z = 1) and fourteen matrix layers (l = 14) were also chosen to be optimal. Using oocytes washed from the PBS (storage buffer) by immersing them for a few seconds in three subsequent drops of ultrapure water before deposition on ITO glass allows us to obtain a higher, more reliable signal, especially in the negative ionization mode in comparison with the oocytes ﬁxed with PFA. g p y We hope that our approach will be useful for lipid analysis in such extraordinary material. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ma16041479/s1, Step-by-step preparation sequence of the Figure 13 from the manuscript and co-registered images of the cell with a few ion maps for the different m/z. Author Contributions: Conceptualization, A.B.-K., P.M. and W.M.; methodology, A.B.-K., W.M., P.M. and P.S.; investigation, A.B.-K., P.M. and D.L.; resources, W.M.; data curation, P.M.; writing—original draft preparation, A.B.-K., W.M., P.M., D.L. and M.S.; writing—review and editing, P.S. and M.S.; visualization, A.B.-K. and M.S.; supervision, A.B.-K.; project administration, M.S.; funding acqui- sition, A.B.-K., W.M., P.M. and P.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Polish National Science Center (NCN) grant number 2018/29/B/NZ4/02243 to A.B.-K., statutory funds from the Maj Institute of Pharmacology at the Polish Academy of Sciences to P.M., subsidy no 16.16.160.557 of the Polish Ministry of Science and Education to A.B.-K. and P.S. Data Availability Statement: Data are available on request from the authors. 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. 5. Conclusions and subvention number SUB-020013-D016 of the Ministry of Sciences and Higher Education to University of Agriculture in Krakow, Poland to W.M. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. 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https://openalex.org/W2006710942	https://zenodo.org/record/1266057/files/article.pdf	English	null	Reorganizing a new generation airline network based on an Ant-Colony Optimization-inspired Small-World network	null	2,012	public-domain	6,131	Reorganizing A New Generation Airline Network Based on An Ant-Colony Optimization-Inspired Small-World Network Hidefumi Sawai Advanced ICT Research Institute, National Institute of Information and Communications Technology Kobe 651-2492 Japan, Email: sawai@nict.go.jp Hidefumi Sawai Advanced ICT Research Institute, National Institute of Information and Communications Tech Kobe 651-2492 Japan, Email: sawai@nict.go.jp Fig. 2. A conventional global airline network.[2] Abstract—We describe a real-world application for a next gen- eration airline network using a novel Small-World (SW) network architecture. An emergent method that creates a new type of Small-World network with less average path-length than that obtained with conventional small-world networks is presented. This method is inspired from an Ant-Colony Optimization (ACO) algorithm. The resultant network architecture becomes a multi- star network, which yields a large clustering coefﬁcient and the shortest average path-length among the conventional complex networks such as the Watts-Strogatz and Barab´asi-Albert models etc., from both a theoretical and an experimental analysis of the properties of those networks. Given the advantageous properties of the multi-star network in real-world applications, it could be used to design a new generation global airline network superseding in terms of efﬁciency and convienience the current, conventional airline network owing to fewer transits and a shorter cruising distance on average from any starting point to any destination on Earth. This will be beneﬁcial not only both to travelers and airline companies, but will also contribute to the reduction of greenhouse gases such as carbon dioxide (CO2) in the near future, while enhancing communications and transportation worldwide. Fig. 2. A conventional global airline network.[2] Typical conventional airline networks are shown in Fig.1 and Fig.2 in the cases of a North America and of a global airline network, respectively. A signiﬁcant characteristic of these network architectures is that they are called “scale-free”, which means that more links tend to connect to a node with more degree (the connection probability is proportional to the number of links possessed by the node). This is called “preferential attachment”. The scale-free networks are often modeled by a Barab´asi-Albert (BA) model [5]. This kind of airline network has so far been considered to be convenient for both travelers and airline companies because people can reach any destination from any starting city if the corresponding ﬂight between the two cities is available. WCCI 2012 IEEE World Congress on Computational Intelligence June, 10-15, 2012 - Brisbane, Australia WCCI 2012 IEEE World Congress on Computational Intelligence June, 10-15, 2012 - Brisbane, Australia IEEE CEC U.S. Government work not protected by U.S. copyright Reorganizing A New Generation Airline Network Based on An Ant-Colony Optimization-Inspired Small-World Network However, have the current airline networks been optimized in terms of the efﬁciency of the cruising distance (i.e., saving jet fuel) and of convenience for travelers (i.e., saving cruising time and minimizing the number of transits) as a whole? Isn’t there any room to increase both the efﬁciency and convenience by changing the architecture or topology of the existing airline networks? Keywords—Ant-Colony Optimization (ACO), Small-World, airline networks, n-star networks, complex networks, average path length B. Background of the Small-World In the 1960’s, S.Milgram performed a famous Small-world experiment using letter delivery in the US [3]. From this study, he coined the term ”Six-degrees of separation”, which means that people are separated from only six persons on average. Later, several researchers additionally performed similar ex- periments for various kinds of communities such as actors in Hollywood, co-authors of mathematical papers, etc. [4], which supported the claim made by Milgram based on his experiment. Recently, Yahoo began a similar experiment using Facebook to verify the ”Six-degrees of separation” hypothesis for the members of Facebook [6]. In this study, we will re-examine the characteristics of the Small-World using several network parameters, such as the number of nodes N, the average degree ⟨k⟩, the average path-length L, the clustering coefﬁcient C, the link exchange probability ρ, etc., through experimental simulations using the Dijkstra method [9], searching for the shortest path in several complex networks. (Barab´asi-Albert) model, the Tree, the WS (Watts-Strogatz) model [4], the hypercube and the complete graph. Following are some network indexes used in this paper; Generally speaking, it is said that the WS (Watts-Strogatz) and BA (Barab´asi-Albert) models yield comparatively shorter average path-lengths than those of the other complex networks. However, we will further try to have new Small-World net- works emerge with a shorter average path-length (it might be the shortest one) in a self-organizing manner. • Number of nodes: N. • Average degree: ⟨k⟩. • Link probability: p = ⟨k⟩/(N −1). • Average path length: L. • Clustering coefﬁcient: C. Next, we will analyze the resultant network architecture theoretically and experimentally. By deﬁning a new network index R, we will show that the degree of the Small-World (we call it ”Small-Worldness”) can be evaluated for several complex networks. Finally, we will show that this new small- world network can be applied to a new generation airline network in the real world. • Link exchange probability: ρ (in the case of the WS model). Comparing the average path-lengths among the complex net- works in Table 1, the order of the average path-length L is O(logN) for the Tree, random graphs, BA models and hypercubes as well as for the real networks, which is compara- tively shorter than for other kinds of networks. The clustering coefﬁcient C is large for real networks, lattices and cycles, but small for Trees, random graphs and hypercubes. B. Background of the Small-World For a WS model, starting from an extended cycle, as the link exchange probability ρ gradually increases from 0, it is well known that the average path length dramatically decreases in the range Table 1. Comparing characteristics in typical complex networks. [7] Table 1. Comparing characteristics in typical complex networks. [7] I. INTRODUCTION A. Conventional Airline Networks I. INTRODUCTION A. Conventional Airline Networks Fig. 1. A conventional airline network in North America.[1] Very recently, a next-generation airplane, the Boeing 787, has been brought into in service [10]. This airplane is middle- sized and has good characteristics that are designed to ensure energy-saving. It can operate a non-stop ﬂight over a distance of 14,200-15,200 km (this distance corresponds to a ﬂight between Tokyo in Japan and Johannesburg in South Africa) because of its light body and energy efﬁciency. Many airline companies have already purchased and/or ordered a number of models of this airplane [10]. Changing the current airline Fig. 1. A conventional airline network in North America.[1] U.S. Government work not protected by U.S. copyright U.S. Government work not protected by U.S. copyright Fig. 3. Typical Complex Networks networks to design more efﬁcient and convenient networks using this airplane will be greatly beneﬁcial for both ﬂying and travelers in terms of reducing jet fuel consumption and travel time because of shorter cruising distances and transit times. To achieve this goal, we explore a novel Small-World network inspired from Ant-Colony Optimization (ACO) [8], whose average path length is shorter than that obtained with any other conventional complex network, such as a Watts- Strogatz model [4] and a Barab´asi-Albert model [5] etc. Thus, travel time and jet fuel consumption of airplanes are reduced by cruising on the shortest path between any starting city and destination on the Earth, which not only increases fuel efﬁciency (i.e., saves energy) and reduces both travel times and the number of transits (to the beneﬁt of passengers), but also reduces the emission of greenhouse gases such as carbon dioxide (CO2) on Earth, while enhancing communications and transportation worldwide. Fig. 3. Typical Complex Networks C. Comparison of Complex Networks In Table 1, we summarize the features of conventional complex networks in terms of the Small-World using several network indexes. Also, in Fig.3, we show some typical exam- ples of complex networks, such as a random graph, the BA Fig. 4. Sorting the order of pheromone concentration on links along the shortest path. an average degree ⟨k⟩. In this case, the generation probability for each link is p = ⟨k⟩/(N −1). (2) Search for the shortest path from a start node Vs to a destination node Vd using the Dijkstra method. Pheromones are spread by ants along the shortest path. In the initial stage, the concentration of pheromones is zero independently of links. (3) Each ant searches for the shortest path, and spreads some amount of pheromones while detecting the higher concentration paths in all combinations of node pairs Vs and Vd. As a result, a difference of pheromone concentration along each path will arise. Namely, the concentration of pheromones will be relatively higher for the paths on which more ants walked than for those on which less ants walked. Fig. 4. Sorting the order of pheromone concentration on links along the shortest path. (4) Sort the links formed in step (3) on the order of high concentration of pheromones. Then, each vertex in two edges is randomly connected (See Fig.4). This operation is based on the fact that pheromone trails with higher concentration tend to be connected with each other. As the average degree ⟨k⟩increases when one or two edges are newly created, simultaneously cut the links with the order of smaller concentration of pheromones to keep the value of ⟨k⟩as constant as possible. Fig. 5. Adjacency Matrix (left) and its corresponding network (right) generated by a method inspired from ACO (Pseudo-Ant-SW Method) when N=100. (5) Calculate the average path-length L and clustering coefﬁcient C for the newly created network in step (4). (6) If the average path-length L is shorter than a pre- determined threshold value Lth, stop the operation. Otherwise, return to step (2) and repeat the steps from (2) to (5). (6) If the average path-length L is shorter than a pre- determined threshold value Lth, stop the operation. Otherwise, return to step (2) and repeat the steps from (2) to (5). C. Comparison of Complex Networks It is very interesting to note that this network architecture obtained through a bottom-up process like ACO is similar to that obtained through an opposite top-down process called the Monte Carlo Bimodal method (See [12]). Fig. 5. Adjacency Matrix (left) and its corresponding network (right) generated by a method inspired from ACO (Pseudo-Ant-SW Method) when N=100. III. SIMULATION EXPERIMENTS AND RESULTS Fig. 6. Multi-star networks with star nodes at the center and their peripheral nodes. The star nodes fully connect with their peripheral nodes, and some peripheral nodes connect with some other peripheral nodes, as denoted by dotted lines. of small values of ρ. However, the WS model converges to a random graph as ρ reaches 1. The only graph that yields the smallest value of L (=1) and the largest value of C (=1) is the complete graph. From a Small-World viewpoint, it is necessary to know which network architecture has an absolutely small value of L, rather than the order of L (for example, O(logN)). II. SELF-ORGANIZING OF A SMALL-WORLD NETWORK USING ACO Given a complex network with a number of nodes N and an average degree ⟨k⟩, one can obtain the average path length L using the Dijkstra method [9] and the clustering coefﬁcient C. Then, we propose a self-organizing method for generating a network architecture with a shorter path-length L than that obtained with conventional complex networks, without increasing the average degree ⟨k⟩. A self-organizing method for creating a Small-World inspired from ACO (Ant-Colony Optimization) [8] is described as follows: A self-organizing method for creating a Small-World inspired from ACO (Ant-Colony Optimization) [8] is described as follows: Fig. 6. Multi-star networks with star nodes at the center and their peripheral nodes. The star nodes fully connect with their peripheral nodes, and some peripheral nodes connect with some other peripheral nodes, as denoted by dotted lines. (1) Generate a random graph with a number of nodes N and (1) Generate a random graph with a number of nodes N and Fig. 7. L vs. ⟨k⟩in several complex networks. length L and clustering coefﬁcient C are calculated as follows; Fig. 7. L vs. ⟨k⟩in several complex networks. ⟨k⟩= n(2N −n −1) N ; n = 1, 2, 3, ..., N. (1) L = 2N(N −n −1) + n(n + 1) N(N −1) ; n = 1, 2, 3, ..., N. (2) C = n{n−1C2+(n−1)(N−n)} N−1C2 + (N −n) N ; n = 2, 3, ..., N. (3) C = 0; n = 1. The relationships between ⟨k⟩and n (Eq.(1)), L and n Fig. 9. ⟨k⟩vs. n (upper left), L vs. n (upper right), C vs. n (bottom left), and L vs. ⟨k⟩(bottom right) in multi-star networks with n-star nodes and their peripheral nodes. Fig. 7. L vs. III. SIMULATION EXPERIMENTS AND RESULTS ⟨k⟩in several complex networks. Fig. 8. R vs. ⟨k⟩in several complex networks. Fig. 9. ⟨k⟩vs. n (upper left), L vs. n (upper right), C vs. n (bottom left), and L vs. ⟨k⟩(bottom right) in multi-star networks with n-star nodes and their peripheral nodes. (Eq.(2)), and between C and n (Eq.(3)) are shown in Figure 9, at the upper left, upper right and bottom left of the ﬁgure, respectively. For n=1, the n-star network becomes a so-called single star network, and the network becomes a complete graph for n = N −1 and N. The average degree ⟨k⟩ monotonically increases from 0 to N. On the other hand, L monotonically decreases from 2 to 1 as n increases. The clustering coefﬁcient C decreases once, reaching a minimum value around ⟨k⟩= 33, and then increases with n. If n is removed using Eqs.(1) and (2) to obtain the relationship between L and ⟨k⟩, we obtain Eq.(4) as follows: Fig. 8. R vs. ⟨k⟩in several complex networks. Figure 5 (left) shows the adjacency matrix reﬂecting pheromone concentration in the Pseudo-Ant-SW method when N=100. The thicker the color, the higher the pheromone concentration. Figure 5 (right) shows the multi-star network corresponding to the adjacency matrix in Figure 5 (left) from two different points of view. From these ﬁgures, we found that the network architecture consists of a few stars with many links and many peripheral nodes with a few links, and these peripheral nodes are mutually connected with a small probability. Furthermore, from the values of the adjacency matrix and the connection conﬁguration around nodes number 41 and 81, we can ﬁnd a multi-star (in a sense, a ”fractal”) structure in the network. This results from the algorithm according to which the higher concentration nodes connect with one another in the Pseudo-Ant-SW method. L = 2N −⟨k⟩−2 N −1 = 2 −p (4) (4) The relationship between L and ⟨k⟩for N =100 is shown in Figure 9 (bottom right). Although Eq.(4) is derived for ⟨k⟩ values, each value corresponding to n = 1, 2, 3, .., N, it can be shown that this equation is also valid for any intermediate values of ⟨k⟩with arbitrary numbers of peripheral edges connected with each other, which are represented by dotted lines in Figure 6. The relationship between L and ⟨k⟩for N =100 is shown in Figure 9 (bottom right). III. SIMULATION EXPERIMENTS AND RESULTS Therefore, we can comparatively evaluate each network as the degree of Small-World (we may call it ”Small- Worldness”) based on this invariant R. The relationships between L and ⟨k⟩, and R and ⟨k⟩are shown in Figures 7 and 8, respectively. The R values are always 2 for n-star networks, as mentioned before. On the other hand, other networks such as the random graph, the WS and BA models have relatively larger R values than 2. Although the BA model possesses a ”scale-free” characteristic with the order of L ∝O(logN), the R value is not so small compared to that obtained in the random graph and WS model. The proposed Pseudo-Ant-SW and Monte Carlo Bimodal models (See [12] for the latter) have remarkably small values of L and R. In particular, the R value in the Monte Carlo Bimodal model almost reaches 2 in n-star networks. This results indicate that these networks achieve the maximum ”Small-Worldness” for a given average degree ⟨k⟩(i.e., the minimum value of L with an appropriate average degree ⟨k⟩) . to map an n-Star network onto a global airline network while considering the above importance ranking of a city etc. We will use the latter method in the following section for realizing a global airline network. It is well known that the current airline network has a ”scale- free” characteristic like the BA model (see also Table 1). As is proved in Sec.III, the L value of the n-star network is much smaller than that of the BA model in the range of a small value of ⟨k⟩. For example, the average path-length L of the n-star network is 24.4% smaller than that of the BA model for ⟨k⟩= 5.88. Very recently, a next-generation airplane, the Boeing 787, has been brought into service [10]. This airplane is middle-sized and has good characteristics that are designed to ensure energy-saving. It can operate a non-stop ﬂight over a distance of 14,200-15,200 km (this distance corresponds to a ﬂight between Tokyo in Japan and Johannesburg in South Africa) because of its light body. The n-star networks could be applied to the design of next-generation global airline networks, as an example of logistics. Figure 10 shows how to calculate the distance between any two cities, P and Q on the surface of the Earth in 3D spherical polar coordinates (r, θ, φ). IV. APPLYING N-STAR NETWORKS TO AIRLINE NETWORKS Fig. 10. Calculation of the cruising distance in global airline networks. III. SIMULATION EXPERIMENTS AND RESULTS A British consulting company (GaWC) ranked important cities in the world [11]. We chose the top 100 cities from this list (see Appendix) and compare in the next section the average path-lengths L among ﬁve kinds of airline networks. We selected ﬁve major cities on ﬁve continents from these top cities that occupy a central place in the world in terms of economy and geography: Tokyo, Chicago, Frankfurt, Sydney and Johannesburg. III. SIMULATION EXPERIMENTS AND RESULTS Although Eq.(4) is derived for ⟨k⟩ values, each value corresponding to n = 1, 2, 3, .., N, it can be shown that this equation is also valid for any intermediate values of ⟨k⟩with arbitrary numbers of peripheral edges connected with each other, which are represented by dotted lines in Figure 6. Next, we analyze the characteristics of several types of complex networks using Lagrange multipliers. In the case of n-star networks, for a given value of ⟨k⟩, we deﬁne the R value as follows in order to obtain the minimum value of L; For the n-star networks (we can call the multi-star network the n-star network), the average degree ⟨k⟩, average path- R ≡L + λ ⟨k⟩ (5) (5) R ≡L + λ ⟨k⟩ By calculating the partial derivative of R with respect to n, we obtain λ = 1/(N −1). Substituting this formula into Eq.(5), we obtain the invariant R ≡2, independent of the values of L and ⟨k⟩. This means that the R value is invariant for n- star networks. Therefore, we can comparatively evaluate each network as the degree of Small-World (we may call it ”Small- Worldness”) based on this invariant R. The relationships between L and ⟨k⟩, and R and ⟨k⟩are shown in Figures 7 and 8, respectively. The R values are always 2 for n-star networks, as mentioned before. On the other hand, other networks such as the random graph, the WS and BA models have relatively larger R values than 2. Although the BA model possesses a ”scale-free” characteristic with the order of L ∝O(logN), the R value is not so small compared to that obtained in the random graph and WS model. The proposed Pseudo-Ant-SW and Monte Carlo Bimodal models (See [12] for the latter) have remarkably small values of L and R. In particular, the R value in the Monte Carlo Bimodal model almost reaches 2 in n-star networks. This results indicate that these networks achieve the maximum ”Small-Worldness” for a given average degree ⟨k⟩(i.e., the minimum value of L with an appropriate average degree ⟨k⟩) . By calculating the partial derivative of R with respect to n, we obtain λ = 1/(N −1). Substituting this formula into Eq.(5), we obtain the invariant R ≡2, independent of the values of L and ⟨k⟩. This means that the R value is invariant for n- star networks. V. SIMULATION EXPERIMENTS AND RESULTS IN SEVERAL AIRLINE NETWORKS We construct the following ﬁve kinds of airline networks based on the n-star networks, and compare their performance in terms of the Small-World, i.e., of efﬁciency and convenience for both ﬂying and travelers. First, we deﬁne the following parameters for constructing several network models: • Average ﬂight service per city: ⟨k⟩. • Population of city i: Po(i). • Spherical distance between city i and city j on the surface of the Earth: dij. Fig. 10. Calculation of the cruising distance in global airline networks. j • Trafﬁc between city i and city j: tij ∝Po(i) · Po(j). In the previous sections, we have found that the n-Star network could be formed in a self-organizing way by an ACO- inspired Small-World network. An essential reason why the n- Star network shows a high degree of “Small-Worldness” is the architecture consisting of its clique with n star nodes and their peripheral nodes with some mutual links. This advantageous architecture can be applied to real-world problems such as lo- gistics, for example, reorganizing an efﬁcient next-generation airline network, while reﬂecting the importance ranking of a city, its population and the distance between any two cities in the world. For that purpose, it might be possible to generate from scratch (i.e., a random graph) an n-Star network for the airline network in a self-organizing way while considering the importance ranking of a city etc. However, it is much easier • Importance of relation between city i and city j (weight value): wij ∝tij/dij ∝Po(i) · Po(j)/dij . j j j j • Physical average distance between any two cities on the surface of the Earth: L(distance). • R value based on the physical average distance L(distance): R(distance). • Average number of steps between any two cities: L(steps) • R value based on the average number of steps L(steps): R(steps). • Threshold for trafﬁc and importance of relation: T h. The spherical distance dij between a city i and a city j is calculated using the equation shown in Fig.10. Trafﬁc tij between a city i and a city j can be proportional to the multiplication of the populations of two cities. The importance of the relation between a city i and a city j can be proportional to the trafﬁc and reversely proportional to the distance between the two cities. V. SIMULATION EXPERIMENTS AND RESULTS IN SEVERAL AIRLINE NETWORKS The peripheral cities connect V. SIMULATION EXPERIMENTS AND RESULTS IN SEVERAL AIRLINE NETWORKS As the 5 star cities, we chose Tokyo in Japan, Sydney in Australia, Chicago in the US, Frankfurt in Germany, and Johannesburg in South Africa, for example, because these cities are located near at the center of each continent, and play important roles in terms of geography and/or economics. There are some peripheral links between some cities around each nearest star city. As the number of peripheral cities increases, the average degree ⟨k⟩increases. This network includes a sub-complete network (i.e., a clique) among the above 5 star cities. (3) An airline network with 5 star cities and their peripheral links (denoted as 5 star + peripheral links around the nearest star” in Figures 11-16): This network consists of 5 star cities with their peripheral city links around the nearest star city. As the 5 star cities, we chose Tokyo in Japan, Sydney in Australia, Chicago in the US, Frankfurt in Germany, and Johannesburg in South Africa, for example, because these cities are located near at the center of each continent, and play important roles in terms of geography and/or economics. There are some peripheral links between some cities around each nearest star city. As the number of peripheral cities increases, the average degree ⟨k⟩increases. This network includes a sub-complete network (i.e., a clique) among the above 5 star cities. Fig. 11. L(distance) versus ⟨k⟩for distance in global airline networks. L is normalized by the radius r of the Earth, 6,356 Km (from the pole to the center) -6,378 Km (from the equator to the center). (4) An airline network with 5 star cities and their peripheral links at random (denoted as 5 star + peripheral links in Figures 11-16): This network consists of the 5 star cities with their peripheral city links. The peripheral cities are selected at random within the non-stop cruising distance of a Boeing 787 (i.e., 14,200-15,200km). As the number of peripheral cities increases, the average degree ⟨k⟩increases as well. Fig. 11. L(distance) versus ⟨k⟩for distance in global airline networks. L is normalized by the radius r of the Earth, 6,356 Km (from the pole to the center) -6,378 Km (from the equator to the center). (5) An airline network with 5 star cities and their peripheral links with city rank (denoted as 5 star + peripheral links+ city rank in Figures 11-16 ): This network also consists of the 5 star cities with peripheral city links. V. SIMULATION EXPERIMENTS AND RESULTS IN SEVERAL AIRLINE NETWORKS (1) An airline network with frequent trafﬁc (denoted as ”Trafﬁc + peripheral links” in Figures 11-16): This airline network is formed based on the population of cities and their trafﬁc in proportion to the multiplication of the population in each city. As the threshold value T h of trafﬁc changes, the average degree ⟨k⟩changes. Namely, if the trafﬁc between two cities i and j is larger than the threshold value T h, the corresponding component aij of an adjacency matrix a is 1, otherwise 0. Therefore, there are some peripheral links between some cities according to the threshold. (2) An airline network with frequent trafﬁc and city rank (denoted as ”Trafﬁc + peripheral links + city rank” in Figures 11-16): This airline network is formed based on the population of cities and their trafﬁc in proportion to the multiplication of the population in each ranked city. Furthermore, the values (i.e., the importance of the relation between two cities) in proportion to each city rank are weighted according to the above evaluation (i.e, wij ∝tij/dij). Similar to the case of (1), if the importance between two cities is larger than the threshold value T h, the corresponding component aij of an adjacency matrix a is 1, otherwise 0. Therefore, there are some peripheral links between some cities according to the threshold. Note that the top ranked few selected cities (e.g., the top ﬁve cities) are not necessarily fully connected by forming a “clique (sub-complete network)” in the cases of (1) and (2). When considering the clustering coefﬁcients C in Fig.15 and the maximum eigenvalues in Fig.16, the network with 5 star cities and their peripheral links around the nearest star (3) is the smallest among the ﬁve kinds of networks, where the values C increase almost in proportion to the increase of ⟨k⟩. Networks (1) and (2) produce some relatively larger values. Network (4) yields an intermediate value among the ﬁve kinds of networks. (3) An airline network with 5 star cities and their peripheral links (denoted as 5 star + peripheral links around the nearest star” in Figures 11-16): This network consists of 5 star cities with their peripheral city links around the nearest star city. V. SIMULATION EXPERIMENTS AND RESULTS IN SEVERAL AIRLINE NETWORKS Namely, when the trafﬁc is larger, and the distance is shorter, the importance of the relation between two cities becomes higher. We will construct the following ﬁve kinds of airline network models based on these parameters. with one another based on each city weight value wij. The city weight value wij is deﬁned in proportion to the trafﬁc tij and in reverse proportion to the distance dij between two cities. We calculated the average distance L(distance) between any two cities i and j, and show the results in Fig.11. From these values of L, we calculated the R(distance) values and show the results in Fig.12. These distances are based on the physical spherical distance on the surface of the Earth. On the other hand, another important issue for airline networks is the number of transits, because the fewer the transits, the more convenient traveling is for passengers. Fig.13 shows the average number of steps L(step) to reach any destination city from any starting city. If the L(step) is less than 2, travelers can reach any destination city with only one transit. In this sense, an average small value L(step) is extremely important as well as the average physical cruising distance L(distance), for airline companies, travelers and Earth’s environment in terms of reducing fuel consumption, travel time and green- house gases such as carbon dioxide. From these values of L(steps), we also calculated the values of R(steps), and show them in Fig.14 as well. From this ﬁgure, we can easily evaluate the “Small-Worldness” for each network, where the networks (4) and (5) have better characteristics in terms of “Small-Worldness”, especially in a relative smaller range of ⟨k⟩, compared to the other networks (1)-(3). (1) An airline network with frequent trafﬁc (denoted as ”Trafﬁc + peripheral links” in Figures 11-16): This airline network is formed based on the population of cities and their trafﬁc in proportion to the multiplication of the population in each city. As the threshold value T h of trafﬁc changes, the average degree ⟨k⟩changes. Namely, if the trafﬁc between two cities i and j is larger than the threshold value T h, the corresponding component aij of an adjacency matrix a is 1, otherwise 0. Therefore, there are some peripheral links between some cities according to the threshold. VI. DISCUSSION We have re-examined the characteristics of the Small-World (SW) in several complex networks from the viewpoint of the Fig. 14. R(steps) versus ⟨k⟩for the number of transits in global airline networks. Fig. 12. R(distance) versus ⟨k⟩for distance in global airline networks. R is normalized by the radius r of the Earth, 6,356 Km (from the pole to the center) -6,378 Km (from the equator to the center). Fig. 12. R(distance) versus ⟨k⟩for distance in global airline networks. R is normalized by the radius r of the Earth, 6,356 Km (from the pole to the center) -6,378 Km (from the equator to the center). Fig. 14. R(steps) versus ⟨k⟩for the number of transits in global airline networks. Fig. 15. C versus ⟨k⟩in global airline networks. Fig. 13. L(steps) versus ⟨k⟩for the number of transits in global airline networks. Fig. 15. C versus ⟨k⟩in global airline networks. Fig. 13. L(steps) versus ⟨k⟩for the number of transits in global airline networks. and travelers because of the shortest cruising distance (e.g., 1.32 r = 8,404 km for ⟨k⟩=13.2) and smallest number of transits (i.e., only one transit is necessary between any two cities for any value of ⟨k⟩) on average, which will also have a beneﬁcial impact on Earth’s environment in terms of reducing greenhouse gases (e.g., carbon dioxide (CO2)), while enhancing communications and transportation worldwide. ”absolute” average path-length, because conventional complex networks known as Small-World are not necessarily SW in a rigorous sense. A method inspired by ACO is proposed to create a new Small-World with the smallest average path- length L. Also, we compared several characteristics in con- ventional typical complex networks from different viewpoints. In particular, we deﬁned a new evaluation criterion, the R- value, in order to minimize the average path-length L when ⟨k⟩is constant. We found that the R-value reaches a minimum value and is invariant for the n-star networks. Several kinds of complex networks can be evaluated based on this R-value in terms of the Small-World (”Small-Worldness”). APPENDIX Fig. 17. Schematic diagram of a new generation global airline network in the near future. Table 2. Top 100 cities (shown in part) in the world. [11] The population is represented in units of ten thousands. Table 2. Top 100 cities (shown in part) in the world. [11] The population is represented in units of ten thousands. No. Rank City Country Pop. 1 A++ London UK 1250 2 A++ New York US 800 3 A+ Hong Kong China 700 4 A+ Paris France 1184 5 A+ Singapore Singapore 507 6 A+ Tokyo Japan 1320 7 A+ Shanghai China 2300 8 A+ Chicago US 300 9 A+ Dubai UAE 226 10 A+ Sydney Australia 434 .. .. ....... ..... ... .. .. ....... ..... ... 44 A- Santiago Chili 467 45 A- Lisbon Portugal 565 46 A- Philadelphia US 580 47 A- Johannesburg South Africa 388 48 B+ Dusseldorf Germany 58 49 B+ Stockholm Sweden 75 50 B+ Prague Czech 120 51 B+ Montreal Canada 162 52 B+ Roma Italy 272 53 B+ Hamburg Germany 174 54 B+ Manila Philippine 166 .. .. ....... ..... ... .. .. ...... ..... ... 91 B- Panama Panama 71 92 B- Helsinki Finland 59 93 B- Shenzhen China 1200 94 B- Guatemala City Guatemala 101 95 B- Stuttgart Germany 60 96 B- Abu Dhabi UAE 143 97 B- Birmingham UK 100 98 B- Kolkata India 458 99 B- Koln Germany 100 100 B- St. Louis US 283 Fig. 17. Schematic diagram of a new generation global airline network in the near future. applications, it could be used to design a new generation global airline network superseding in terms of efﬁciency and convenience the current, conventional airline network owing to fewer transits and a shorter cruising distance on average from any starting point to any destination on Earth. This will be beneﬁcial not only both to travelers and airline companies, but will also contribute to the reduction of greenhouse gases such as carbon dioxide (CO2) in the near future, while enhancing communications and transportation worldwide.. VII. CONCLUSION We described a new generation airline network as a real- world application of a novel Small-World (SW) network archi- tecture. An emergent method that creates a new type of Small- World network with less average path-length than that obtained with conventional small-world networks was presented. This method is inspired by an Ant-Colony Optimization (ACO) algorithm. The resultant network architecture becomes a multi- star network, which yields a large clustering coefﬁcient and the shortest average path-length among the conventional complex networks such as a Watts-Strogatz model and a Barab´asi- Albert model etc., from both a theoretical and an experimental analysis of the properties of those networks. Considering the advantageous properties of the multi-star network in real-world On the other hand, based on the above knowledge, we could also design an optimized next-generation airline network, as shown in Fig.17, where 5 star cities are completely connected (i.e., forming a “clique”) and their peripheral cities are ade- quately connected based on demand (i.e., trafﬁc, importance and value of relation between two cities). This network ar- chitecture has not only the merit of saving mileage, but also is more efﬁcient and convenient to both airline companies Fig. 16. Eigenvalue versus ⟨k⟩in global airline networks. [5] A.-L. Bar´abasi, R, Albert, Emergence of Scaling in Random Networks, Science, Vol.286,pp.509-512, 1999. pp [6] Yahoo’s SW experiment using Facebook, http://www.zdnet.com/blog/facebook/yahoo-facebook-test-8220six- degrees-of-separation-8221-idea/2678. g p [7] N. Masuda, N. Konno, Complex Networks, From Fundamentals to Appli- cations, Kindai Kagaku-sha (in Japanese), 2010. g p [7] N. Masuda, N. Konno, Complex Networks, From F cations, Kindai Kagaku-sha (in Japanese), 2010. [8] E. Bonabeau, M. Dorigo, G. Theraulaz, Swarm Intelligence, From Natural to Artiﬁcial Systems, Oxford University Press, 1999. [8] E. Bonabeau, M. Dorigo, G. Theraulaz, Swarm Inte to Artiﬁcial Systems, Oxford University Press, 1999. [9] E.W. Dijkstra, A Note on Two Problems in Connexion with Graphs, Numerishe Mathematik 1, pp.269-271, 1959. pp [10] Boeing 787 Dream Liner, http://www.newairplane.com/. [11] J.V. Beaverstock, R.G. Smith, P.J. Taylor, A Roster of World Cities, pp.445-458, vol.15, issue 6, Cities, Dec.1999. [12] H. Sawai, Emergence and Analysis of A New Small-World Network, Japanese Society for Artiﬁcial Intelligence (JSAI) Joint-Technical Meet- ing and Symposium, Tokyo, Dec.2011. (in Japanese) Fig. 16. Eigenvalue versus ⟨k⟩in global airline networks. ACKNOWLEDGEMENT The author would like to express his gratitude to Drs. H. Ohsaki, K. Leibnitz, H. Suzuki and F. Peper for fruitful discussions and their useful comments on this study, and to Dr.J-C. Terrillon for his proof-reading this paper with some useful comments. [1] http://www.unitedairlines.co.jp/core/download/UAL NA 10 09.pdf [3] S. Milgram, The Small-World Problem, pp.61-67, Psychology Today, 1967. [4] J.D. Watts, Small Worlds, The Dynamics of Networks between Order and Randomness, Princeton University Press, 1999. REFERENCES [1] http://www.unitedairlines.co.jp/core/download/UAL NA 10 09.pdf http://www.unitedairlines.co.jp/core/download/UAL World [3] S. Milgram, The Small-World Problem, pp.61-67, Psychology Today, 1967. [4] J.D. Watts, Small Worlds, The Dynamics of Networks between Order and Randomness, Princeton University Press, 1999. [4] J.D. Watts, Small Worlds, The Dynamics of Networks between Order and Randomness, Princeton University Press, 1999.
W2035075734.txt	https://www.biodiversitylibrary.org/partpdf/161029	fr		Mousses du Sahara	Bulletin de la Société botanique de France	1,911	public-domain	1,824	500 SEANCE M. F. DU Camus 13 OCTOBRE la communication lit Mousses du PAR Au de cours de France, en certain nombre resultat fut reservee un Mousses par M. M. dont Flahault Cependant pour un examen ulterieur velles dont Jules confia 1'examen. dans le Rapport sur Tune et de une a cette espece. Ce est assez grand etant ce sujet. Cardot Grimmia me realite bryologique La a pour bien et presente Rapport voulu me l'etude de ces Card, et Copp. Mairei sont plantes fut nominee avec obtusifolia, n'ap- deux especes donnees l'insuffisance Note au her- ces var. sera la de par en M. nou- pauvrete nos done, publie Le les autre, flavicans desertique Societe botanique ■ avait recueilli un il en l'interet la Maire Desmatodon complement competence R. doute a un de de flore naissances M. extraordinaire Algerie. j COPPEY. Societe. pas sorte, en A. la partient la 1906, suivante Sahara; de point de Session publie borisations M. la de 1911. con- quelque Flahault. plantes. sp. now Dioica (plantis masculis et femineis intermixtis), dense cespitulosa, xr -~~„^ I11UQV.UUO ci icmiiicia unci iiiiA-tioy, uv«^v L i<ipa r.fliiloc tT»£»r»I ^o«^^ j:~:~: v jm _n: n^i:* «^^f^i»tic<iiiTl/l. ^isea. Gaules erecti, parce divisi, 5-15 mm. alti. Folia confertissima, ITltl" magnsea d ida patenti-erecta, sicca appressa, oblonga, concava, subcarinata, media 0,8■« 1 m ™m. longa, 0,4-0,5 lata, inferiora obtusa, superiora pilifera, marginibus uious pi planis vel subreflexis, integris, costa 30 p basi lata, in foliis HOTrioribus et mediis plerumque sub apice dilatata ibique evanida, in tolas superioribus in pilum hyalinum laevem vel parce et minute denticulatum excurrente, cellulis inferioribus juxta costam breviter rectangulis, ceteris rotundato-quadratis, obscuris, Iambus, parietibus inaequaliter incrassatis, ubique unistratosis. Folia perichrctialia caulinis duplo fere longiora, Pj ' usque 2,5 mm. longo et capsulam superante preedita. Capsula in pediceii arcuatulo, 2-3 mm. longo, fusco-rubra, sicca plicatula, 1,5 mm. long'; 1 mm. lata, operculo apiculato, annulo triplici, persistente, penstom dentibus rubris, papilloso-spinulosis, usque ad basim in 3-5 crura m qualia et plus minus cohaerentia divisis. Spone !a;ves, lutescentes, dia 10-15 ix. Calyptra ignota. Cette dediee, espece sur l'Extreme-Sud les a ete recueillie rocliers de Oranais. Elle se par M. Rene Maire, a gres du ravin de Ben trouve la en qui compagnie elle est Zireg, dans du Tot- A. COPPEY. (Sm.) Lindb. tula atrovirens ex parte, Bruch) la precedente A details Jur., MOUSSES ■ du Crossidi et especes de diagnose, deja touffes d'ailleurs pour les rogames, dans le sont 50i nm chloronotos (Brid. region tres completement autres sable tres mediterraneenne. detaillee, ■ j'ajouterai les grise de la surface, due aux peut attirer un regard, toutefois qu'en est touftes certain doivent depourvues de plus ces longs la capsule femelles et toute la Sur cette derniere les tres sur court; les riformes, les paraphyses antheridies qui ont, Les tiges devel heaucoup de Phane- pour la desagregation facilement. une une teintc des feuilles supe- peu pluvieuse, les doute,a l'etat blancs saison attentif. un sans blancs qui n'existent manquent a la que base jeune, sur les des tiges un poil male. superieures ontrarement perigoniales sontovales, courtes, sont nombreuses ainsi vides, des Seule tres mais et de poils feuilles presentent et mime plante feuilles arrive longs poils entourant cela si visibles, feuilles comme resulte transporte etre enfouies, qui que 11 vent fin et rieures, le Mousses gr&8 peu une longueur un de faisceau 0,35 axial cochleaque les mm. (Gentralstrang) bien oppe. Les cellules foliaires rectangulaires sur nervure, 20 de parois sont hyalines a un espace restreint situe 30 t a tres inferieur. minces, Ailleurs, lisses, a moins u sur sur les parois 10; une cellules la les a de deux a sont rangees carrees, epaissies parois chaque laterales sont irregulierement base, cote de presque dans la carrees, le arrondics, et minces, quart opaques, contour plus ou embrassantes el sinueux. Les feuilles \|>ourvues de •lans la monte «le SAHARA. suivants. Les a la DU perichetiales sont legerement cellules rectangulaires, hyalines, a parois ou tiers Le long poil qui est blanc, moitie le inferieur. s'en detache brusquement; quelques petites dents ou papilles. peu saillante et seulement deux rangees de grandes La nervure, formee de a lisse, la face cellules les ou sur pourvu inferieure, internes, est separees P 1icr. Les il minces archegones des fleurs femelles sont peu nombreux, les 5 °2 SEANCE paraphyses ment rares arque. DU ou nulles. Peut-etre La capsule est nombre de a Les 16 atteignent 13 8 0,2 cylindriques Le 10, en pedicelle capsule est legere- ordinairement courbe a l'etat jeune. de superficiels une rangee peristome, de longueur aigues ou « Les affinites de bien des tatonnemenls, de stomaies du mm. 19H. la est-il pourvue dents OCTOBHE transversale dressees, et sont cette je court, un peu nervure amincie d'ailleurs considerablement par superieures beaucoup quement contractees anneau tres Malgre large, les plis assez superficiels, Grimmia sms Tortula Autoica, au etc. incurve, a on stnVt Apres la Hpe, ses a dont et rappelle par peristomiales tres tissu; elle son ses non puisse qu'elle dents base on mais feuilles ou inferieures plus larges, brus- peu carenees, son mais » longitudinaux humillima dense arenaria sommet, l etablir. espece pedicelle les papilles qu'une son obtuses, de vois ne Grimmia mutiques, couvertes difficiles le diflere base. ajourees sont c'est en sa curieusement plante rapprocher, sa vers au ramifiees. la perforees, disposes, de peut classer l.imnr Card, cespitosa, et humillima, la capsule, visiblesasec, cette plante dans Copp., sp. nov. 1-2 alta. Folia mm. la section madida erecto- patentia, sicca appressa, ovalia, concava, apice abrupte et breviter apiculata vel mucronata, rarius rotundata, media 0,75 mm. longa, 0,4 lata, margimbus integris valde revolutis, costa superne dilatata et granulosa, imque 60 >x crassa, plerumque paulo infra apicem desinente, cellulis in dimidio mferiore hyalinis, la?vibus, rectangulis, in parte superiore obscuns, utraque pagina dense papillosis, quadratis vel irregulariter hexagonis, 10-18 10-15 u ii latis latis.Fnli Folia - a peric ^ cha}tialia cau i inis a liquanto majora. Capsula in pedicello rubente, 5 mm.n. ., longo, erecta, ovalis, 1 mm. longa, operculo breviter et Spor;e irregulares cucullata. oblique rostrato, " a * A„„„„,„ , annulo peristomioque deficientibus. et grosse verrucoso-pap coso-papillosa?, diam. 20-22 ,u. CalypU' Cette espece a ete egalement dans le Sahara exposes au Nord, au croit en compagnie Br. E., Crossidium squamigerum {Entosthodon 1. Cardot(J.), (Viv.) decouverte Jur. litt. de Bryum et M. sur l'oued Maire des rochers Beni-Ounif, de arqenteum L. Grimmia orbicularis, (Brid., Bruch) Jur., Crossidium Lindb.). attenuata palmeraie. £res la Funaria et a de chloronotos Templetoni in algerien, regard de par (Dicks.) Elle Lindb. A. De meme COPPEY. que de fructifications attirent un sont Les feuilles cellules a en parois assez a peu qui physes En coupe, est au lisse pr6s le dans qui de de cette touttes ou fin, les jusque sous jjl , et y longueur. le compris Les para- sommet. Les le tiers inferieur, 2i k \k sur et tres papilleuses du base, depasse ;x 3G n'a 45 [x a la sa face que non 15 papilleuses, ;jl et passent GO superieure. est tres papilleuse a couches de 2 contraire sur le stereides. 4 rangees degrandes dos, oil elle formee Le pedicelle est legerement la gauche L'opercule a de une superieure a de m'assurer 140 opaques et de 50J regard. cellules epaissies forme les sable le atteignent tiers parois vers dans seules revolutees aux elle deux peu rectangulaires, moitie presente le hyalines nervure, la ensevelis nombreuses, sont rapidement dans sont occupe sont La decrite, SAHARA. rudimentaires. minces, sommet. precedernment celle-ci antheridies, pedoncule DU Tespece gazonnements Les MOUSSES tordu est (ligne de ou elle cellules; elle de cellules torsion a montant l'observateur). longueur totale rangees de cellules nettement de l'absence de peu 0,2 mm., et son bee est ou non obliques. J'ai pu totale d'anneau et de la base. peris- tome. La capsule Cette minuscule nettement de un doute. Limpr., tissu des rangee espece, malgre la doit prendre pr6s feuilles par une Tortula; Elle strict. plement possede sa du est de structure tr£s taille, sa des nettement de peristome la des rochers trachytiques du plus allongees, longuement non dilatee dans rapproche le gees, etc. ' > i. Cardot [iXin memo litt. haut, du est laisser la section Tortula sens, (Sm.) Elle ses Tortula jeunes « dont com- feuilles mucro[/absence (Phil.) inais celui-ci la nervure inferieures le distingue piliferes. Buyssoni piliferes, cellules Lindb., s'en brievement Mont-Dore les peristome, saurait comparable. tivs de ne dans capsule, a feuilles atrovirens nees feuilles ou l'absence place, Tortula stomates plus Broth., a les mince, allon- 504 SEANCE DU 13 OCTOB11E Explication des Planche 1911. planches. XV. a. Grimmia Mairei; gr. nat. b. — ; gr. 10. c. — , coupe de la tige a Torigine d'une feuille; gr. 250. d-e. — , coupe de feuille; gr. 250. f. — y feuille moyenne; gr. 30 . g. — , feuille moyenne; tissu de la partie superieure du h. — i. j. k. /. — — — — , tissu de limbe; gr. la partie 250. moyenne du limbe; tissu de Tangle inferieur; gr. feuille superieure ; gr. 30. , feuille perichetiale; gr. 30. , tissu de la precedente dans 250. , , gr. 250. la partie superieure; gr. 250. m. — , Stomate; gr. 280. n-o-p. Tortula humillima, feuilles; gr. 40. q. — , sommet des feuilles mucron6es; r. — , tissu de la moitie superieure du gr. limbe; 300. gr. s. — region £. u. V- — — — w x VV'%. z gr. 250. opercule ; gr. 60. , spores; gr. 250. capsule et coiffe; gr. 13. — , antherid6e; gr. 120. Plagiothecium curvifolium, feuilles; gr. 30. — , tissu de la region moyenne , coupe feuille dans la gr. 70. coupe de la nervure vers la coupe de la nervure vers le coupe de la nervure dans , 9 , — d'une — — , 300. moyenne; base; gr. 250^ milieu; gr. 250. le tiers supSrieur; , des feuilles; gr. 200. Planche XVI. a. Grimmia Mairei; gr. 7. b - c d. — Tortula e f- — g-h. Plagiothecium l — - , plante , dents humillima, — , — 1. (Test par grossissement. gr. 7. du peristome; feuille moyenne; ; gr. capsule , <?; 7. avec gr. gr. 170. 30. opercule se detachant visible; gr. 30. curvifolium, capsule a sec, opercule; capsule erreur que a sec, la planche desoperculSe; porle 40/1 et gr. gr. comme spora 7. '• indication du Coppey, M A . 1911. "Mousses du Sahara." Bulletin de la Société botanique de France 58, 500–504. https://doi.org/10.1080/00378941.1911.10832341. View This Item Online: https://www.biodiversitylibrary.org/item/8680 DOI: https://doi.org/10.1080/00378941.1911.10832341 Permalink: https://www.biodiversitylibrary.org/partpdf/161029 Holding Institution Missouri Botanical Garden, Peter H. Raven Library Sponsored by Missouri Botanical Garden Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's largest open access digital library for biodiversity literature and archives. Visit BHL at https://www.biodiversitylibrary.org. This file was generated 18 September 2023 at 07:05 UTC
https://openalex.org/W4393174028	https://ojs.transpublika.com/index.php/PRIMA/article/download/724/630	Indonesian	null	PERENCANAAN BISNIS PADA UMKM VEZARA COLLECTION	Portal Riset dan Inovasi Pengabdian Masyarakat	2,023	cc-by	2,490	Abstrak Tujuan dari pengabdian ini adalah memberikan pelatihan pembuatan rencana bisnis bagi wirausahaan pemula. Business plan adalah suatu dokumen cetak yang dibuat oleh pengusaha dengan menggambarkan atau memperhitungkan unsur eksternal dan internal dalam memulai usaha baru.Secara umum, hal yang dituangkan dalam dokumen business plan adalah tujuan utama dan strategi sebuah perusahaan. Keterbatasan SDM yang memahami bidang tersebut menjadikan penyusunan rencana bisnis yang dibuat UMKM hanya berdasarkan kebutuhan saja, bukan menjadi sebuah rencana yang matang. Sehingga muncul permasalahan yang dihadapi pebisnis atau umkm di kemudian hari pada saat bisnis tersebut dijalankan. Oleh karena itu Tim PKM perlu memberikan pelatihan cara membuat perencanaan bisnis (bussines plan) agar membantu Ibu Desy sebagai wirausahaan muda yang memiliki usaha di PGC dalam menata masa depan usahanya agar sukses dan menjadikan rencana bisnis sebagai kebutuhan bagi wirausaha untuk menjadikan bisnisnya sukses. Kata kunci: Perencanaan Bisnis, Wirausaha, SDM Kata kunci: Perencanaan Bisnis, Wirausaha, SDM PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT (PRIMA) Volume 2 Issue 2 (2023) PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT (PRIMA) Volume 2 Issue 2 (2023) Keywords: Business Plan, Entrepreneur, HR Keywords: Business Plan, Entrepreneur, HR PERENCANAAN BISNIS PADA UMKM VEZARA COLLECTION Yusbardini1*, Marcello2, Said Ashadi Cahyadi3 1-3 Fakultas Ekonomi dan Bisnis, Universitas Tarumanagara E-mail: 1) yusbardini@fe.untar.ac.id Abstract The purpose of this service is to provide training in creating business plans for novice entrepreneurs. A business plan is a written document that outlines the external and internal elements involved in starting a new business. Typically, a business plan includes the company's main objectives and strategies. However, many MSMEs (Micro, Small, and Medium Enterprises) face challenges due to limited human resources with expertise in this area. As a result, their business plans are often created based on immediate needs rather than being thoroughly developed. Consequently, business owners, especially young entrepreneurs like Mrs. Desy, may encounter difficulties in the future as they run their businesses. To address this issue, the PKM Team aims to provide training on how to create a comprehensive business plan, offering valuable support to Mrs. Desy and other entrepreneurs in PGC. A well-crafted business plan is essential for achieving success in business ventures. 1. PENDAHULUAN Membuka sebuah bisnis adalah hal yang membutuhkan kesungguhan dan kematangan langkah. Salah satu bentuk kesungguhan yang perlu ditunjukkan pebisnis adalah dengan membuat sebuah business plan. Namun, sayangnya tidak semua orang memahami cara membuat business plan yang baik dan benar. Business plan atau rencana bisnis adalah suatu dokumen tertulis yang perlu dipersiapkan oleh setiap pengusaha sebelum menjalankan bisnisnya. Umumnya, isi dokumen tersebut berisi tujuan, strategi, serta berbagai unsur eksternal dan internal yang perlu diperhitungkan dalam menjalankan PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 217 PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 PERENCANAN BISINIS PADA UMKM VEZARA COLLECTION Yusbardini, Marcello, Said Ashadi Cahyadi bisnis pada masa mendatang. Menurut Hisrich and Peters (1995), “business plan adalah suatu dokumen cetak yang dibuat oleh pengusaha dengan menggambarkan atau memperhitungkan unsur eksternal dan internal dalam memulai usaha baru.Secara umum, hal yang dituangkan dalam dokumen business plan adalah tujuan utama dan strategi sebuah perusahaan. Tujuan utama harus mampu ditunjukkan secara realistis dengan menjabarkan berbagai strategi di bawahnya. Misalnya adalah strategi SDM, pemasaran, keuangan, produksi, pengelolaan infrastruktur dan sebagainya.” Bagi sebagian besar calon wirausaha membuat dan menyusun rencana bisnis sangat sulit, lain halnya bagi wirausaha yang sudah lama bergaul dengan dunia usaha menyusun rencana bisnis merupakan hal yang sangat mudah. Rencana bisnis yang baik menurut (Suryana : 2009) harus memuat aspek manajemen, pemasaran, keuangan, SDM, dan aspek teknis. Menurut penelitian Mahendra (2009: 345) menyatakan bahwa “seorang wirausaha yang baik adalah jika membuat rencana bisnis dengan matang akan meningkatkan keberhasilan usahanya. Karena rencana bisnis yang dibuat secara matang akan memberi manfaat bagi wirausaha itu sendiri di masa datang.” Sedangkan, menurut (Suryana: 2009)” Perencanaan usaha sebagai persiapan awal memiliki dua fungsi penting yaitu: (1) sebagai pedoman untuk mencapai keberhasilan manajemen usaha, dan (2) sebagai alat untuk mengajukan kebutuhan permodalan yang bersumber dari luar.” Dengan demikian, rencana bisnis yang dibuat akan memberikan kemudahan dalam mengembangkan usaha di masa datang. Banyaknya aspek yang akan dibuat dalam membuat rencana bisnis, membuat sebagian wirausaha UMKM menganggap bahwa menyusun rencana bisnis bukan menjadi keharusan karenanya mereka lebih memilih menjalankan usaha berdasarkan pengalaman mereka di dunia usaha, sehingga pembuatan dan penyusunan rencana usaha sering diabaikan. 1. PENDAHULUAN Keterbatasan SDM yang memahami bidang tersebut menjadikan penyusunan rencana bisnis yang dibuat hanya berdasarkan kebutuhan saja, bukan menjadi sebuah rencana yang matang. Sehingga muncul permasalahan yang dihadapi pebisnis atau umkm di kemudian hari pada saat bisnis tersebut dijalankan. Begitu pula yang selama ini bisnis yang dijalankan oleh ibu Desy di PGC. Bisnis pakaian muslim yang ada di pertokoan Pusat Glosir Cililitan lantai 2 Zona Kuning merupakan salah satu UMKM yang belum memahami betul langkah-langkah dalam membuat rencana bisnis, cara untuk meningkatkan keberhasilan usaha, dan bagaimana peran rencana bisnis sebagai alat untuk meningkatkan keberhasilan usaha. Oleh karena itu Tim PKM perlu melakukan pelatihan cara membuat perencanaan bisnis (bussines plan) agar membantu Ibu Desy sebagai wirausahaan dalam menata masa depan usahanya agar sukses dan menjadikan rencana bisnis sebagai kebutuhan bagi wirausaha untuk menjadikan bisnisnya sukses. Komponen Business Plan Terpenting yang perlu dibuat agar mempermudah bisnis dijalankan. Berikut ini komponen penting dari Business Plan yang perlu diperhatikan adalah : 1. Ringkasan Eksekutif .Ringkasan eksekutif ini seperti sebuah poster film yang menggambarkan seperti apa suasananya. Dalam konteks ini, ringkasan eksekutif perlu menjelaskan nama perusahaan serta gambaran makro bisnis yang hendak dijalankan. Misalnya seperti akan bergerak di bidang apa dan apa visi misi yang ingin Anda raih. 2. Pendahuluan .Sebagai pendahuluan, perlu menjelaskan terkait latar belakang perusahaan. Misalnya seperti orang-orang yang bekerja, struktur perusahaan, PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 218 https://ojs.transpublika.com/index.php/PRI E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT (PRIMA) PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT (PRIMA) pemilik saham, dan sebagainya. Dapat juga menjelaskan visi, misi, tujuan jangka pendek dan jangka panjang dalam kurun waktu tertentu. Gambar 1. Mitra UKM VEZARA COLLECTION di PGC Cililitan Jak-Tim Gambar 1. Mitra UKM VEZARA COLLECTION di PGC Cililitan Jak-Tim 3. Analisis Produksi/Jasa (Operational).Model bisnis secara umum terbagi menjadi dua, yaitu produk dan jasa. Jika hendak menjual produk, maka perlu menganalisis apa inti produknya dan bagaimana kemasannya. Dapat juga menentukan cara memproduksinya, apakah harus menciptakan barang baru atau memberikan nilai tambah pada sebuah barang tanpa mengubah bentuknya.Jika menyediakan jasa kepada pelanggan, maka perlu membuat sistem pemberian jasa, mulai dari persiapan hingga jasa selesai diberikan pada pelanggan.Jika menyediakan jasa kepada pelanggan, maka perlu membuat sistem pemberian jasa, mulai dari persiapan hingga jasa selesai diberikan pada pelanggan.Prinsipnya adalah menciptakan sistem produksi dan penyediaan jasa yang menarik, punya daya saing, dan relevan dengan pelanggan agar pelanggan bisa menikmatinya. 4. Analisis Pasar danPemasaran. Perlu menjelaskan analisis pasar yang telah ditargetkan. Di samping itu dapat juga menjelaskan bagaimana persaingan yang mungkin terjadi dengan kompetitor. Terkait bagian pemasaran, menjelaskan tentang bagaimana tujuan dan strategi pemasaran yang relevan dengan perusahaan. 5. Analisis SDM Setiap perusahaan pasti membutuhkan tenaga kerja agar mampu beroperasi. Ada berbagai divisi yang dibutuhkan dalam perusahaan dan setiap divisi membutuhkan orang-orang yang berbeda. Orang-orang dengan beragam PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 219 PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 219 https://ojs.transpublika.com/index.php/PR E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 PERENCANAN BISINIS PADA UMKM VEZARA COLLECTION Yusbardini, Marcello, Said Ashadi Cahyadi kemampuan juga perlu dijelaskan agar bisa mengetahui bagaimana sistem operasi perusahaan. p p 6. Analisis Keuangan.Menjalankan bisnis membutuhkan modal, baik itu berupa uang, infrastruktur, dan lainnya. Perlu melakukan analisis keuangan untuk bisa mengukur kebutuhan modal, prediksi pemasukan, pengembalian modal, pengembalian investasi, dan juga kalkulasi daya ungkit dalam menjalankan bisnis. p p 6. Analisis Keuangan.Menjalankan bisnis membutuhkan modal, baik itu berupa uang, infrastruktur, dan lainnya. Perlu melakukan analisis keuangan untuk bisa mengukur kebutuhan modal, prediksi pemasukan, pengembalian modal, pengembalian investasi, dan juga kalkulasi daya ungkit dalam menjalankan bisnis. 6. Analisis Keuangan.Menjalankan bisnis membutuhkan modal, baik itu berupa uang, infrastruktur, dan lainnya. PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT (PRIMA) Perlu melakukan analisis keuangan untuk bisa mengukur kebutuhan modal, prediksi pemasukan, pengembalian modal, pengembalian investasi, dan juga kalkulasi daya ungkit dalam menjalankan bisnis. 7. Peluang Bisnis (SWOT) dan Strategi Setiap peluang pasti menghadirkan masalah yang wajib dihadapi. Pada poin ini, perlu menjelaskan masalah apa saja yang mungkin ditemui dan bagaimana cara menyelesaikannya. Dan Stategi yang perlu dilakukan untuk memenangkan persaingan. 7. Peluang Bisnis (SWOT) dan Strategi Setiap peluang pasti menghadirkan masalah yang wajib dihadapi. Pada poin ini, perlu menjelaskan masalah apa saja yang mungkin ditemui dan bagaimana cara menyelesaikannya. Dan Stategi yang perlu dilakukan untuk memenangkan persaingan. 8. Rencana Eksekusi. Pada poin ini, perlu menjelaskan bagaimana mengeksekusi atau menjalankan rencana yang sudah dibuat. Mulai dari awal membuat, produksi, pemasaran, hingga mendapatkan keuntungan. 8. Rencana Eksekusi. Pada poin ini, perlu menjelaskan bagaimana mengeksekusi atau menjalankan rencana yang sudah dibuat. Mulai dari awal membuat, produksi, pemasaran, hingga mendapatkan keuntungan. 9. Lampiran .Pada poin ini, dapat dijelaskan legalitas perusahaan, sertifikat, dan dokumen pendukung lain yang semakin bisa meyakinkan kualitas business plan. 9. Lampiran .Pada poin ini, dapat dijelaskan legalitas perusahaan, sertifikat, dan dokumen pendukung lain yang semakin bisa meyakinkan kualitas business plan. 9. Lampiran .Pada poin ini, dapat dijelaskan legalitas perusahaan, sertifikat, dan dokumen pendukung lain yang semakin bisa meyakinkan kualitas business plan. Gambar 2. Komponen Penting Dalam Bussines Plan ( Perencanaan Bisnis) Gambar 2. Komponen Penting Dalam Bussines Plan ( Perencanaan Bisnis) METODE PENELITIAN Pelaksanaan pengabdian ini dilakukan dengan tahapan sebagai berikut : 1. Tahap awal pengabdian ini yaitu tahapan persiapan. Pada tahap ini tim pelaksana kegiatan melakukan konsultasi dengan mitrayang lain. Kemudian melakukan survey dan observasi terhadap kegiatan yang selama ini dilakukan mitra yang bersangkutan. 220 PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 220 PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT (PRIMA) 2. Tahap kedua yaitu tahap pelaksanaan kegiatan pengabdian. Pada tahapan ini tim PKM melaksanakan kegiatan pkm secara daring, tim Tim PKM langsung memberikan materi bagaimana cara membuat bussines plan yang baik dan benar. Setiap peserta pengabdian mendengarkan paparan dari narasumber selama proses pelatihan berlangsung. 3. Tahap ketiga yaitu tahap tanya jawab Pada tahap ini peserta pelatihan diperbolehkan menanyakan berbagai macam masalah sekitar pembuatan bussines plan. Factor factor apa saja yang perlu dipersiapkan agar bisnisnya lancer dan berhasil. 3. HASIL DAN PEMBAHASAN Kegiatan pengabdian kepada masyarakat ini dilakukan pada tanggal 2 Juli 2023 untuk mencari solusi akan permasalahan yang timbul dari UMKM Vezara Collection tentang kendala kendala dalam membuat perencanaan bisnis sehingga akan meningkatkan keberhasilan usahanya. Karena rencana bisnis yang dibuat secara matang akan memberi manfaat bagi wirausaha itu sendiri di masa datang. PemilikVezara Collection juga belum paham tentang bagaimana caranya perencanaan bisnis yang baik karena adanya keterbatasan SDM yang memahami bidang tersebut menjadikan penyusunan rencana bisnis yang dibuat hanya berdasarkan kebutuhan saja, bukan menjadi sebuah rencana yang matang. Sehingga muncul permasalahan yang dihadapi pebisnis atau umkm di kemudian hari pada saat bisnis tersebut dijalankan.Untuk itu Tim PKM memberikan penjelasan kepada ibu Desy sebagai pemilik Vezara Collection untuk memahami pentingnya pembuatan rencana bisnis yang berguna untuk mengembangan bisnis di masa yang akan datang. Pada kegaiatan PKM ini TIM PKM mencoba membantu ibu Desy sebagai pemilik UMKM vezara dengan memberikan materi tentang bagaimana membuat perencanaan bisnis Usaha pakaian muslim yang berlokasi di pertokoan PGC jakarta timur. Berikut pemberian materi mengenai Bussiness Plan melalui Zoom metting yg dilakukan tim PKM : PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 221 PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 PERENCANAN BISINIS PADA UMKM VEZARA COLLECTION Yusbardini, Marcello, Said Ashadi Cahyadi Gambar 3. Materi Bussiness Plan Gambar 3. Materi Bussiness Plan Gambar 3. Materi Bussiness Plan Pada saat pemberian materi ibu Desy cukup antusias mendengarkan dan langsung melakukan tanya jawab. Ibu Desy merasa bahwa sebagian perencanaan bisnis sudah di buat walaupun itu masih terdapat kekurangan seperti analisis kondisi pasar kaitanya dengan seberapa besar permintaan busana muslim di Indonesia kuhusnya di daerah jakarta timur yang mayoritas penduduknya beragama islam. Stategi pemasaran pun telah di terapkan seperti mengunakan media sosial, internet, histagram dll. Analisis teknik dan produksi pun mulai akan di terapkan sepert model maju muslim yang mengikuti tran desain produk yang sedang digandrungi remaja sekarang. Sedangan untuk analisis Financial dan resiko masih butuh membenahan mengenai pencatatan dan pelaporan semua transaksinya. Keinginan yang cukup besar dalam membuat perencanaan bisnis yang baik mendorong ibu Desy untuk dilakukannya pendampingan oleh tim PKM. Hal inilah yang masih di rundingkan Tim PKM. Setelah memberikan materi tim PKM menyarankan kepada ibu Desy untuk mencoba membuat bisnis plan dengan tahapan sebagai berikut yang merupakan dasar pembuatan bussines plan atau perencanaan bisnis. 222 PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 4. KESIMPULAN Perencanaan bisnis sangat dibutuhkan bagi pemula bisnis karena dapat dijadikan pedoman dalam menjalankan bisnis dimasa yang akan datang. Selain itu dapat digunakan untuk mendapatkan tambahan modal pada lembaga keuangan. Dengan membuat bussiness plan strategi strategi berbisnis dibuat dengan baik seperti stategi pemasaran, strategi produksi, strategi keuangan, strategi SDM sehingga memudahkan dalam pengawasan dan pengendalian bisnis. Namun, Diperlukan pendampingan berkelanjutan oleh tim PKM dalam merancang Bussines Plan secara detail agar membantu para pemilik UKM dalam membuat bussiness plan yang baik dan benar. DAFTAR PUSTAKA Aris Rahmana, Helman(2011), Strategi Menyusun Rencana Bisnis Yang Berkualitas, http://pengusahamuslim.com/strategi-menyusun-rencana- 1372/#.U7oY30DHLMw, diakses 7 juli 2014 Hisrich, Peters. 2002 Entrepreunership, fifth edition. New York, USA : Mc Graw Hill inc4. Hisrich, Peters. 2002 Entrepreunership, fifth edition. New York, USA : Mc Graw Hill inc4. Irmayanti (2021) Strategi Penerapan Business Plan Dalam Meningkatkan Penjualan Menurut Perspektif Ekonomi Islam- Jurnal Akunsyah: Jurnal Akuntansi & Keuangan Syariah, 1(2), 2021, 65 - 80 Irmayanti (2021) Strategi Penerapan Business Plan Dalam Meningkatkan Penjualan Menurut Perspektif Ekonomi Islam- Jurnal Akunsyah: Jurnal Akuntansi & Keuangan Syariah, 1(2), 2021, 65 - 80 g y Sull,Donald N., 2004, Formulate (and Reformulate) Your Business Plan, http://www.myownbusiness.org/s2/#4, access Sull,Donald N., 2004, Formulate (and Reformulate) Your Business Plan, http://www.myownbusiness.org/s2/#4, access PORTAL RISET DAN INOVASI PENGABDIAN MASYARAKAT \| PRIMA https://ojs.transpublika.com/index.php/PRIMA/ E-ISSN : 2809-7939 \| P-ISSN : 2809-8218 223 DAFTAR PUSTAKA Aris Rahmana, Helman(2011), Strategi Menyusun Rencana Bisnis Yang Berkualitas, http://pengusahamuslim.com/strategi-menyusun-rencana- 1372/#.U7oY30DHLMw, diakses 7 juli 2014 Hisrich, Peters. 2002 Entrepreunership, fifth edition. New York, USA : Mc Graw Hill inc4. Irmayanti (2021) Strategi Penerapan Business Plan Dalam Meningkatkan Penjualan Menurut Perspektif Ekonomi Islam- Jurnal Akunsyah: Jurnal Akuntansi & Keuangan Syariah, 1(2), 2021, 65 - 80 Sull,Donald N., 2004, Formulate (and Reformulate) Your Business Plan, http://www.myownbusiness.org/s2/#4, access
https://openalex.org/W986185544	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0134058&type=printable	English	null	Impact of Prior Abdominal Surgery on Rates of Conversion to Open Surgery and Short-Term Outcomes after Laparoscopic Surgery for Colorectal Cancer	PloS one	2,015	cc-by	7,559	RESEARCH ARTICLE Impact of Prior Abdominal Surgery on Rates of Conversion to Open Surgery and Short- Term Outcomes after Laparoscopic Surgery for Colorectal Cancer Ik Yong Kim1, Bo Ra Kim2, Young Wan Kim1* 1 Department of Surgery, Division of Gastrointestinal Surgery, Yonsei University Wonju College of Medicine, Wonju, Korea, 2 Department of Internal Medicine, Division of Gastroenterology, Yonsei University Wonju College of Medicine, Wonju, Korea * youngwkim@yonsei.ac.kr * youngwkim@yonsei.ac.kr * youngwkim@yonsei.ac.kr Methods We compared three groups as follows: colon cancer patients with no PAS (n = 272), major PAS (n = 24), and minor PAS (n = 33), and rectal cancer patients with no PAS (n = 282), major PAS (n=16), and minor PAS (n = 26). Editor: Andreas Krieg, Heinrich-Heine-University and University Hospital Duesseldorf, GERMANY Editor: Andreas Krieg, Heinrich-Heine-University and University Hospital Duesseldorf, GERMANY Received: April 17, 2015 Accepted: July 3, 2015 Published: July 24, 2015 University Hospital Duesseldorf, GERMANY Received: April 17, 2015 Accepted: July 3, 2015 Published: July 24, 2015 OPEN ACCESS Citation: Kim IY, Kim BR, Kim YW (2015) Impact of Prior Abdominal Surgery on Rates of Conversion to Open Surgery and Short-Term Outcomes after Laparoscopic Surgery for Colorectal Cancer. PLoS ONE 10(7): e0134058. doi:10.1371/journal. pone.0134058 Purpose To evaluate the impact of prior abdominal surgery (PAS) on rates of conversion to open sur- gery and short-term outcomes after laparoscopic surgery for colon and rectal cancers. Conclusions Our results suggest that colorectal cancer patients with minor PAS or patients with prior rad- ical hysterectomy can be effectively managed with a laparoscopic approach. In addition, laparoscopy can be selected as the primary surgical approach even in patients with major PAS (prior gastrectomy or colectomy) given the assumption of a higher conversion rate. Results In patients with colon and rectal cancers, the rate of conversion to open surgery was signifi- cantly higher in the major PAS group (25% and 25%) compared with the no PAS group (8.1% and 8.9%), while the conversion rate was similar between the no PAS and minor PAS groups (15.2% and 15.4%). The 30-day complication rate did not differ among the three groups (28.7% and 29.1% in the no PAS group, 29.2% and 25% in the major PAS group, and 27.3% and 26.9% in the minor PAS group). The mean operative time did not differ among the three groups (188 min and 227 min in the no PAS group, 191 min and 210 min in the major PAS group, and 192 min and 248 min in the minor PAS group). The rate of conver- sion to open surgery was significantly higher in patients with prior gastrectomy or colectomy compared with the no PAS group, while the conversion rate was similar between the no PAS group and patients with prior radical hysterectomy in patients with colon and rectal cancers. Copyright: © 2015 Kim 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: Ethical restrictions make data unsuitable for public deposition. Requests for data may be sent to the corresponding author. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. 1 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 Prior Abdominal Surgery Introduction Despite accumulating evidence for the short-term clinical advantages of laparoscopic colorectal surgery and its oncologic equivalence to open procedures [1–4], this minimally invasive surgi- cal approach has not been widely adopted in patients with a history of prior abdominal surgery (PAS). The main reasons are technical difficulties in adhesiolysis and prolonged operative time [5]. A previous abdominal operation inevitably results in intra-abdominal adhesions, and the ergonomic constraints of laparoscopic instruments make it difficult to achieve sufficient adhe- siolysis. In addition, laparoscopic adhesiolysis increases the risk of inadvertent bowel injury [6,7]. To date, a history of PAS is no longer considered a relative contraindication for laparoscopy; however, PAS is likely to increase the complexity of the procedure [8]. There are conflicting data available with regard to laparoscopic colorectal surgery in patients with PAS. Law et al. [9] reported that a history of PAS did not negatively influence short-term clinical outcomes with respect to postoperative complication rate, ileus, open conversion rate, and duration of hospital stay after laparoscopic colorectal surgery. In contrast, Yamamoto et al. [10] showed higher rates of intraoperative enterotomy and postoperative ileus, and delayed time to diet in patients with PAS. We hypothesized that laparoscopy for colorectal cancer is feasible and safe for selected patients with PAS, and specific types of PAS may guide preoperative patient selection for lapa- roscopic colorectal cancer resection. To date, there are studies comparing outcomes between patients with and without PAS after laparoscopic surgery for benign and malignant colorectal diseases [5,9–16]. The current study included only malignant conditions and compared the conversion rates and short-term outcomes of the three groups as follows: patients with no PAS, those with major PAS, and those with minor PAS undergoing laparoscopy for colon and rectal cancer. PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 Methods Patients This retrospective cohort study was conducted at a tertiary referral center. All clinical investiga- tions were conducted according to the principles expressed in the Declaration of Helsinki. Informed consent was obtained from all patients before surgery. All participants provided written informed consent and the ethics committee (the institutional review board of Wonju Severance Christian Hospital) approved this retrospective study (YWMR-155013). All patient records/information was de-identified prior to analysis. This observational study was con- ducted using the STROBE (STrengthening the Reporting of OBservational studies in Epidemi- ology) guidelines [17]. Eligibility criteria included having histologically confirmed colorectal cancer and undergoing major laparoscopic colorectal resection between March 1, 2008 and February 28, 2014. Patients undergoing palliative non-resectional or bypass surgery, emergent 2 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 Prior Abdominal Surgery operation, trans-anal local excision, or multi-visceral resection were not eligible for this study. Among 653 patients, 99 patients (15.2%) had a history of any type of abdominal surgery. Among these, 40 patients (6.1%) had major PAS. We compared conversion rates and short- term outcomes among the three groups as follows: colon cancer patients with no PAS (n = 272), major PAS (n = 24), and minor PAS (n = 33) undergoing laparoscopy for colon can- cer, and rectal cancer patients with no PAS (n = 282), major PAS (n = 16), and minor PAS (n = 26) undergoing laparoscopy for rectal cancer. Study endpoints The primary endpoint was to investigate the rate of conversion to open surgery after laparo- scopic surgery for colon and rectal cancer among patients without PAS, with minor PAS, and with major PAS. Secondary endpoints were to evaluate short-term outcomes (operative time, inadvertent enterotomy, postoperative complication rate, hospital stay, and time to diet) after laparoscopic surgery for colon and rectal cancer among patients without PAS, with minor PAS, and with major PAS. Classification of prior abdominal surgery Prior abdominal surgery (PAS) was defined as any previous abdominal surgery by either lapa- roscopy or laparotomy, and was classified as major and minor PAS. Major PAS was defined as abdominal surgery involving more than one abdominal quadrant accessed through midline incision from the xiphoid to the umbilicus or from the umbilicus to the symphysis pubis. Minor PAS was defined as abdominal surgery involving one abdominal quadrant through a right subcostal, paramedian, Rockey-Davis or Pfannenstiel incision. Low midline incisions for minor or benign gynecologic surgery, such as oophorectomy, salphingectomy, Cesarean sec- tion, or abdominal hysterectomy for myoma, were classified as minor PAS. Laparoscopic abdominal surgeries, such as cholecystectomy or appendectomy, were classified as minor PAS. PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 Patient characteristics Baseline characteristics of the patients in the 3 groups are presented in Table 1. The major PAS (66.7%) and minor PAS (69.7%) groups had a significantly higher proportion of female patients compared to the no PAS group (39.3%) among patients with colon cancer. Although not significant, the major PAS (43.8%) and minor PAS (42.3%) groups had a higher proportion of female patients compared to the no PAS group (27.7%) in patients with rectal cancer. Age, body mass index, American Society of Anesthesiologists (ASA) score, and the incidence of pT4 tumor did not differ among the three groups. Outcome measures All laparoscopy data were analyzed according to the intention-to-treat principle. Postoperative complications were defined as events that required additional treatment within 30 days of sur- gery. Wound infection was defined according to the definition of the Centers for Disease Con- trol (CDC) as superficial or deep incisional surgical site infection occurring in the surgical wound [18]. All postoperative events were graded based on the Clavien-Dindo classification [19]. Conversion to open surgery was defined as stopping the laparoscopic approach and using a conventional laparotomy incision to complete the planned surgical procedure. Sample size calculation Sample size calculation was performed using PASS 2008 ver. 8.0 (NCSS, LLC, Kaysville, UT, USA). Arteaga Gonzalez et al.[12] reported conversion rates in patients with (26.1%) or with- out prior abdominal surgery (5.1%, p = .02) after laparoscopic colorectal surgery. Thus, our sample size was calculated using these results. Group sample sizes of 41 in no PAS group and 41 in the PAS group two achieve 80% power to detect a difference between the group propor- tions of .2100. The proportion in group one (the PAS group) is assumed to be .0510 under the null hypothesis and .2610 under the alternative hypothesis. The proportion in group two (the no PAS group) is .0510. For the test statistic, we used the two-sided Z test with pooled variance. The significance level of the test was targeted at .0500. The significance level actually achieved by this design was .0456. The final sample size was 41 patients in each group. We finally enrolled 57 colon cancer patients with PAS and 42 rectal cancer patients with PAS. Surgery, adjuvant therapy and follow-up All operations were performed by two experienced colorectal surgeons. One surgeon had over 700 cases of laparoscopic experience, and the other had over 200 cases of laparoscopic experi- ence. Before surgery, the type of surgical approach—laparoscopy or open procedure—was dis- cussed with patients and their families. Patients with right colon cancer did not receive any mechanical bowel preparation and fasted only from midnight the night before the surgery. Patients with left colon and rectal cancers received mechanical bowel preparation with 4 L of polyethylene glycol solution. A glycerin enema was performed twice, once in the afternoon and once in the evening on the day before the surgery. First-generation cephalosporin (cefazolin) was used as a prophylactic antibiotic and was administered just before the start of surgery. Postoperative antibiotic treatment was continued for 24 to 48 hours. After standardized preop- erative preparation, complete mesocolic excision with central vascular ligation was performed in patients with colon cancer. Tumor-specific mesorectal excision including high ligation of the inferior mesenteric artery was performed for rectal cancer. The operative principles under- lying the laparoscopic approach were basically the same as those of complete mesocolic exci- sion or tumor-specific mesorectal excision through laparotomy. After recovery from surgery, chemotherapy was recommended for patients with stage II, III and IV disease according to National Comprehensive Cancer Network (NCCN) guidelines. All patients were registered in an electronic colorectal database and were followed until death or December 31, 2014. Median follow-up period was 45.6 months (interquartile range, 21.8 to 67.2 months). 3 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 Prior Abdominal Surgery Statistical analysis All statistical analyses were performed using MedCalc Statistical Software version 15.2.2 (Med- Calc Software bvba, Ostend, Belgium) and IBM SPSS Statistics for Windows, version 22.0 (IBM, Armonk, NY, USA). The Student’s t-test was used for comparison of continuous vari- ables, and the Chi-squared test (or Fisher's exact test) was used for comparison of categorical variables. A p-value <0.05 was considered to be statistically significant. Prior Abdominal Surgery Table 1. Patient characteristics. Colon cancer (n = 329) No PAS (n = 272) Major PAS (n = 24) Minor PAS (n = 33) N (%), mean (SD) N(%), mean(SD) p* N(%), mean(SD) p† Age (years) 68(11.1) 67(12.0) .644 64(12.3) .052 Sex (female) 107(39.3) 16(66.7) .009 23(69.7) .001 Body mass index (kg/m2) 23.9(3.2) 23.4(3.7) .483 23.9(3.5) .978 ASA score (3) 44(16.2) 1(4.2) .116 2(6.1) .125 pT4 (+) 48(17.6) 2(8.3) .243 10(30.3) .080 Operation Right colectomy 163(59.9) 11(45.8) .090 13(39.4) .020 Sigmoid, left colectomy 99(36.4) 10(41.7) 20(60.6) Other 10(3.7) 3(12.5) 0(.0) Rectal cancer (n = 324) No PAS (n = 282) Major PAS (n = 16) Minor PAS (n = 26) N(%), mean(SD) N(%), mean(SD) p* N(%), mean(SD) p† Age (years) 68(10.8) 67(10.3) .904 68(9.0) .978 Sex (female) 78(27.7) 7(43.8) .166 11(42.3) .115 Body mass index (kg/m2) 23.7(3.5) 22.8(4.5) .359 25(2.8) .067 ASA score (3) 34(12.1) 2(12.5) .958 4(15.4) .622 pT4 (+) 21(7.4) 3(18.8) .106 1(3.8) .495 Preoperative radiation (+) 73(25.9) 3(18.8) .524 8(30.8) .588 Operation Abdominoperineal resection 35(12.4) 0(.0) .035 2(7.7) .703 Low anterior resection 245(86.9) 15(93.8) 24(92.3) Other 2(.7) 1(6.3) 0(.0) PAS, prior abdominal surgery; SD, standard deviation; ASA, American Society of Anesthesiologists * Comparison between the no PAS and major PAS groups. † Comparison between the no PAS and minor PAS groups. Table 1. Patient characteristics. PAS, prior abdominal surgery; SD, standard deviation; ASA, American Society of Anesthesiologists * Comparison between the no PAS and major PAS groups. PAS, prior abdominal surgery; SD, standard deviation; ASA, American Society of Anesthesiologists * Comparison between the no PAS and major PAS groups. † Comparison between the no PAS and minor PAS groups. doi:10.1371/journal.pone.0134058.t001 gynecologic cancer (18.8%). In the minor PAS group, appendectomy was most common (50.0%), followed by gynecologic procedures (7.7%). Reasons for prior abdominal surgery The detailed reasons for PAS are presented in Table 2. In colon cancer patients, radical hyster- ectomy with extended lymph node dissection for gynecologic cancer was most common (41.7%) in the major PAS group, followed by left colectomy and proctectomy (16.7%), and gas- trectomy (8.3%). In the minor PAS group, appendectomy was most common (45.5%), followed by gynecologic procedures (27.3%). In rectal cancer patients, gastrectomy was most common (43.8%), followed by radical hysterectomy with extended lymph node dissection for 4 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 Table 2. Reasons for prior abdominal surgery. Table 2. Reasons for prior abdominal surgery. Colon cancer (n = 57) Major PAS (n = 24) N(%) Minor PAS (n = 33) N(%) Gynecologic surgery (radical hysterectomy with extended lymph node dissection)* 10(41.7) Appendectomy 15(45.5) Gastrectomy 2(8.3) Gynecologic surgery 9(27.3) Left colectomy, proctectomy 4(16.7) Cholecystectomy 6(18.2) Right colectomy 3(12.5) Other 3(9.1) Small bowel resection 2(8.3) Urologic surgery (bladder, kidney) 1(4.2) Hepatobiliary surgery 2(8.4) Rectal cancer (n = 42) Major PAS (n = 16) N(%) Minor PAS (n = 26) N(%) Gynecologic surgery (radical hysterectomy with extended lymph node dissection)* 3(18.8) Appendectomy 13(50.0) Gastrectomy 7(43.8) Gynecologic surgery 2(7.7) Left colectomy 1(6.3) Cholecystectomy 3(11.5) Right colectomy 2(12.5) Stoma creation 1(3.8) Small bowel resection 2(12.5) Other 7(26.9) Urologic surgery (bladder) 1(6.3) PAS, prior abdominal surgery * Cesarean section (n = 4), abdominal hysterectomy for uterine myoma (n = 2), and laparoscopic gynecologic procedures (n = 5) were excluded. PAS, prior abdominal surgery * Cesarean section (n = 4), abdominal hysterectomy for uterine myoma (n = 2), and laparoscopic gynecologic procedures (n = 5) were excluded. minal hysterectomy for uterine myoma (n = 2), and laparoscopic gynecologic procedures (n = 5) were excluded. Pathologic results with respect to distal margin, number of lymph nodes retrieved, and tumor size were all comparable in the major and minor PAS groups compared with the no PAS group. When comparing TNM classification, stage IV disease was more common in the minor PAS group (18.2%) compared with the no PAS group (3.7%) (Table 3). Short-term outcomes of rectal cancer patients In patients with rectal cancer, the rate of conversion to open surgery was significantly higher in the major PAS group (25%) compared with the no PAS group (8.9%), while the conversion rate was similar between the no PAS and minor PAS groups (15.4%). Adhesion-related conver- sion was more common in the major PAS (50%) and minor PAS (25%) groups, when com- pared to no PAS group (8%). The 30-day complication rate was 29.1% in the no PAS group, 25% in the major PAS group, and 26.9% in the minor PAS group, respectively. The wound infection rate was 4.3% in the no PAS group, 6.3% in the major PAS group, and 11.5% in the minor PAS group, respectively. Mean operative time was 227 min in the no PAS group, 210 min in the major PAS group, and 248 min in the minor PAS group, respectively. The intrao- perative enterotomy rate was 2.1% in the no PAS group, 6.3% in the major PAS group, and 3.8% in the minor PAS group, respectively. Other intraoperative and postoperative outcomes such as blood loss, mortality, severity of complications (Clavien-Dindo grade), time to soft diet, hospital stay, and time to chemotherapy initiation did not differ among the groups (Table 4). Short-term outcomes of colon cancer patients In patients with colon cancer, the rate of conversion to open surgery was significantly higher in the major PAS group (25%) compared with the no PAS group (8.1%), while the conversion rate was similar between the no PAS and minor PAS groups (15.2%). Adhesion-related conver- sion was more common in the major PAS (50%) and minor PAS (20%) groups when compared to no PAS group (9.1%). The 30-day complication rate did not differ among the three groups (28.7% in the no PAS group, 29.2% in the major PAS group, and 27.3% in the minor PAS group). The wound infection rate did not differ among the three groups (8.1% in the no PAS group, 8.3% in the major PAS group, and 9.1% in the minor PAS group). Mean operative time did not differ among the three groups (188 min in the no PAS group, 191 min in the major PAS group, and 192 min in the minor PAS group). The intraoperative enterotomy rate did not differ among the three groups (2.6% in the no PAS group, 8.3% in the major PAS group, and 3.0% in the minor PAS group). Other intraoperative and postoperative outcomes such as blood loss, mortality, severity of complications (Clavien-Dindo grade), time to soft diet, hospital stay, and time to chemotherapy initiation did not differ among the groups. 5 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 Prior Abdominal Surgery Table 2. Reasons for prior abdominal surgery. Table 2. Reasons for prior abdominal surgery. doi:10.1371/journal.pone.0134058.t002 Prior Abdominal Surgery Table 3. Short-term outcomes of colon cancer patients (n = 329). No PAS (n = 272) Major PAS (n = 24) Minor PAS (n = 33) N(%), mean(SD) N(%), mean(SD) p* N(%), mean(SD) p† Conversion to open surgery(+) 22(8.1) 6(25.0) .007 5(15.2) .177 Adhesion-related 2(9.1) 3(50.0) 1(20.0) Tumor ﬁxation (locally advanced tumor) 17(77.3) 3(50.0) 3(60.0) Technical reason (bleeding) 3(13.6) 0(.0) 1(20.0) 30-day complication rate (+) 78(28.7) 7(29.2) .959 9(27.3) .866 Wound infection 22(8.1) 2(8.3) 3(9.1) Anastomotic leakage 8(2.9) 1(4.2) 1(3.0) Postoperative ileus 15(5.5) 0(.0) 1(3.0) Operative time (min) 188(62) 191(66) .827 192(55) .752 Median 178 180 190 Range 69, 450 105, 345 95, 310 Interquartile range 145, 220 138, 235 148, 225 Blood loss (ml) 106(209) 100(140) .877 83(84) .535 Intraoperative enterotomy (+) 7(2.6) 2(8.3) .115 1(3.0) .877 30-day mortality (+) 2(.7) 0(.0) .673 0(.0) .621 Clavien-Dindo score (3) 23(8.5) 2(8.3) .983 4(12.1) .484 Mean time to soft diet (days) 6(3) 7(7) .295 6(2) .730 Mean hospital stay (days) 12(7) 12(10) .799 12(5) .873 Time to chemotherapy (days) 33.1(9) 32.8(4) .924 30.6(5) .263 TNM classiﬁcation 0, I 69(25.4) 8(33.3) .071 8(24.2) .003 II 88(32.4) 11(45.8) 6(18.2) III 105(38.6) 3(12.5) 13(39.4) IV 10(3.7) 2(8.3) 6(18.2) Lymph node harvest (number) 23(11) 21(12) .566 27(13) .052 Distal margin (cm) 11.8(10) 12.7(9) .663 15.3(9) .063 Tumor size (cm) 4.6(2) 3.9(2) .113 4.2(2) .178 PAS i bd i l SD t d d d i ti Table 3. Short-term outcomes of colon cancer patients (n = 329). doi:10.1371/journal.pone.0134058.t003 compared with the no PAS group (8.1%), while the conversion rate was similar between the no PAS group and patients with prior radical hysterectomy (20.0%). There were no differences among the three groups with respect to 30-day complication rates (28.7% in the no PAS group, 11.1% in patients with prior gastrectomy or colectomy, and 40% in patients with radical hysterectomy) and operative time (188 min in the no PAS group, 203 min in patients with prior gastrectomy or colectomy, and 191 min in patients with radical hysterectomy). Other postoperative outcomes such as time to diet, duration of hospital stay, and lymph node harvest were similar among the three groups. In patients with rectal cancer, outcomes were compared between the no PAS group and patients with prior gastrectomy or colectomy (n = 10), and patients with prior radical hysterec- tomy with extended lymph node dissection for gynecologic cancer (n = 3). Outcomes according to types of major prior abdominal surgery In colon cancer patients, outcomes were compared between the no PAS group and patients with prior gastrectomy or colectomy (n = 9), and patients with prior radical hysterectomy with extended lymph node dissection for gynecologic cancer (n = 10). The rate of conversion to open surgery was significantly higher in patients with prior gastrectomy or colectomy (33.3%) 6 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 Prior Abdominal Surgery Table 4. Short-term outcomes of rectal cancer patients (n = 324). No PAS (n = 282) Major PAS (n = 16) Minor PAS (n = 26) N(%), mean(SD) N(%), mean(SD) p* N(%), mean(SD) p† Conversion to open surgery(+) 25(8.9) 4(25.0) .034 4(15.4) .276 Adhesion-related 2(8.0) 2(50.0) 1(25.0) Tumor ﬁxation (locally advanced tumor) 20(80.0) 2(50.0) 2(50.0) Technical reason (bleeding) 3(12.0) 0(.0) 1(25.0) 30-day complication rate (+) 82(29.1) 4(25.0) .726 7(26.9) .817 Wound infection 12(4.3) 1(6.3) 3(11.5) Anastomotic leakage 15(5.3) 1(6.3) 1(3.8) Postoperative ileus 16(5.7) 1(6.3) 2(7.7) Operative time (min) 227(84) 210(60) .414 248(75) .236 Median 213 195 235 Range 90, 625 135, 360 130, 420 Interquartile range 170, 270 163, 245 205, 300 Blood loss (ml) 174(274) 89(115) .233 233(305) .313 Intraoperative enterotomy (+) 6(2.1) 1(6.3) .290 1(3.8) .574 30-day mortality (+) 1(.4) 0(.0) .811 0(.0) .761 Clavien-Dindo score (3) 47(16.7) 2(12.5) .662 3(11.5) .497 Mean time to soft diet (days) 5(4) 6(6) .637 5(3) .551 Mean hospital stay (days) 14(11) 13(9) .590 13(8) .593 Time to chemotherapy (days) 37.3(15) 48.6(50) .518 50.9(49) .282 TNM classiﬁcation 0, I 84(29.8) 7(43.8) .452 8(30.8) .714 II 83(29.4) 3(18.8) 5(19.2) III 97(34.4) 6(37.5) 11(42.3) IV 18(6.4) 0(.0) 2(7.7) Circumferential margin (+) 26(9.3) 2(12.5) .669 0(.0) .104 Lymph node harvest (number) 18(9) 18(7) .839 17(7) .507 Distal margin (cm) 3.2(3) 2.7(2) .510 3.1(5) .859 Tumor size (cm) 4(2) 3.5(2) .373 3.4(2) .160 PAS i bd i l SD t d d d i ti Table 4. Short-term outcomes of rectal cancer patients (n = 324). between the no PAS and patients with prior radical hysterectomy (0.0%). There were no differ- ences among the three groups with respect to 30-day complication rates (29.1% in the no PAS group, 20.0% in patients with prior gastrectomy or colectomy, and 33.3% in patients with radi- cal hysterectomy) and operative time (227 min in the no PAS group, 199 min in patients with prior gastrectomy or colectomy, and 200 min in patients with radical hysterectomy). Other postoperative outcomes such as time to diet, duration of hospital stay, and lymph node harvest were similar among the three groups (Table 5). The rate of conver- sion to open surgery was significantly higher in patients with prior gastrectomy or colectomy (30.0%) compared with the no PAS group (8.9%), while the conversion rate was similar 7 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 doi:10.1371/journal.pone.0134058.t004 Prior Abdominal Surgery Table 5. Outcomes according to type of major prior abdominal surgery. Colon cancer No PAS (n = 272) Prior gastrectomy or colectomy (n = 9) Prior gynecologic surgery (n = 10) N(%), mean(SD) N(%), mean(SD) p* N(%), mean(SD) p† Age (years) 68(11) 73(6) .041 60.8(12) .040 Sex (female) 107(39.3) 4(44.4) .758 10(100.0) < .001 Conversion to open surgery 22(8.1) 3(33.3) .009 2(20.0) .185 30-day complication rate (+) 78(28.7) 1(11.1) .249 4(40.0) .439 Operative time (min) 188(62) 203(83) .508 191(58) .884 Time to soft diet (days) 6(3) 5(2) .599 5.1(2) .572 Hospital stay (days) 12(7) 9(2) .327 11.1(3) .772 Lymph node harvest (number) 23(11) 19(14) .424 22(12) .888 Rectal cancer No PAS (n = 282) Prior gastrectomy or colectomy (n = 10) Prior gynecologic surgery (n = 3) Age (years) 68(11) 68(10) .979 66(14) .786 Sex (female) 78(27.7) 3(30.0) .871 3(100.0) .006 Conversion to open surgery 25(8.9) 3(30.0) .026 0(.0) .589 30-day complication rate (+) 82(29.1) 2(20.0) .533 1(33.3) .872 Operative time (min) 227(84) 199(65) .282 200(57) .573 Time to soft diet (days) 5(4) 7(8) .343 4(2) .585 Hospital stay (days) 14(11) 15(11) .974 10(3) .507 Lymph node harvest (number) 18(9) 17(8) .696 20(8) .784 PAS, prior abdominal surgery; SD, standard deviation * Comparison between the no PAS and major PAS groups (patients with prior gastrectomy or colectomy) † Comparison between the no PAS and major PAS groups (patients with prior gynecologic surgery) doi:10.1371/journal.pone.0134058.t005 Table 5. Outcomes according to type of major prior abdominal surgery. stay did not differ among the groups. Intraoperative enterotomy was more frequent in non- converted patients with major PAS (11.1%) than in patients with no PAS (2.6%, p = 0.043). In rectal cancer patients, outcomes were compared between the no PAS and major PAS groups who completed laparoscopy (n = 12) and those who underwent conversion to laparot- omy (n = 4). Thirty-day complication rate, operative time, intraoperative enterotomy time to diet, and duration of hospital stay did not differ among the groups (Table 6). Outcomes of converted patients with major prior abdominal surgery In colon cancer patients, outcomes were compared between the no PAS and major PAS groups who completed laparoscopy (n = 18) and those who underwent conversion to laparotomy (n = 6). The 30-day complication rate, operative time, time to diet, and duration of hospital 8 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 doi:10.1371/journal.pone.0134058.t005 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 PAS, prior abdominal surgery; SD, standard deviation Prior Abdominal Surgery Table 6. Outcomes of converted patients with major prior abdominal surgery. Colon cancer No PAS (n = 272) Converted patients with major PAS (n = 6) Non-converted patients with major PAS (n = 18) N(%), mean(SD) N(%), mean(SD) p* N(%), mean(SD) p† 30-day complication rate (+) 78(28.7) 3(50.0) .256 4(22.2) .556 Operative time (min) 188(62) 237(73) .063 176(58) .421 Intraoperative enterotomy (+) 7(2.6) 0(.0) .691 2(11.1) .043 Time to soft diet (days) 6(3) 6(2) .712 7(8) .639 Hospital stay (days) 12(7) 12(3) .902 12(12) .820 Lymph node harvest (number) 23(11) 14(7) .076 23(13) .749 Rectal cancer No PAS (n = 282) Converted patients with major PAS (n = 4) Non-converted patients with major PAS (n = 12) 30-day complication rate (+) 82(29.1) 1(25.0) .858 3(25.0) .760 Operative time (min) 227(84) 259(73) .460 194(49) .169 Intraoperative enterotomy (+) 6(2.1) 0(.0) .768 1(8.3) .167 Time to soft diet (days) 5(4) 5(1) .528 6(7) .372 Hospital stay (days) 14(11) 16(10) .745 12(9) .417 Lymph node harvest (number) 18(9) 15(7) .467 19(8) .858 * Comparison between the no PAS and converted patients of major PAS groups † Comparison between the no PAS and non-converted patients of major PAS groups d i 10 1371/j l 0134058 t006 Table 6. Outcomes of converted patients with major prior abdominal surgery. were most common in the minor PAS group. When compared with the no PAS group, there were more women in the major and minor PAS groups, likely due to the high incidence of gynecologic surgery. In the present study, conversion rates and short-term outcomes did not differ between the no PAS and minor PAS groups. Minor PAS (abdominal surgical procedures involving one quadrant of the abdominal cavity) is unlikely to cause widespread adhesions or influence the clinical outcomes of subsequent surgery [20]. Thus, colorectal cancer patients with minor PAS may be safely managed with a laparoscopic approach. y y g p p pp In the current study, conversion to open surgery was more frequent in the major PAS group compared to the no PAS group. Furthermore, adhesion-related conversion was more common in the major PAS group (50% in colon cancer and 50% in rectal cancer, respectively) when compared to the no PAS group (9.1% in colon cancer and 8% in rectal cancer) and minor PAS group (20% in colon cancer and 25% in rectal cancer). Arteaga Gonzalez et al. Discussion The major findings of this study were that a higher rate of conversion to open surgery was observed in the major PAS group compared with the no PAS group in patients with colon can- cer and rectal cancers, respectively. Specific types of major PAS (prior gastrectomy or colect- omy) increased the conversion rates in patients with colon and rectal cancers, respectively. However, the converted patients with major PAS did not have worse clinical outcomes in terms of operative time, postoperative complication rate, and time to diet. Between the no PAS and major PAS groups, other short-term outcomes did not differ. In addition, all short-term outcomes in the minor PAS group were comparable to those in the no PAS group. In patients with previous abdominal operations, intra-abdominal adhesions are trouble- some and must be cleared for oncologic resection. Moreover, it is difficult to predict the sever- ity and degree of adhesions preoperatively, complicating patient selection for a laparoscopic approach in patients with PAS. Thus, we categorized types of PAS to predict preoperative suit- ability for laparoscopy. Data on prior operations showed that gynecologic surgery was most common in the major PAS group in colon cancer patients and gastrectomy was most common in the major PAS group in rectal cancer patients, while appendectomy and gynecologic surgery PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 9 / 14 doi:10.1371/journal.pone.0134058.t006 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 [12] reported that a history of prior surgery was associated with higher conversion rates (26.1% vs. 5.1%), and Franko et al. [13] also observed a higher rate of conversion in the prior surgery group (19.6% vs. 11.4%). However, other investigators did not observe this difference in conversion rate [5,9–11,15,20]. We observed that the conversion rate was not different between the no PAS and minor PAS groups. In colon and rectal cancer patients, the most common reason for conversion was tumor fixation due to locally advanced tumor in the no PAS and minor PAS groups followed by technical issues such as uncontrolled bleeding. Thus, the higher rate of con- version in the major PAS group may be attributed to resultant adhesions. Patient-related fac- tors (obesity or adhesion) and tumor-related factors (locally advanced or bulky tumors) are well-known risk factors for conversion to open surgery [1,3,21]. Similarly, we observed that a history of major PAS was a significant risk factor for conversion. Specifically, higher conversion rates were observed in patients with prior gastrectomy or colectomy, but not in patients with prior radical hysterectomy with extended lymph node dissection for gynecologic cancer. 10 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 Prior Abdominal Surgery We also evaluated outcomes of converted patients with major PAS because several studies reported adverse outcomes in converted patients, including higher morbidity, increased trans- fusion, longer operative time, prolonged hospital stay [14,22,23]. In our study, short-term out- comes were not worse in converted patients. Operative time, intraoperative enterotomy, 30-day complication rate, time to diet, and duration of hospital stay were the same in converted and laparoscopic patients. In colon and rectal cancer patients with major PAS, converted patients had a lengthy operative time when compared to patients with no PAS or non-con- verted patients with major PAS. However, these differences did not reach statistical significance due to the small sample size. We believe that appropriate surgical decision-making regarding conversion to an open approach is important for patient safety and oncologic results during laparoscopic procedures. Timely conversion to open surgery may avoid prolonged operative time or prevent unnecessary tumor manipulation and tumor cell spillage, thus reducing the potential risk of adverse clinical and oncologic outcomes [1,24]. Indeed, Belizon et al. [22] sug- gested that prompt conversion within 30 minutes of the start of surgery may reduce adverse postoperative outcomes. Unfortunately, we could not assess the time point of conversion to open surgery due to the retrospective nature of data collection. Thus, conversion should be regarded as another strategy to solve the technical difficulties of laparoscopy for colorectal cancer. On review of literature, short-term outcomes in patients with PAS are variable. Beck et al. [25] demonstrated that previous abdominal surgery significantly prolongs time to opening the abdomen. Vignali et al. [5] reported longer operative times in patients with PAS (218 min vs. 192 min) in their case-matched study. However, other investigators observed similar operative times between patients with PAS and those without, as was observed in our series [9–12,15,20]. Subsequent surgery in patients with major PAS increases operative time because adhesiolysis in patients who have been operated on in four quadrants can take a substantial amount of time. In the current study, although statistically nonsignificant, patients with major PAS had shorter operative times. A test for normality with the Shapiro-Wilk method was used to deter- mine whether operative time data were normally distributed. Although the sample size was small, operative time data showed a normal distribution pattern: colon cancer patients with major PAS (p = .087) and minor PAS (p = .348), and rectal cancer patients with major PAS (p = .258) and minor PAS (p = .383). There could be two reasons for shorter operative times in patients with major PAS in this study. First, operative time is mainly affected by surgical proce- dure type, and, accordingly, differences among the no PAS, major PAS, and minor PAS groups probably influence operative time. In colon cancer patients, right colectomy was more fre- quently performed in the no PAS group when compared to major PAS and minor PAS groups. At our institution, right colectomy for right-sided colon cancer was routinely performed based on complete mesocolic excision with central vascular ligation, which generally necessitates a lengthy operative time. In rectal cancer patients, abdominoperineal resection was commonly performed in the no PAS group when compared to major PAS and minor PAS groups. Opera- tive time for abdominoperineal resection is generally longer than that for low anterior resection due to perineal dissection. Second, to reduce operative time in patients with major PAS, adhe- sions irrelevant to the planned surgical procedures were not removed. A higher rate of inadvertent enterotomy has been reported in patients with a history of prior surgery [10,13]; however, PAS did not influence risk of inadvertent enterotomy in this study and others [20]. In the literature, the intraoperative enterotomy rate ranges from 0.1 to 0.2% in the no PAS group and 0.9 to 5.5% in the PAS group [10,13,26]. The rather high rate of enterotomy in this study was partly due to inclusion of colon and rectal cancer patients. Unlike laparoscopy for benign colorectal diseases, laparoscopic surgery for colorectal cancer focuses on oncologic clearance and radicality. Laparoscopic complete mesocolic excision for colon PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 11 / 14 Prior Abdominal Surgery cancer or laparoscopic total mesorectal excision for rectal cancer are procedures with increased technical complexity. Franko et al. [13] observed a higher incidence of postoperative ileus and reoperation in patients with PAS, and Aytac et al. [20] reported a higher postoperative compli- cation rate in the PAS group. However, we observed similar postoperative complication rates in the major and minor PAS groups compared with the no PAS group [5,9–12,15]. Yamamoto et al. observed that PAS increased time to diet significantly, but most other studies, including ours, reported that PAS did not increase time to diet [9,12]. In this study, hospital stay was sim- ilar in the PAS groups compared with the no PAS group. In previous literature, PAS did not influence length of hospital stay [5,9–12,15,20]. Favorable short-term outcomes of the major and minor PAS groups are in line with other published data [9,11,12,15]. We believe that suffi- cient surgeon experience and surgical team training positively influenced these findings [27]. Rates of wound infection after laparoscopic colorectal surgery have been reported to range from 4 to 12% [4,28,29]; wound infection rates in our subgroups varied from 4.3 to 11.5%, which is within this reported range. This study was limited by its retrospective nature and the relatively small number of patients included in a single center. We included only malignant cases, categorized types of PAS such as minor and major, and evaluated the impact of types of PAS on outcomes after laparoscopic surgery for colon and rectal cancers. The incidence of PAS ranges from 21 to 46% in the litera- ture [5,9–13,15] and the incidence in this study was 15%. Acknowledgments The authors thank Hyun Jun Kwon for management of the colorectal database without which this study would not have been possible. There are ethical restrictions with regard to data dis- tribution imposed by our Institutional Review Board and these restrictions are in line with the approved consent procedure. Accordingly, interested researchers may submit requests for our data and all data are available upon request from the corresponding author. The low rate of prior surgery may be related to the retrospective nature of the data collection and the possibility that the patient's surgical history was missed in the history taking before surgery. In addition, surgeon preference might have influenced the choice of laparoscopy for patients with previous history of abdomi- nal surgery. In summary, if technical difficulties are anticipated during surgery, surgeons tend to decline a laparoscopic approach [30]. Our results suggest that colorectal cancer patients with minor PAS or patients with prior radical hysterectomy with extended lymph node dissection for gyne- cologic cancer can be effectively managed with a laparoscopic approach. Specific types of major PAS (prior gastrectomy or colectomy) increased conversion rates in patients with colon and rectal cancers. The higher rate of conversion observed in the major PAS group was partly due to the underlying adhesions related to major PAS. However, other short-term outcomes of the major PAS group are comparable to the no PAS group. Moreover, short-term outcomes were not worse in the converted patients with major PAS. Thus, laparoscopy can be selected as the primary surgical approach even in patients with major PAS (prior gastrectomy or colect- omy) given the assumption of a higher conversion rate. In the future, it would be helpful to for- mulate a management pathway according to types of PAS. References 1. Jayne DG, Guillou PJ, Thorpe H, Quirke P, Copeland J, et al. (2007) Randomized trial of laparoscopic- assisted resection of colorectal carcinoma: 3-year results of the UK MRC CLASICC Trial Group. J Clin Oncol 25: 3061–3068. PMID: 17634484 2. Kang SB, Park JW, Jeong SY, Nam BH, Choi HS, et al. (2010) Open versus laparoscopic surgery for mid or low rectal cancer after neoadjuvant chemoradiotherapy (COREAN trial): short-term outcomes of an open-label randomised controlled trial. Lancet Oncol 11: 637–645. doi: 10.1016/S1470-2045(10) 70131-5 PMID: 20610322 3. Clinical Outcomes of Surgical Therapy Study Group (2004) A comparison of laparoscopically assisted and open colectomy for colon cancer. N Engl J Med 350: 2050–2059. PMID: 15141043 4. Guillou PJ, Quirke P, Thorpe H, Walker J, Jayne DG, et al. (2005) Short-term endpoints of conventional versus laparoscopic-assisted surgery in patients with colorectal cancer (MRC CLASICC trial): multicen- tre, randomised controlled trial. Lancet 365: 1718–1726. PMID: 15894098 5. Vignali A, Di Palo S, De Nardi P, Radaelli G, Orsenigo E, et al. (2007) Impact of previous abdominal sur- gery on the outcome of laparoscopic colectomy: a case-matched control study. Tech Coloproctol 11: 241–246. PMID: 17676267 6. Coleman MG, McLain AD, Moran BJ (2000) Impact of previous surgery on time taken for incision and division of adhesions during laparotomy. Dis Colon Rectum 43: 1297–1299. PMID: 11005501 7. van der Voort M, Heijnsdijk EA, Gouma DJ (2004) Bowel injury as a complication of laparoscopy. Br J Surg 91: 1253–1258. PMID: 15376204 8. Miskovic D, Ni M, Wyles SM, Tekkis P, Hanna GB (2012) Learning curve and case selection in laparo- scopic colorectal surgery: systematic review and international multicenter analysis of 4852 cases. Dis Colon Rectum 55: 1300–1310. doi: 10.1097/DCR.0b013e31826ab4dd PMID: 23135590 9. Law WL, Lee YM, Chu KW (2005) Previous abdominal operations do not affect the outcomes of laparo- scopic colorectal surgery. Surg Endosc 19: 326–330. PMID: 15624064 10. Yamamoto M, Okuda J, Tanaka K, Kondo K, Asai K, et al. (2013) Effect of previous abdominal surgery on outcomes following laparoscopic colorectal surgery. Dis Colon Rectum 56: 336–342. doi: 10.1097/ DCR.0b013e31827ba103 PMID: 23392148 11. Hamel CT, Pikarsky AJ, Weiss E, Nogueras J, Wexner SD (2000) Do prior abdominal operations alter the outcome of laparoscopically assisted right hemicolectomy? Surg Endosc 14: 853–857. PMID: 11000368 12. Arteaga Gonzalez I, Martin Malagon A, Lopez-Tomassetti Fernandez EM, Arranz Duran J, Diaz Luis H, et al. Author Contributions Performed the experiments: BRK YWK. Analyzed the data: BRK YWK. Contributed reagents/ materials/analysis tools: BRK YWK. Wrote the paper: YWK IYK. Participated in study concep- tion and design: YWK IYK. Read and approved the final manuscript: IYK BRK YWK. 12 / 14 PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 Prior Abdominal Surgery PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 References (2006) Impact of previous abdominal surgery on colorectal laparoscopy results: a comparative clinical study. Surg Laparosc Endosc Percutan Tech 16: 8–11. PMID: 16552371 13. Franko J, O'Connell BG, Mehall JR, Harper SG, Nejman JH, et al. (2006) The influence of prior abdomi- nal operations on conversion and complication rates in laparoscopic colorectal surgery. Jsls 10: 169– 175. PMID: 16882414 14. Chan AC, Poon JT, Fan JK, Lo SH, Law WL (2008) Impact of conversion on the long-term outcome in laparoscopic resection of colorectal cancer. Surg Endosc 22: 2625–2630. doi: 10.1007/s00464-008- 9813-3 PMID: 18297346 15. Offodile AC 2nd, Lee SW, Yoo J, Whelan RL, Moradi D, et al. 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PLOS ONE \| DOI:10.1371/journal.pone.0134058 July 24, 2015 References Allaix ME, Degiuli M, Arezzo A, Arolfo S, Morino M (2013) Does conversion affect short-term and onco- logic outcomes after laparoscopy for colorectal cancer? Surg Endosc 27: 4596–4607. doi: 10.1007/ s00464-013-3072-7 PMID: 23846368 25. Beck DE, Ferguson MA, Opelka FG, Fleshman JW, Gervaz P, et al. (2000) Effect of previous surgery on abdominal opening time. Dis Colon Rectum 43: 1749–1753. PMID: 11156462 26. Swank DJ, Van Erp WF, Repelaer Van Driel OJ, Hop WC, Bonjer HJ, et al. (2003) A prospective analy- sis of predictive factors on the results of laparoscopic adhesiolysis in patients with chronic abdominal pain. Surg Laparosc Endosc Percutan Tech 13: 88–94. PMID: 12709613 27. Kim YW, Min BS, Kim NK, Kim JY, Hur H, et al. (2010) The impact of incorporating of a novice assistant into a laparoscopic team on operative outcomes in laparoscopic sigmoidectomy: a prospective study. Surg Laparosc Endosc Percutan Tech 20: 36–41. doi: 10.1097/SLE.0b013e3181cdb762 PMID: 20173619 28. Braga M, Frasson M, Vignali A, Zuliani W, Capretti G, et al. (2007) Laparoscopic resection in rectal can- cer patients: outcome and cost-benefit analysis. Dis Colon Rectum 50: 464–471. PMID: 17195085 29. Lacy AM, Garcia-Valdecasas JC, Delgado S, Castells A, Taura P, et al. (2002) Laparoscopy-assisted colectomy versus open colectomy for treatment of non-metastatic colon cancer: a randomised trial. Lancet 359: 2224–2229. PMID: 12103285 30. Marks JH, Kawun UB, Hamdan W, Marks G (2008) Redefining contraindications to laparoscopic colo- rectal resection for high-risk patients. Surg Endosc 22: 1899–1904. doi: 10.1007/s00464-008-9828-9 PMID: 18347862 14 / 14
https://openalex.org/W4382343337	https://dergipark.org.tr/tr/download/article-file/3055594	English	null	Morphometric analysis and clinical significance of the os sacrum	DergiPark (Istanbul University)	2,023	cc-by	5,578	MEDICINE PALLIATIVE CARE DOI: 10.47582/jompac.1276050 J Med Palliat Care. 2023;4(3):211-218 PALLIATIVE CARE ABSTRACT Aims: The purpose of this study is to assess the architecture and clinical importance of the sacrum, which features the dorsal and pelvic nerves. Methods: 32 os sacrum of adult Anatolians of undetermined gender were measured for this investigation. Sacrum maximum length, os sacrum maximum width, sacrum I vertebral body antero-posterior width, sacrum I vertebral body transverse width, sacral index, Auricular surface short arm, auricular surface long arm and auricular surface oblique arm, the measurements of pelvic surface linea transverse length and, the measurements of dorsal surface linea transverse length and the sacrum height from the dorsal surface are evaluated. Results: Sacrum maximum length 103.30±10.03 mm, sacrum maximum width 108.40±6.10 mm, sacrum I vertebral body transverse width 47.00±5.00 mm, sacrum I vertebral body antero-posterior width 28.30±3.50 mm, sacral index 104.00±9.00, Auricular surface short arm 31.90±4.20 mm, Auricular surface long arm 39.40±4.80 mm, Auricular surface oblique arm 49.10±6.00 mm, the length measurements of dorsal surface distance respectively as mm; 36.72±0.37, 29.75±0.31, 26.53±0.33, 26.56±0.39, the length measurements of dorsal surface distance respectively as mm; 29.16±0.36, 27.16±0.33, 24.50±0.26, 24.38±0.24 and the sacrum height from the dorsal surface as 103.4±9.70 mm were calculated. Conclusion: Clinically stated, understanding the architecture of the sacrum and taking morphometric measures of it are crucial to avoiding difficulties and the surgical intervention that will be used to treat disorders associated to the sacrum. Keywords: Anatomy, morphometry, sacrum This study was presented as a poster presentation at the ”Anthropology, Radiology & Anatomy Congress” held in Ankara University, Ankara between Accepted: 24.05.2023 Accepted: 24.05.2023 Published: 28.06.2023 Corresponding Author: Gamze Taşkın Şenol, rumeysagamzetaskin@ibu.edu.tr This work is licensed under a Creative Commons Attribution 4.0 International License. Original Article Original Article Morphometric analysis and clinical significance of the os sacrum Ayşe Gül Kabakcı1, Gamze Taşkın Şenol2 1Department of Anatomy, Faculty of Medicine, Çukurova University, Adana, Turkey 1Department of Anatomy, Faculty of Medicine, Çukurova University, Adana, Turkey 2D t t f A t F lt f M di i B l Ab t I t B l U i it B p y y Ç y y 2Department of Anatomy, Faculty of Medicine, Bolu Abant Izzet Baysal University, Bolu, Turkey partment of Anatomy, Faculty of Medicine, Bolu Abant Izzet Baysa Kabakcı AG, Taşkın Şenol G. Morphometric analysis and clinical significance of the os sacrum. J Med Palliat Car Cite this article as: Kabakcı AG, Taşkın Şenol G. Morphometric analysis and clinical significance of the os sacr 2023;4(3):211-218. Cite this article as: Kabakcı AG, Taşkın Şenol G. Morphometric analysis and clinical significance of the os sacrum. J Med Palliat Care. 2023;4(3):211-218. INTRODUCTION The five sacral vertebrae come together to form the huge triangular bone known as the os sacrum, which is located in the posterior upper portion of the pelvic cavity (cavitas pelvis). Above is its base (basis ossis sacri), and below is its apex (apex ossis sacri). Facies pelvica and facies dorsalis are the names of the sacrum’s anterior and rear surfaces, respectively.1-5 The lateral side of the os sacrum forms a “L”-shaped joint with the facies auricularis of the ilium. The promontorium, or base of the sacrum, protrudes anteriorly. The union of the processus transversus additionally forms the crista sacralis lateralis. The ala ossis sacri, which resembles a pair of wings, is visible on the upper portion of the crista sacralis lateralis. Additionally, the sacrum’s posterior surface is convex and its anterior surface is concave. There are four transverse lines, or linea transversae, on the face of the os sacrum, also known as the facies pelvica, which connect the corpus vertebrae. The foramina sacralia pelvica, eight holes on the right and left sides of these lines contain the anterior branches of the spinal nerves. Additionally, the aa. and vv. sacralis lateralis passes through these openings. The facies dorsalis refers to the dorsal surface of the os sacrum. There are eight foramina sacralia posteriora on the right and left posterior surfaces of the os sacrum, through which the posterior branches of the spinal nerves continue.4,6 Through the foramen intervertebrales, these holes join the canalis sacralis.7 In terms of protecting neural tissues during surgical interventions to this area and caudal anesthesia, foramina sacralia anteriora and foramina sacralia posteriora are clinically significant.8 The pelvic girdle has a solid joint system that allows weight to be transferred from the trunk to the lower extremities. The spine lumbalis transmits body weight to the os sacrum, which travels through the sacroiliac joint to the os coxae and os femoris.5 The number of bones that make up the sacrum can change in some circumstances.9 In the treatment of lumbosacral, sacral, and sacroiliac anomalies or injuries, the sacrum is a vital bone for Kabakcı et al. Os sacrum morphometric analysis J Med Palliat Care. 2023;4(3):211-218 out by drawing lines between the two reference points. stability and fusion. Therefore, it is crucial to understand the complicated anatomical nature of the os sacrum before performing any surgery there. METHODS Since the study was performed on dry bones, ethics committee approval is not required, institutional approval was obtained. All procedures were carried out in accordance with the ethical rules and the principles. In this study, 32 pieces of dry bone os sacrum of unknown age and gender were measured. Partially broken, fragmented or damaged bones were excluded from the study. All procedures were carried out in accordance with the ethical rules and the principles of the Declaration of Helsinki. We measured maximum length of os sacrum, maximum width of os sacrum, anterior posterior width of os sacrum corpus vertebrae, transverse width of os sacrum corpus vertebrae, sacral index, facies auricularis short arm length, facies auricularis long arm length, facies auricularis oblique arm length, measurements of os sacrum facies pelvica, measurements of facies dorsalis and the height of the os sacrum on the dorsal (Figure 1-5). Measurements of os sacrum’s includes facies pelvica length between foramina sacralia anteriora I, length between foramina sacralia anteriora II, length between foramina sacralia anteriora III, length between foramina sacralia anteriora IV parameters (Figure 5). In addition, the measurements of the facies dorsalis of the os sacrum are the length between foramina sacralia posteriora I, length between foramina sacralia posteriora II, length between foramina sacralia posteriora III, between foramina sacralia posteriora IV length parameters (Figure 1). Figure 1. Measurements of os sacrum facies dorsalis (FDL1; length between foramina sacralia posteriora 1, FDL2; length between foramina sacralia posteriora 2, FDL3; length between foramina sacralia posteriora 3, FDL4; length between foramina sacralia posteriora 4). Height of os sacrum (SH): The distance between the highest point of the crista sacralis media from the os sacrum facies dorsalis on the dorsal aspect and the cornu sacrale in the mid-sagittal plane was taken as reference.11 Anterior-posterior width of the os sacrum corpus vertebrae (CVAPW): The anterior-posterior width of the corpus vertebrae of the os sacrum was measured (Figure 2).9 Figure 2. Measurements of os sacrum I corpus vertebrae (CVAPW; anterior posterior width of os sacrum corpus vertebrae, CVTW; transverse width of os sacrum corpus vertebrae). Measurements Parameters Measurements of the dorsal surface (facies dorsalis) of the os sacrum (FDL1-4): The length between the right foramina sacralia posteriora and the left foramina sacralia posteriora was measured (Figure 1).11 This study, it is aimed to reveal the anatomical structure of the os sacrum with morphometric measurements and to emphasize its clinical importance. Thus, the results obtained in this study will guide clinical research and applications. Figure 1. Measurements of os sacrum facies dorsalis (FDL1; length between foramina sacralia posteriora 1, FDL2; length between foramina sacralia posteriora 2, FDL3; length between foramina sacralia posteriora 3, FDL4; length between foramina sacralia posteriora 4). INTRODUCTION The architecture of the os sacrum must be understood to provide adequate fixation and prevent neurovascular damage.6,9-11 100 Maximum width of os sacrum (SW): The distance between the most lateral right and left parts of the ala ossis sacri on the most upper part of the os sacrum facies auricularis from the anterior facies was measured Maximum length of the os sacrum Measurement Design Each bone was photographed from a distance of 100 cm under artificial light. The photographic system was set up by fixing the camera at a distance of 100 cm with an adjustable tripod. The tripod height was also positioned and fixed in accordance with the position of the bones. Photographs of dry bones were taken with a Digital SLR camera with fixed photo shooting settings (Canon EOS 80D; ISO 100 f/4.5) from the right-left, pelvic-dorsal side. The photos taken were transferred to the computer and uploaded to the Sketchup design program. The reference points of the parameters to be measured were determined and the length measurements were carried i Photographs of dry bones were taken with a Digital SLR camera with fixed photo shooting settings (Canon EOS 80D; ISO 100 f/4.5) from the right-left, pelvic-dorsal side. The photos taken were transferred to the computer and uploaded to the Sketchup design program. The reference points of the parameters to be measured were determined and the length measurements were carried Figure 2. Measurements of os sacrum I corpus vertebrae (CVAPW; anterior posterior width of os sacrum corpus vertebrae, CVTW; transverse width of os sacrum corpus vertebrae). 212 Kabakcı et al. Os sacrum morphometric analysis J Med Palliat Care. 2023;4(3):211-218 Figure 4. Length and width measurements of the os sacrum (SL; maximum length of os sacrum, SW; maximum width of os sacrum). Figure 5. Measurements of os sacrum facies pelvica (FPL1; length between foramina sacralia anteriora 1, FPL2; length between foramina sacralia anteriora 2, FPL3; length between foramina sacralia anteriora 3, FPL4; length between foramina sacralia anteriora) Sacral index (SI):12-14 Maximum width of os sacrum × 100 Maximum length of the os sacrum Measurements of the os sacrum facies pelvica (FPL1- 4): The length of the linea transversa between the right Figure 4. Length and width measurements of the os sacrum (SL; maximum length of os sacrum, SW; maximum width of os sacrum). Transverse width of the os sacrum corpus vertebrae (CVTW): The os sacrum corpus vertebrae was measured horizontally from right lateral to left lateral (Figure 2).9 Transverse width of the os sacrum corpus vertebrae (CVTW): The os sacrum corpus vertebrae was measured horizontally from right lateral to left lateral (Figure 2).9 Figure 4. Length and width measurements of the os sacrum (SL; maximum length of os sacrum, SW; maximum width of os sacrum). Facies auricularis short arm length (SAL) of Os sacrum: Measurements were made from the right side and the distance from point A to point B was calculated (Figure 3).11 Figure 3. Measurements of facies auricularis of os sacrum (SAL; facies auricularis short arm length, LAL; facies auricularis long arm length, OAL; facies auricularis oblique arm length). Figure 4. Length and width measurements of the os sacrum (SL; maximum length of os sacrum, SW; maximum width of os sacrum). Figure 4. Length and width measurements of the os sacrum (SL; maximum length of os sacrum, SW; maximum width of os sacrum). Figure 5. Measurements of os sacrum facies pelvica (FPL1; length between foramina sacralia anteriora 1, FPL2; length between foramina sacralia anteriora 2, FPL3; length between foramina sacralia anteriora 3, FPL4; length between foramina sacralia anteriora) Figure 3. Measurements of facies auricularis of os sacrum (SAL; facies auricularis short arm length, LAL; facies auricularis long arm length, OAL; facies auricularis oblique arm length). Facies auricularis long arm length (LAL) of Os sacrum: Measurements were taken from the right side and the distance from point B to point C was measured (Figure 3).11 Os sacrum’s facies auricularis oblique arm length (OAL): Measurements were made from the right side and the distance from point A to point C was measured (Figure 3).11 Figure 5. Measurements of os sacrum facies pelvica (FPL1; length between foramina sacralia anteriora 1, FPL2; length between foramina sacralia anteriora 2, FPL3; length between foramina sacralia anteriora 3, FPL4; length between foramina sacralia anteriora) Maximum length of the os sacrum (SL): The distance between the promontorium and the lowest point of the anterior edge of the os sacrum was calculated in the mid- sagittal plane (Figure 4).12-14 Sacral index (SI):12-14 Maximum width of os sacrum × 100 Sacral index (SI):12-14 Maximum width of os sacrum × 100 Statistical Analysis Table 1. Mean (μ), standard deviation (σ), minimum (min.) and maximum (max.) values of the measurements of os sacrum Parameters (mm) μ σ Minimum Maximum SW 108.40 6.10 91.50 120.00 SL 103.30 10.03 80.00 125.00 CVTW 47.00 5.00 37.00 56.00 CVAPW 28.30 3.50 21.50 35.00 SI 104.00 9.00 78.00 133.00 SAL 31.90 4.20 21.00 42.00 LAL 39.40 4.80 31.00 49.00 OAL 49.10 6.00 38.00 58.00 FDL1 36.72 0.37 28.0 40.3 FDL2 29.75 0.31 24.0 30.6 FDL3 26.53 0.33 22.0 30.3 FDL4 26.56 0.39 20.0 30.5 FPL1 29.16 0.36 23.0 30.6 FPL2 27.16 0.33 19.0 30.3 FPL3 24.50 0.26 20.0 30.0 FPL4 24.38 0.24 20.0 30.1 SH 103.40 9.70 82.0 126.0 SW; maximum width of os sacrum, SL; maximum length of os sacrum, CVTW; transverse width of os sacrum corpus vertebrae, CVAPW; anterior posterior width of os sacrum corpus vertebrae, SI; sacral index, SAL; facies auricularis short arm length, LAL; facies auricularis long arm length, OAL; facies auricularis oblique arm length, FDL1; length between foramina sacralia posteriora 1, FDL2; length between foramina sacralia posteriora 2, FDL3; length between foramina sacralia posteriora 3, FDL4; length between foramina sacralia posteriora 4, FPL1; length between foramina sacralia anteriora 1, FPL2; length between foramina sacralia anteriora 2, FPL3; length between foramina sacralia anteriora 3, FPL4; length between foramina sacralia anteriora 4, SH; Facies dorsalis height of os sacrum. μ; mean, σ: Standard deviation. Table 1. Mean (μ), standard deviation (σ), minimum (min.) and maximum (max.) values of the measurements of os sacrum The SPSS 22 package program was used for statistical analysis and statistically significant degree were considered as p<0.05. Also, the normal distribution of the data distribution were identified by Skewness and kurtosis statistics (between +1.5 and -1.5). After statistical evaluation of the measurement results, mean (μ), standard deviation (σ), minimum (min.) and maximum (max.) values were calculated. Maximum length of the os sacrum Measurements of the os sacrum facies pelvica (FPL1- 4): The length of the linea transversa between the right foramina sacralia anteriora and the left foramina sacralia anteriora was measured (Figure 4).11 (Figure 4).12-16 213 J Med Palliat Care. 2023;4(3):211-218 Table 1. Mean (μ), standard deviation (σ), minimum (min.) and maximum (max.) values of the measurements of os sacrum Parameters (mm) μ σ Minimum Maximum SW 108.40 6.10 91.50 120.00 SL 103.30 10.03 80.00 125.00 CVTW 47.00 5.00 37.00 56.00 CVAPW 28.30 3.50 21.50 35.00 SI 104.00 9.00 78.00 133.00 SAL 31.90 4.20 21.00 42.00 LAL 39.40 4.80 31.00 49.00 OAL 49.10 6.00 38.00 58.00 FDL1 36.72 0.37 28.0 40.3 FDL2 29.75 0.31 24.0 30.6 FDL3 26.53 0.33 22.0 30.3 FDL4 26.56 0.39 20.0 30.5 FPL1 29.16 0.36 23.0 30.6 FPL2 27.16 0.33 19.0 30.3 FPL3 24.50 0.26 20.0 30.0 FPL4 24.38 0.24 20.0 30.1 SH 103.40 9.70 82.0 126.0 SW; maximum width of os sacrum, SL; maximum length of os sacrum, CVTW; transverse width of os sacrum corpus vertebrae, CVAPW; anterior posterior width of os sacrum corpus vertebrae, SI; sacral index, SAL; facies auricularis short arm length, LAL; facies auricularis long arm length, OAL; facies auricularis oblique arm length, FDL1; length between foramina sacralia posteriora 1, FDL2; length between foramina sacralia posteriora 2, FDL3; length between foramina sacralia posteriora 3, FDL4; length between foramina sacralia posteriora 4, FPL1; length between foramina sacralia anteriora 1, FPL2; length between foramina sacralia anteriora 2, FPL3; length between foramina sacralia anteriora 3, FPL4; length between foramina sacralia anteriora 4, SH; Facies dorsalis height of os sacrum. μ; mean, σ: Standard deviation. of the os sacrum with the literature Literature Measurement type Population/Year Koç et al.11 Dry os Turkey/2014 Duman.6 Computed Tomography Turkey/2009 This study Dry os Turkey/2018 alia posteriora 2, FDL3; length between foramina sacralia posteriora 3, FDL4; length 1, FPL2; length between foramina sacralia anteriora 2, FPL3; length between foramina ard deviation. J Med Palliat Care. 2023;4(3):211-218 Kabakcı et al. Os sacrum morphometric analysis RESULTS Our study includes the following findings; width of os sacrum, length of os sacrum, transverse width of os sacrum corpus vertebrae, sacral index, anterior posterior width of os sacrum corpus vertebrae, facies auricularis short arm length, facies auricularis long arm length, facies auricularis oblique arm length, length between foramina sacralia posteriora 1, 2, 3, 4, length between foramina sacralia anteriora 1, 2, 3, 4 and facies dorsalis height of os sacrum. The minimum, maximum, mean and standard deviation values of the linear measurements of 32 dry bone os sacrum are shown in Table 1. In addition, we compared the results of these parameters in our study with the literature findings (Table 2, Table 3 and Table 4). Table 2. Comparison of anterior and posterior surface measurements of the os sacrum with the literature Parameters μ±σ Literature Measurement type Population/Year FDL1 38.68±4.03 mm Koç et al.11 Dry os Turkey/2014 FDL2 31.45±4.13 mm FDL3 26.10±3.68 mm FDL4 26.04±3.31 mm FPL1 30.01±5.03 mm FPL2 28.33±4.48 mm FPL3 26.51±4.82 mm FPL4 24.81±3.93 mm FPL1 Erkek Kadın Duman.6 Computed Tomography Turkey/2009 31.1±3.6 mm 28.0±3.1 mm FPL2 28.8±3.4 mm 26.1±2 mm FPL3 26.5±2.3 mm 24.6±2.4 mm FPL4 24.6±2.4 mm 23.2±2.4 mm FDL1 FDL2 FDL3 FDL4 FPL1 FPL2 FPL3 FPL4 36.72±0.37 mm 29.75±0.31 mm 26.53±0.33 mm 26.56±0.39 mm 29.16±0.36 mm 27.16±0.33 mm 24.50±0.26 mm 24.38±0.24 mm This study Dry os Turkey/2018 FDL1; length between foramina sacralia posteriora 1, FDL2; length between foramina sacralia posteriora 2, FDL3; length between foramina sacralia posteriora 3, FDL4; length between foramina sacralia posteriora 4, FPL1; length between foramina sacralia anteriora 1, FPL2; length between foramina sacralia anteriora 2, FPL3; length between foramina sacralia anteriora 3, FPL4; length between foramina sacralia anteriora 4, μ; mean, σ: Standard deviation. FDL1; length between foramina sacralia posteriora 1, FDL2; length between foramina sacralia posteriora 2, FDL3; length between foramina sacralia posteriora 3, FDL4; length between foramina sacralia posteriora 4, FPL1; length between foramina sacralia anteriora 1, FPL2; length between foramina sacralia anteriora 2, FPL3; length between foramina sacralia anteriora 3, FPL4; length between foramina sacralia anteriora 4, μ; mean, σ: Standard deviation. 214 Kabakcı et al. Os sacrum morphometric analysis J Med Palliat Care. 2023;4(3):211-218 Table 3. DISCUSSION failure to fuse or develop that can be seen in the lamina vertebral arch of one or more vertebrae, may occur, as well as sacral hiatus variants, caudal agenesis, or developmental abnormalities.7,23 On the other hand, since it is situated close to where the common iliac is divided into its terminal branches, the promontorium is a crucial point of reference in laparoscopic surgery.7 The five sacral vertebrae fuse to form the triangle- shaped sacrum, which functions as the anterior border of the pelvic cavity.1-5,21,22 The sacral vertebrae begin connecting after puberty. The development of the sacral canal and laminae is incomplete in the presence of any recognized formational defect. Spina bifida and cystitis are co-occurring conditions with this illness, and neurological issues might be seen in this scenario.21 The median sacral crest is created when the spinous processes unite at the midlineOn either side of the median sacral crest lies the sacral groove. The sacral groove is made up of sacral laminae. The sacral groove serves as the origin of back muscles such the multifidi, sacrospinal muscle, and erector spinae. Without the formation of the second sacral lamina, these muscles won’t be able to properly insert to the dorsal surface of the sacrum. Dorsal agenesis is brought on by the sacral lamina’s failure to fuse, and hidden spina bifida is brought on by a defect in the sacral canal’s posterior wall at the level of the second sacral vertebra.21 Caudal anesthesia refers to the injection of the anesthetic into the sacral canal through the sacral hiatus during surgical procedures to be done to the uterine cervix and perineum after birth. Surgery can be done while under anesthesia if an anesthetic solution is applied to the roots of the sacral and coccygeal segments in the sacral canal. Knowing the sacrum morphometry inside and out is crucial in this regard.7 Accordingly, it is claimed that effective caudal epidural anesthesia and analgesia require knowledge of the usual anatomical structure, morphometric values, and variations of the sacrum.6,21,22 Additionally, the sacrum and coccyx are the vertebrae most frequently affected by malformations of the vertebrae connected to the number. A sixth lumbar vertebra, known as lumbarization, can be formed when the S1 vertebra separates from the sacrum and combines with the L5. RESULTS Comparison of measurements of corpus vertebrae and facies auricularis of os sacrum with literature Parameters (mm) μ Literature Population/Year CVAPW 29.71 mm Koç et al.11 Turkey/2004 31.10 mm (female) 29.50 mm (male) Kothapalli et al.12 India/2012 28.50 mm (female) 31.50 mm (male) Sachdeva et al.15 India/2011 29.40 mm Singh et al.16 India/2017 30.50 mm (female) 30.60 mm (male) Kumar and Wishwakarma17 Oman /2015 28.30 mm This study Turkey/2018 CVTW 49.33 mm Koç et al.11 Turkey/2004 47.20 mm (female) 44.60 mm (male) Kothapalli et al.12 India/2012 45.50 mm (female) 47.60 mm (male) Sachdeva et al.15 India/2011 48.00 mm Singh et al.16 India/2017 51.40 mm (female) 53.00 mm (male) Kumar and Wishwakarma17 Oman/2015 47.00 mm This study Turkey/2018 Facies auricularis lengths 32.08 mm (short arm) 52.47 mm (long arm) 59.60 mm (oblique arm) Koç et al.11 Turkey/2004 31.9 mm (short arm) 39.4 mm (long arm) 49.1 mm (oblique arm) This study Turkey/2018 CVAPW; Anterior-posterior width of the corpus vertebrae, CVTW: Transverse width of the corpus vertebrae, μ;mean Table 4. Comparison of os sacrum length, width and index results with the literature... Parameters (mm) μ Literature Population/Year SL 93.49 mm Koç et al.11 Turkey/2014 100.10 mm (female) 98.50 mm (male) Kothapalli et al.12 India/2012 100.10 mm (female) 98.50 mm (male) Janipati et al.13 India/2014 94.46 mm (female) 109.47 mm (male) Patel et al.14 India/2014 106.10 mm Singh et al.16 India/2017 91.80 mm (female) 104.10 mm (male) Sachdeva et al.15 India/2011 93.50 mm (female) 102.70 mm (male) Kumar and Wishwakarma17 Oman/2015 92.50 mm (female) 115.40 mm (male) Mustafa et al.18 Egypt/2012 90.58 mm (female) 107.53 mm (male) Mishra et al.19 India/2003 101.80 mm (female) 103.10 mm (male) Sinha et al.20 India/2013 103.30 mm This study Turkey/2018 SW 108.40 mm (female) 102.20 mm (male) Başaoğlu et al.9 Turkey/2005 111.67 mm Koç et al.11 Turkey/2004 108.20 (female) 101.10 (male) Janipati et al.13 India/2014 106.45 mm (female) 106.42 mm (male) Patel et al.14 India/2014 101.70 mm (female) 103.10 mm (male) Sachdeva et al.15 India/2011 103.10 mm Singh et al.16 India/2017 109.50 mm (female) 99.90 mm (male) Kumar and Wishwakarma17 Oman/2015 115.00 mm (female) 113.60 mm (male) Mustafa et al.18 Egypt/2012 105.79 mm (female) 105.34 mm (male) Mishra et al.19 India/2003 108.40 mm This study Turkey/2018 108.20 mm (female) 101.10 mm (male) Kothapalli et al.12 India/2012 215 J Med Palliat Care. 2023;4(3):211-218 Kabakcı et al. Os sacrum morphometric analysis Table 4. RESULTS Comparison of os sacrum length, width and index results with the literature...(cont.) Parameters (mm) μ Literature Population/Year SI 115.72 (female) 104.08 (male) Kothapalli et al.12 India/2012 115.72 (female) 104.08 (male) Janipati et al.13 India/2014 113.40 (female) 97.61 (male) Patel et al.14 India/2014 111.74 (female) 100.24 (male) Sachdeva et al.15 India/2011 117.35 (female) 97.51 (male) Kumar and Wishwakarma17 Oman/2015 121.70 (female) 100.20 (male) Mustafa et al.18 Egypt/2012 104 This study Turkey/2018 SW; maximum width of os sacrum, SL; maximum length of os sacrum, SI; sacral index, μ: mean able 4. Comparison of os sacrum length, width and index results with the literature...(cont.) ETHICAL DECLARATIONS Ethics Committee Approval: Since the study was performed on dry bones, ethics committee approval is not required, institutional approval was obtained. Informed Consent: Since the study was performed on dry bones, no written informed consent was obtained. Our mean auricular surface of the sacrum length measurements were found to be lower than those found in studies conducted in Turkey. On the other hand, the SL was found to be higher than the measurements in Turkish,11 Oman17 and Indian12,13,20 populations. When the parameter of the SW was compared with the literature, it was found to be higher than the results found in studies conducted by Patel et al.14 Mishra et al.19, Sachdeva et al.15 and Singh et al.16; similar to the results found in studies conducted by Janipati et al.13 and Başaoğlu et al.9 and lower than the results found in studies conducted by Mustafa et al.18 and Koç et al.11 Referee Evaluation Process: Externally peer-reviewed. Conflict of Interest Statement: The authors have no conflicts of interest to declare. Financial Disclosure: The authors declared that this study has received no financial support. Author Contributions: All of the authors declare that they have all participated in the design, execution, and analysis of the paper, and that they have approved the final version. CONCLUSION that is most frequently utilized to determine gender and that best demonstrates gender differences is the sacrum.15 The sacrum morphometric data, according to Kumar and Viscwakarma, are valuable metrics for predicting gender in broken, incomplete, and damaged dry human bones.17 It is thought that factors such as race, gender, age, nutritional status, geographical conditions and the measurement methods used are effective in differences between the data obtained from studies conducted in literature. In the present study, the relationship between vertebral column and pelvis and the sacrum and morphometric values of the sacrum were determined. It can be said that a good knowledge of the variations related with the sacrum, which is at a critical point, will increase the success of surgical interventions in this region in the future and prevent the emergence of future clinical complications. It is also clear that the data obtained as a result of the study will contribute to basic and clinical research that will be conducted on sacrum in the future. In our study, it was found that the FPL1-4 were higher than the FPL1-4. These values were found to be lower than the data in Koç et al.’s11 study in all parameters, except for FDL3 and FDL4 parameters. When our FPL1-4 measurement values were compared with the results found in the study conducted by Duman T6 by using computed tomography, it was found that all parameters except for FPL3 parameter were among the mean values found in female and male population. In our study, mean vertebral body anterior posterior diameter of the sacrum was found to be lower than that of Indian,12,15,16 Oman17 and Turkish11 population. Mean of the CVTW was found to be lower than those found in studies by Koç et al.11 Kumar and Viscwakarma17 and Singh et al.16 DISCUSSION On the other hand, sacralization and the fusion of L5 with the sacrum are both possible.4,5,7,23 Spina bifida or concealed spina bifida, which emerge as a result of Clinicians such as anesthesiologists, radiologists, orthopaedists, and obstetricians should have an extensive knowledge of normal anatomy, morphometric measures, and variations of the sacrum. A structural difference in the sacrum helps in the analysis of sacral spina radiography by radiologists, the estimation of age, gender, and height by forensic scientists, the diagnosis of low back pain by orthopaedists, and the detection of spondylolisthesis by obstetricians.6,21,22 Additionally, accurate diagnosis and administration of treatment in clinical disorders affecting the lumbosacral and sacrococcygeal regions depend on a detailed understanding of sacrum variations.22 Measurement results of the sacrum have been observed to vary between males and women in the literature.6,15,23-25 In terms of gender, it is stated that the bones of men are heavier and larger. The reason why the SI value obtained as a result of dividing the width of the SW is lower in men is due to the fact that the sacrum is longer in men than in women. It has been reported that the sacrum is wider and shorter in women, while promontorium is smaller.5,26 Differences in age, climatic conditions, race, and gender are thought to be the root causes of these sacral variations.15,23,24 The fact that the ala of sacrum is wider in women is due to the increased pelvic volume and the need for a wider pelvic outlet during pregnancy, in addition to an increased sacrovertebral angle and the shifting of apex of sacrum to the posterior at delivery.23 In addition, measurements such as sacral index and subpubic angle were found to be very useful measurements in determining gender differences due to the effect of sex hormones.24,25 One of the bones 216 Kabakcı et al. Os sacrum morphometric analysis J Med Palliat Care. 2023;4(3):211-218 REFERENCES Morphometric study of sexual dimorphism in adult sacra of South Indian population. Int J of Biol Med Res. 2012;3:2076-81. 13. Janipati P, Kothapalli J, Shamsunder RV. Study of sacral index: comparison between different regional populations of India and abroad. Int J Anat Res. 2014;2(4):640-644. 14. Patel S, Nigam M, Mishra P and Waghmare CS. A study of sacral index and its interpretation in sex determination in Madhya Pradesh. J Indian Acad Forensic Med. 2014;36:146-149. 15. Sachdeva K, Singla RK, Kalsey G and Sharma G. Role of sacrum in sexual dimorphisim A morphometric study. J Indian Acad Forensic Med. 2011;33:206-210. 16. Singh SN, Sharma A, Magotra R. Morphological and morphometric study of dry human sacra in Jammu region. JK Sci. 2017;19:154-156. 17. Kumar A, Wishwakarma N. An anthropometric analysis of dry human sacrum:gender discrimination. Int J Sci Res. 2015;4:1305- 1310. 18. Mustafa MS, Mahmoud OM, El Raouf HHA and Atef H. Morphometric study of sacral hiatus in adult human Egyptian sacra: Their significiance in caudal epidural anesthesia. Saudi J Anaesth. 2012;6:350-357. 19. Mishra SR, Singh PJ, Agrawal AK. Identification of sex of sacrum of Agra region. J Anat Soc India. 2003;52:132-136. 20. Sinha MB, Rathore M, Trivedi S and Siddiqui AU. Morphometry of first pedicle of sacrum and its clinical relevance. Int J Healthc Biomed Res. 2013;1:234-240. 21. Vasuki AKM, Sundaram KK, Nirmaladevi, Jamuna M, Hebzibah DJ, Fenn TKA. Anatomical variations of sacrum and its clinical significance. Int J Anat Res. 2016;4:1859-1863 22. Laishram D, Ghosh A, Shastri D. A study on the variations of sacrum and its clinical significance. J Dent Med Sci. 2016;15:8-14. 23. Cheng JS, Song JK. Anatomy of the sacrum. Neurosurg Focus. 2003;15:1-4. 24. Isaac UE, Ekanem TB, Igiri AO. Gender differentiation in the adult human sacrum and the subpubic angle among indigenes of cross river and Akwa Ibom States of Nigeria using radiographic films. Anatomy J Africa. 2014;3:294-307. 25. Patel MM, Gupta BD, Singel TC. Sexing of sacrum by sacral index and kimura’s base-wing index. JIAFM. 2005;27:5-9. 26. McGrath MC, Stringer MD. Bony landmarks in the sacral region:the posterior superior iliac spine and the second dorsal sacral foramina:a potential guide for sonography. Surg Radiol Anat. 2011;33:279–286. 27. Dubory A, Bouloussa H, Riouallon G, et al. Wolff S. A computed tomographic anatomical study of the upper sacrum. Application for a user guide of pelvic fixation with iliosacral screws in adult spinal deformity. Int Orthop. 2017;41(12):2543-2553. doi:10.1007/ s00264-017-3580-5. REFERENCES In addition, when we examine the current studies comparing CT and osteometry, Dubory et al. found that the thickness of the lower part of the Sacral 1 vertebrae body increased in male patients (p<0.001). While no significant difference was found between vertebral body width in men and women in this study, they found a significant difference (p<0.001) in both sexes in terms of vertebral body median diameter and vertebral body height parameters in both osteometry and CT scans.27 In another study, the transverse and vertical diameters of the auricular surface had significant differences (p<0.001) in males and females in both osteometry and CT scans.28 1. Taner D. Fonksiyonel anatomi ekstremiteler ve sırt bölgesi, 4. Baskı. Ankara: HYB Basım Yayın; 2009. 1. Taner D. Fonksiyonel anatomi ekstremiteler ve sırt bölgesi, 4. Baskı. Ankara: HYB Basım Yayın; 2009. 2. Simriti NS, Ashwani Sharma RM. Morphological and morphometric study of dry human sacra in Jammu region. JK Science. 2017;19:154-156. 3. Arıncı K. Elhan A. Anatomi. 3. Baskı. Ankara: Güneş Kitabevi; 2001. 4. Stranding S. Gray’s Anatomy. The anatomical basis of clinical practice. 41st Ed. London: Elsevier; 2016. 5. Hansen JT. Netter’s clinical anatomy. 4th Ed. Newyork: Elsevier; 2018. 6. Duman T. Yetişkinlerde os sacrum’un çok kesitli bilgisayarlı tomografi ile morfometrik incelenmesi. Yüksek Lisans Tezi. Selçuk Üniversitesi, Konya; 2009. 7. Gövsa Gökmen F. Sistematik Anatomi. 1. Baskı. İzmir: Güven Kitabevi; 2003. The limitations of our study were the fact that 32 dry sacrum were used in the study since partly broken, fragmented and damaged bones were not included in the study and the study was conducted with only one department. We recommend such studies to be conducted by bringing together many departments or by using more sacrum. 8. Yıldırım M. Topografik anatomi. 1. Baskı. İstanbul: Nobel Tıp Kitabevi; 2000. 9. Başaloğlu H, Turgut M, Taşer FA, Ceylan T, Başaloğlu HK, Ceylan AA. Morphometry of the sacrum for clinical use. Surg Radiol Anat. 2005;27(6):467-471. doi:10.1007/s00276-005-0036-1 10. Peretz AM, Hipp JA, Heggeness MH. The internal bony architecture of the sacrum. Spine (Phila Pa 1976). 1998;23(9):971- 974. doi:10.1097/00007632-199805010-00001 217 Kabakcı et al. Os sacrum morphometric analysis J Med Palliat Care. 2023;4(3):211-218 11. Koç T, Ertekin T, Acer N, Cinar S. Os sacrum kemiğinin morfometrik değerlendirilmesi ve eklem yüzey alanlarının hesaplanması. Erciyes Üniv Sağ Bil Derg. 2014;23:67-73. 12. Kothapalli J, Velichety SD, Desai V and Zameer MR. REFERENCES 28. Banik S, Mohakud S, Sahoo S, Tripathy PR, Sidhu S, Gaikwad MR. Comparative morphometry of the sacrum and its clinical implications: a retrospective study of osteometry in dry bones and CT scan images in patients presenting with lumbosacral pathologies. Cureus. 2022;14(2):e22306. doi:10.7759/cureus.22306. 218
https://openalex.org/W4388442149	https://iiste.org/Journals/index.php/JEP/article/download/61682/63671	English	null	Engaging Specialized Information and Communication Technology (ICT) Tools in Combating Examination Malpractice in Educational Institutions	null	2,023	cc-by	4,548	www.iiste.org www.iiste.org iste.org Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.29, 2023 Engaging Specialized Information and Communication Technology (ICT) Tools in Combating Examination Malpractice in Educational Institutions Johnson, Idorenyin Edet Department of Curriculum and Teaching (Educational Technology Unit) Faculty of Educational Foundation Studies University of CalabaR, Calabar +2348066128242 E-mail: irandyjohnny@unical.edu.ng Imoke, John Eteng Department of Curriculum and Teaching (Educational Technology Unit) Faculty of Educational Foundation Studies University of Calabar, Calabar +2347033923536 imokeeteng@unical.edu.ng Ugah, Joseph Chuckwudi Arts Education Department Faculty of Education University of Nigeria, Nsukka +2347034597493 Ugaj4dyear@gmail.com Ugah, Joseph Chuckwudi Arts Education Department Faculty of Education University of Nigeria, Nsukka +2347034597493 Ugaj4dyear@gmail.com Ugah, Joseph Chuckwudi Arts Education Department Faculty of Education University of Nigeria, Nsukka +2347034597493 Ugaj4dyear@gmail.com Abstract Educational corruption has both short-and-long term effects on the educational system, and the society at large. It has many ways of manifesting, one of which is examination malpractice. This paper conceptualizes examination malpractice and considers its types, causes and effects. It further puts forward a number of specialized ICT tools that, if well utilized, will help checkmate examination malpractice in our educational institutions to include biometric machine, Closed Circuit Television (CCTV), body scanner, metal detector, signal detector, among others. words:Engaging, specialized Information and Communication Technology (ICT) tools, combatin ination malpractice, educational institutions. DOI: 10.7176/JEP/14-29-06 Publication date:October 31st 2023 1. Introduction. Therefore, examination malpractice refers to any act of compromise perpetuated by any of the examination stake holders, at any point in the examination process, to criminally affect examination outcomes and favor or disfavor candidates of choice. compromise of any kind by the examination stake holders, at any stage of the examination process. Ebong (2022) further pointed out that examination malpractice is a situation where a student, teacher, supervisor, invigilator, officer or parent engages in any illegal act before, during or after a given examination, to the end that some candidates may obtain undeserved scores. Therefore, examination malpractice refers to any act of compromise perpetuated by any of the examination stake holders, at any point in the examination process, to criminally affect examination outcomes and favor or disfavor candidates of choice. This criminal practice has over the years been carried out through both manual and digital means. Bonner (2015) advanced that examination malpractice is no longer only about copying from neighbors, using notes, textbooks and key points, copying on paper, palm, laps and cloths; but now adopts mobile technologies like mobile phones, scientific calculators, invisible ink pens, wrist watches, Mp3 players, wireless receivers ear piece, and printed labels. Examination malpractice looks down on the examination ethics, truth, honor, integrity, credibility and essence, and has brought unpleasant lingering consequences on the students, teachers, educational institutions and system, and the society at large. y y g In view of the immediate and long-term effects of examination malpractice, the government, concerned organizations, groups and individuals have tried frantically to find sustainable and replicable solutions to the problem, by way of setting and enforcing strict rules and other practical strategies, but not so much has changed over time. More worrisome is the advancement in the strategies of examination malpractice which desperately yearns for more proactive measures that can sustainably militate against it. The revolution in Information and Communication Technology (ICT) has contributed immensely to solving problems in every sphere of human endeavor. ICT has been considered to incorporate electronic technologies and techniques to manage information and knowledge, including information-handling tools used to produce, process, store, distribute and exchange information (Johnson & Udo, 2020). According to UNESCO (2023), ICT refers to the scientific, technical, and engineering discipline and management technics used in the management of information, its application and engagement with social, economic and cultural matters. 1. Introduction. Many scholars tend to limit the scope of ICT to strictly information production, storage, processing, distribution and exchange. p y p g p g g However, the influence of ICT is seriously felt across education, business, socio-political and other spheres of human endeavors. Johnson (2017) and Johnson et al., (2021) pointed out that there are ICT tools specially designed for surveillance, data capturing, interpretation, management, storage and sharing of information. This implies therefore that there are specialized ICT tools for combating examination malpractices. Considering the potentialities associated with ICT, one is hopeful that, if ICT tools for combating examination malpractice are adopted and effectively utilized, in addition to the already established measures, they could yield the much- desired change. This paper therefore examines the types, causes and effects of examination malpractice, and recommends specialized ICT tools for checkmating them. 1. Introduction. Instructional evaluation is a pivotal step in the teaching-learning process. It is a means by which the extent of success or otherwise of an instructional exercise is determined. The learning outcomes could be measured at a formative (intermittently) or summative (comprehensively at the end of a course program) level. While formative evaluation could be less cumbersome because of its short-term generalization, summative evaluation which could take the form of internal or external examinations come with so much pressure, challenges and general restiveness, which tend to culminate in some level of compromise by the parties concerned. Umanah (2014) noted that, when summative evaluation is compromised, it makes it unreliable to pass valid judgement about the level of achievement of educational goals and objectives. Naturally, many students nurse phobia for examination, no matter how prepared they may be. The fear of both internal or external examinations, puts students under some kind of pressure, especially when their level of preparation does not guarantee them passing independently. The whole restiveness exhibited before, during and after examinations by students, teachers, school administrators, parents, examination invigilators, custodians of examination materials, officials of the examination and security operatives could culminate in tendencies of examination malpractice (Onabanjo, 2016). According to Onyeto (2014) examination malpractice has assumed an alarming proportion that has almost become a norm across all levels of education. Tyokyaa (2014) added that examination malpractice has become a booming business to the extent that there are fraudsters who make brisk business by engaging in the shameful act. The concept of examination malpractice has been variously defined by different scholars, but with a unanimous line of thought. According to Kingsley (2019) it connotes all forms of cheating which directly or indirectly falsify the ability of the student in or outside the examination hall. Tanah (2021) defined it as an act of 29 Journal of Education and Practice ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.29, 2023 Journal of Education and Practice iste.org ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.29, 2023 compromise of any kind by the examination stake holders, at any stage of the examination process. Ebong (2022) further pointed out that examination malpractice is a situation where a student, teacher, supervisor, invigilator, officer or parent engages in any illegal act before, during or after a given examination, to the end that some candidates may obtain undeserved scores. 1.2. Types of Examination Malpractice yp p For the purpose of this discourse, the types of examination malpractice are broken into before, during and after examination. he purpose of this discourse, the types of examination malpractice are broken into before, during and aft ination. examination. a. Examination malpractice before examination: This refers to irregularities and rules breaking activities that get exhibited prior to the execution of a given examination. They include the following: i. Falsification of candidate’s data and examination entry information. ii. Hiring of mercenary to impersonate and write an examination for a candidate. iii. Breaking into an officer’s office to obtain examination questions or related materials. iv. Assigning examination center of choice to favored candidates for a fee. v. Assigning compromised invigilators and supervisors to favored centers. vi. Selling and buying of examination questions before the examinations. vii. Preparing manual and electronic cheating materials to enhance smooth cheating. viii. Offering money to or terrorizing invigilators or supervisor to ensure compromise. b. Examination malpractice during examination: Many irregularities happen at the instance of an examination to enable candidates gain more marks than they can merit, such as the following: i. Instigating issues to disrupt an examination atmosphere. ii. Sneaking bookish or electronic materials into examination hall to aid copying of answers. iii. Collaborating or copying from the works of others. iv. Giving or receiving assistance to copy. v. Writing answers on the board or reading same to students. vi. Moving dull students that have paid money to sit close to intelligent students and copy. vii. Exchanging and transferring roll numbers and answer booklet to favor some candidates for a fee. viii. Refusal to submit answer booklets at the end of an examination. ix. Exchanging answer scripts during examinations. a. Examination malpractice before examination: This refers to irregularities and rules breaking activities that get exhibited prior to the execution of a given examination They include the following: a. Examination malpractice before examination: This refers to irregularities and rules breaking activities that get exhibited prior to the execution of a given examination. They include the following: i. Falsification of candidate’s data and examination entry information. ii. Hiring of mercenary to impersonate and write an examination for a candidate. iii. Breaking into an officer’s office to obtain examination questions or related materials. iv. Assigning examination center of choice to favored candidates for a fee. v. Assigning compromised invigilators and supervisors to favored centers. vi. 1.4. Effects of Examination Malpractice Apart from the immediate penalties of failure or termination of the students’ academic pursuit, and suspension or total dismissal for staff, there are both short-term and long-term effects of examination malpractice which the student, parents, teachers, academic institutions and system, and the society at large suffers both directly and indirectly. Below are such effects: Apart from the immediate penalties of failure or termination of the students’ academic pursuit, and suspension or total dismissal for staff, there are both short-term and long-term effects of examination malpractice which the student, parents, teachers, academic institutions and system, and the society at large suffers both directly and indirectly. Below are such effects: i. Incompetence and quackery: Many graduates today cannot live up to the expectations of the certificates they hold because they never followed due process to acquire mastery, but cheated and came out with grades and certificates they cannot defend. Challenges always expose their emptiness as they are bound to make terrible blunders. ii. Unemployability: Many graduates are unemployable today because all they have is certificates without commensurate expertise. Due to this, they attend interviews upon interviews but never qualify for a job. Even those who get employed by chance get dropped due to their inability to satisfy the needs they are employed to meet. iii. Inability to fit into the society: A lot of certificate holders end up as societal misfits due to examination malpractice. They get a certificate without the accompanying enlightenment. They can’t even communicate with simple and correct expressions. iv. Tendency for post-criminalities: Most contractors who use substandard materials to execute contracts; most politicians who steal in public offices, and; most persons engaging in corrupt practices later in life must have engaged in examination malpractice at some point while in school (Agbatogun, 2017). This claim is so true as examination malpractice could sow seeds of corruption in individuals, which keep manifesting later in life. g v. Distrust in the educational system: Presently, even after meeting the standard qualifications required by a tertiary institution for an admission or, in an organization for an employment; one must still write an aptitude test or go through an interview because there is no trust in the processes of awarding certificates – a sad situation caused by examination malpractice. v. 1.3. Causes of Examination Malpractice 1.3. Causes of Examination Malpractice There are a number of factors that act as remote and immediate causes of examination malpractice. Alutu and Aluede (2015) observed that race, religious affiliation, nationality and economic status have very weak connection with examination malpractice, but pointed out some of the following as causes of examination malpractice: p i. Parental and peer approval and support for examination malpractice. i. Parental and peer approval and support for examination malpractice. ii. Poor and unclear instructional delivery by teachers. iii. Excessive engagement in extra-curricular activities by students which affects their studies. iv. Poor teaching environment and strategies. v. Conducive environment for cheating before, during and after examinations. vi. Societal pressure on good grades and certificate at the expense of merit. vii. Laziness on the part of the students to read and prepare for examinations. viii. Fear of failure and resultant stigmatization by family and peers. ix. Lack of self-confidence on the part of the students. x. Addiction to cheating by students as the only assurance for passing an examination. xi. Poor class attendance and engagement in academic activities by students. xii. Poor remunerations and incentives for teachers which push them into compromise and mounting pressure on students to pay money for marks. xiii. Insatiable greed by examination officers to keep exploiting examination candidates. xiv. Desperation by students to make high grades. y y iii. Excessive engagement in extra-curricular activities by students which affects their studies. Conducive environment for cheating before, during and after examinations. Societal pressure on good grades and certificate at the expense of merit. Laziness on the part of the students to read and prepare for examinations. Addiction to cheating by students as the only assurance for passing an examination. Addiction to cheating by students as the only assurance for passing an exam Poor class attendance and engagement in academic activities by students. Poor remunerations and incentives for teachers which push them into compromise and mounting pressure on students to pay money for marks. p p y y xiii. Insatiable greed by examination officers to keep exploiting examination candidates. 1.2. Types of Examination Malpractice Conscious omission or misplacement of a candidate’s script. v. Receiving money and sex to exonerate candidate caught in examination malpractice. vi. Award of certificates to underserving persons. 1.2. Types of Examination Malpractice Selling and buying of examination questions before the examinations. vii. Preparing manual and electronic cheating materials to enhance smooth cheating. viii. Offering money to or terrorizing invigilators or supervisor to ensure compromise. b. Examination malpractice during examination: Many irregularities happen at the instance of an examination to enable candidates gain more marks than they can merit, such as the following: i. Instigating issues to disrupt an examination atmosphere. ii. Sneaking bookish or electronic materials into examination hall to aid copying of answers. iii. Collaborating or copying from the works of others. iv. Giving or receiving assistance to copy. v. Writing answers on the board or reading same to students. vi. Moving dull students that have paid money to sit close to intelligent students and copy. vii. Exchanging and transferring roll numbers and answer booklet to favor some candidates for a fee. viii. Refusal to submit answer booklets at the end of an examination. ix. Exchanging answer scripts during examinations. 30 Journal of Education and Practice x. Removal of answer scripts from examination hall for purpose of filling answers. xi. Submitting prepared answer booklets by substitution for the original script. xii Asking and sharing answers among candidates x. Removal of answer scripts from examination hall for purpose of filling answers. xi. Submitting prepared answer booklets by substitution for the original script. xii. Asking and sharing answers among candidates. x. Removal of answer scripts from examination hall for purpose of filling answers. xi. Submitting prepared answer booklets by substitution for the original script. c. Examination malpractice after examination: When an examination is concluded, there are certain “behind the scene” corrupt practices that occur and still end up affecting the outcomes of a given examination. Below are a number of them: i. Offering money or sex to examiners to upgrade the score of candidates. ii. Changing of grades with officers in charge of results after submission by examiners. iii. Generating scores and grades for candidates who never took the examination. iv. Conscious omission or misplacement of a candidate’s script. v. Receiving money and sex to exonerate candidate caught in examination malpractice. vi. Award of certificates to underserving persons. i. Offering money or sex to examiners to upgrade the score of candidates. ii. Changing of grades with officers in charge of results after submission by examiners. iii. Generating scores and grades for candidates who never took the examination. iv. 1.5. Specialized Information and Communication Technology (ICT) Tools for Checking Examination Malpractice Installation of Closed-Circuit Television (CCTV): This is a camera that records every activity within its area of coverage. Such recordings are viewed live or stored up for later playback. If this device is installed and connected to a monitor, an invigilator could be assigned to monitor it while the examination lasts. If candidates know they are being monitored, they will not want to cheat, and in case they do, they will be caught and dealt with according to laid down penalties. vii. Installation of Closed-Circuit Television (CCTV): This is a camera that records every activity within its area of coverage. Such recordings are viewed live or stored up for later playback. If this device is installed and connected to a monitor, an invigilator could be assigned to monitor it while the examination lasts. If candidates know they are being monitored, they will not want to cheat, and in case they do, they will be caught and dealt with according to laid down penalties. y y g g Boom Mics installation: Boom mic is known for amplifying silent sounds. If this device is installed at strategic positions in an examination hall, the most silent whispers by candidates will be amplified and the act of whispering answers discouraged. Boom Mics installation: Boom mic is known for amplifying silent sounds. If this device is installed at strategic positions in an examination hall, the most silent whispers by candidates will be amplified and the act of whispering answers discouraged. Provision of sophisticated ICT equipped examination hall: Most halls designated for examinations are just very conducive for cheating. The school management should build and equip examination halls with partitions and sitting arrangements that make it impossible for candidates to interact or cheat freely. Each seat should have a desktop computer that is functional to allow for computer-based examinations. Installation of CCTV cameras and boom mics at strategic positions in the hall and arrangement for light and ventilation will make the hall examination ready. Again, having only one entrance and one exit makes it easier for screening of candidates for a given examination. Conclusively, examination malpractice, being as old as examination itself has done enormous damage to the students, teachers, parents, educational system, and the society at large. 1.5. Specialized Information and Communication Technology (ICT) Tools for Checking Examination Malpractice Use of biometric equipment to capture images and biometric data of candidates, and issuance of permanent identity cards: This makes it possible for the biometric and facial details of the candidate to appear on a customized identity card issued by the examination authority, which must accompany the students to the examination venue as an examination pass. With this approach, it is impossible to impersonate as every candidate must only be admitted to an examination hall after screening and ascertaining the biometric details. of permanent identity cards: This makes it possible for the biometric and facial details of the candidate to appear on a customized identity card issued by the examination authority, which must accompany the students to the examination venue as an examination pass. With this approach, it is impossible to impersonate as every candidate must only be admitted to an examination hall after screening and ascertaining the biometric details. iv. Utilization of body scanner and metal detector: As a pre-examination screening exercise, every candidate should be checked using the body scanner and metal detector. These devices will help to reveal any mobile device hidden anywhere on the candidate’s body. iv. Utilization of body scanner and metal detector: As a pre-examination screening exercise, every candidate should be checked using the body scanner and metal detector. These devices will help to reveal any mobile device hidden anywhere on the candidate’s body. v. Provision of approved calculator for candidates: Since some scientific calculators have other specialized functions that could aid examination malpractice, simplified calculators should be provided for the candidates to use in examinations that require calculations. v. Provision of approved calculator for candidates: Since some scientific calculators have other specialized functions that could aid examination malpractice, simplified calculators should be provided for the candidates to use in examinations that require calculations. vi. Installation of electronic signal detector: This device helps to detect and prevent any form of electronic signal in form of SMS, phone call or social media chats. With this in place, no mobile gadget inside an examination hall can communicate with anyone outside. vi. Installation of electronic signal detector: This device helps to detect and prevent any form of electronic signal in form of SMS, phone call or social media chats. With this in place, no mobile gadget inside an examination hall can communicate with anyone outside. 1.4. Effects of Examination Malpractice Distrust in the educational system: Presently, even after meeting the standard qualifications required by a tertiary institution for an admission or, in an organization for an employment; one must still write an aptitude test or go through an interview because there is no trust in the processes of awarding certificates – a sad situation caused by examination malpractice. 31 Journal of Education and Practice vi. Distrust in professionalism: Due to examination malpractice also, many people lack trust in professions like medicine, engineering, law and many others. This explains why a person who studies abroad is considered more valuable than a person who studies locally. Also, where professional services are required, one prefers to seek them outside the country, especially when the means of travelling out is available. This is because there is a general belief that those who study in developed climes are likely more professional than those who study at home, due to the high rate of educational corruption at home. 1.5. Specialized Information and Communication Technology (ICT) Tools for Checking Examination Malpractice 1.5. Specialized Information and Communication Technology (ICT) Tools for Checking Examination Malpractice ICT has an inexhaustible list of tools that specialize in carrying out various specialized tasks, either by design or utilization. The following, if well utilized, could help to effectively checkmate examination malpractice in educational institutions: i. Making examination/assessment computer-based: Every examination should be computer-based. Under this arrangement, the examiners must not be responsible for marking and scoring, but should submit their marking guides with scores allocated to the right answers. This will make it impossible for the markers and candidates to have any kind of contact. All assessment must also be submitted to an approved e-mail address to maintain the avoidance of contact between the markers and the students. This will help keep examination questions secret as well. i. Making examination/assessment computer-based: Every examination should be computer-based. Under this arrangement, the examiners must not be responsible for marking and scoring, but should submit their marking guides with scores allocated to the right answers. This will make it impossible for the markers and candidates to have any kind of contact. All assessment must also be submitted to an approved e-mail address to maintain the avoidance of contact between the markers and the students. This will help keep examination questions secret as well. p p q ii. Provision of centralized database for storage of results: To avoid any kind of alteration, results should be readily uploaded on a central database and made available to every quarter so that any alternation at any point will render the result questionable. ii. Provision of centralized database for storage of results: To avoid any kind of alteration, results should be readily uploaded on a central database and made available to every quarter so that any alternation at any point will render the result questionable. should be readily uploaded on a central database and made available to every quarter so that any alternation at any point will render the result questionable. iii. Use of biometric equipment to capture images and biometric data of candidates, and issuance of permanent identity cards: This makes it possible for the biometric and facial details of the candidate to appear on a customized identity card issued by the examination authority, which must accompany the students to the examination venue as an examination pass. With this approach, it is impossible to impersonate as every candidate must only be admitted to an examination hall after screening and ascertaining the biometric details. iii. 1.5. Specialized Information and Communication Technology (ICT) Tools for Checking Examination Malpractice Bearing in mind that it has become a safe means of acquiring certificates for lazy and desperate students and a good business for corrupt officers and teachers, it would take very effective and deliberate effort to combat it. In view of the enormous potentials associated with ICT tools, they could be helpful, if well utilized in taking proactive measures to checkmate every type of examination malpractice. 32 Journal of Education and Practice www.iiste.org iste.org ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.29, 2023 ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.29, 2023 ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.29, 2023 REFERENCES Afutu, A. N. & Afuebe, O. (2015). Secondary school perception of education malpractice ethics. Journal of Humanity and Ecology, 26(6), 195-200. ISSN 2222-1735 (Paper) ISSN 2222-288X (Online) Vol.14, No.29, 2023 REFERENCES y gy ( ) Agbatogun, A. O. (2017). Use and misuses of information and communication technology in examination malpractices. Journal of African Educational Research Network, 9(3), 75-83. Bonner, K. (2015). Digital text, distance education and dishonesty: Faculty and administration perceptions and responses. Journal of Educational Network Technologists, 16(4), 162-170. p f g ( ) Ebong, O. H. (2022). Combating examination malpractice. Journal of global education p f g ( ) g, O. H. (2022). Combating examination malpractice. Journal of global educational issues. 25(6), 155-163 Johnson, I. E. (2017). Digital environmental for information and communication technology in education. Education for Today, Journal of faculty of education, university of Calabar. 13(3), 146-154. f y, y , y ( ), Johnson, I. E. and Udo, A. L. (2020). Adopting Information and Communication Technologies for Effective Inclusive Education. Journal of education and practice, 11 (24), 137-141 Johnson, I. E., Nkanu, C. U. and Udo, A. L. (2021). Checkmating the weaknesses associated with information and communication technologies in education for improved effectiveness and efficiency. Journal of education and practice, 12 (8), 80-85. p ( ) Kingsley, T. T. (2019). The two sides of examination malpractice. Journal of global educational issues. 14(8), 169-176. Onabanjo, I. O. (2016). Examination malpractice: The bane of educational quality in Nigeria. Journal of interactive media in education, 26(3), 58-67. Onyito, O. (2014). Examination fraud and half-baked graduates. Journal of global educational issues. 19(4), 89- 96. Tanah, N. D. (2021). An examination of examination malpractices in Nigerian schools: Issues and challenges. Journal of Public Administration, 43(3), 72-81. yaa, M. K. (2014). Causes and remedies of examination irregularities among external candidates in Niger Journal of the Nigeria society for educational psychologists, 4(2), 233-243. Umanah, U. B. (2014). Instructional evaluation. Lagos: Kasvid Press. (2014). Instructional evaluation. Lagos: Kasvid Pre ( ) g UNESCO (2023). 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https://openalex.org/W2053175161	https://europepmc.org/articles/pmc3547840?pdf=render	English	null	Adaptive Immunity Alters Distinct Host Feeding Pathways during Nematode Induced Inflammation, a Novel Mechanism in Parasite Expulsion	PLOS pathogens	2,013	cc-by	8,850	Abstract This is an open-access article distributed under the terms of the Creative Commons Att unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by a BBSRC PhD studentship and Wellcome Trust grant 068634/Z/02/Z. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: This work was funded by a BBSRC PhD studentship and Wellcome Trust grant 068634/Z/02/Z. The funders had no role and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: john.worthington@manchester.ac.uk Adaptive Immunity Alters Distinct Host Feeding Pathways during Nematode Induced Inflammation, a Novel Mechanism in Parasite Expulsion John J. Worthington1, Linda C. Samuelson2, Richard K. Grencis1, John T. McLaughlin3 1 Manchester Immunology Group, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom, 2 Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America, 3 School of Translational Medicine, School of Medicine, University of Manchester, Manchester, United Kingdom 1 Manchester Immunology Group, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom, 2 Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America, 3 School of Translational Medicine, School of Medicine, University of Manchester, Manchester, United Kingdom PLOS Pathogens \| www.plospathogens.org January 2013 \| Volume 9 \| Issue 1 \| e1003122 Abstract Gastrointestinal infection is often associated with hypophagia and weight loss; however, the precise mechanisms governing these responses remain poorly defined. Furthermore, the possibility that alterations in feeding during infection may be beneficial to the host requires further study. We used the nematode Trichinella spiralis, which transiently inhabits the small intestine before migrating to skeletal muscle, as a biphasic model of infection to determine the cellular and molecular pathways controlling feeding during enteric and peripheral inflammation. Through the infection of genetically modified mice lacking cholecystokinin, Tumor necrosis factor a receptors and T and B-cells, we observed a biphasic hypophagic response to infection resulting from two separate immune-driven mechanisms. The enteroendocrine I-cell derived hormone cholecystokinin is an essential mediator of initial hypophagia and is induced by CD4+ T-cells during enteritis. In contrast, the second hypophagic response is extra-intestinal and due to the anorectic effects of TNFa during peripheral infection of the muscle. Moreover, via maintaining naive levels of the adipose secreted hormone leptin throughout infection we demonstrate a novel feedback loop in the immunoendocrine axis. Immune driven I-cell hyperplasia and resultant weight loss leads to a reduction in the inflammatory adipokine leptin, which in turn heightens protective immunity during infection. These results characterize specific immune mediated mechanisms which reduce feeding during intestinal or peripheral inflammation. Importantly, the molecular mediators of each phase are entirely separate. The data also introduce the first evidence that I-cell hyperplasia is an adaptively driven immune response that directly impinges on the outcome to infection. Citation: Worthington JJ, Samuelson LC, Grencis RK, McLaughlin JT (2013) Adaptive Immunity Alters Distinct Host Feeding Pathways during Nematode Induced Inflammation, a Novel Mechanism in Parasite Expulsion. PLoS Pathog 9(1): e1003122. doi:10.1371/journal.ppat.1003122 Citation: Worthington JJ, Samuelson LC, Grencis RK, McLaughlin JT (2013) Adaptive Immunity Alters Distinct Host Feeding Pat Inflammation, a Novel Mechanism in Parasite Expulsion. PLoS Pathog 9(1): e1003122. doi:10.1371/journal.ppat.1003122 Editor: Judith A. Appleton, Cornell University, James A. Baker Institute of Animal Health, United States of America Editor: Judith A. Appleton, Cornell University, James A. Baker Institute of Animal Health, United States of America Received October 19, 2012; Accepted November 26, 2012; Published January 17, 2013 Copyright: 2013 Worthington et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2013 Worthington et al. Author Summary Infection with intestinal parasites often results in a period of reduced appetite which can result in weight loss; however the factors which control these feeding alter- ations and the reason why they occur is unknown. We used the nematode parasite Trichinella spiralis, which during its life cycle causes intestinal and muscular inflammation, as a mouse infection model to study the factors which alter feeding during infection. We found that the mouse immune response to the parasite was driving two periods of reduced feeding by two distinct immune mediators during the intestinal and muscular periods of infection. Interestingly, the immune system was utilizing a hormone which usually terminates feeding during our daily meals to cause a reduction in weight and fat deposits. Furthermore, we found that a reduction in these fat deposits and their associated hormones actually helped the mouse expel the parasite from the intestine. Hence the immune driven weight loss was actually beneficial to the mouse’s ability to resolve an infection. Our study provides novel insights into how the immune system interacts with feeding pathways during intestinal inflammation and may help us design new strategies for helping people with parasitic infections of the gut. Figure 1. T. spiralis induced enteritis and peripheral myositis produces a CCK dependent and independent bi-phasic hypo- phagia. Food intake of naı¨ve and infected wild-type (A) and CCKlacZ (B) mice, derived via weighing chow daily. Representative CCK (C) and lacZ (D) I-cell staining from day 9 p.i. wild-type and CCKlacZ duodenum respectively. Black arrows indicate I-cells. Black bar = 100 mm. (E) Number of CCK/LacZ positive I-cells/20 VCU in wild-type and CCKlacZ mice as determined from immunohistochemistry or immunofluores- cence respectively. Data (n = 8–10 mice/group) from two independent experiments. , P,0.05; , P,0.01; or , P,0.005 between naı¨ve and infected groups, error bars represent SE of means. doi:10.1371/journal.ppat.1003122.g001 immune-EEC driven alteration in feeding also contributes a protective role during gastrointestinal infection. The hypophagia and resulting weight loss causes a reduction in fat secreted leptin, and the reduction in this hormone, which also acts as an inflammatory adipokine, augments the protective Th2 immune response aiding parasite expulsion. These results highlight the importance of the immunoendocrine axis in the gut during infection induced immunity and provide a biological function and associated mechanism for commonly associated infection induced weight loss. Author Summary These data have wide-acting implications for the biology of gut infection and inflammation, and may inform new leptin-derived therapeutic strategies. Furthermore, the T. spiralis infected mouse presents a novel preclinical platform to study the biological mechanisms affecting food intake in inflammatory disorders, and has the unique potential to experimentally dissociate gastrointestinal from peripheral signals in an individual model. PLOS Pathogens \| www.plospathogens.org Introduction intestine culminating in worm expulsion via a mast cell dependent process [6]. Recently we have observed a hypophagic response during the Th2 driven enteritis induced by T. spiralis infection [7]. However, the full mechanisms controlling hypophagia during enteritis and the precise effects reduced feeding have on immunity to intestinal infection require further elucidation. T. spiralis is experimentally highly attractive since the enteritis fully resolves, but is closely followed by a peripheral inflammatory phase characterized by skeletal muscle invasion and myositis as part of the parasite’s life cycle. Intestinal inflammation is commonly associated with reduced feeding (hypophagia) and weight loss [1,2], yet the mechanisms and underlying principles of these responses is unknown. Infection with the intestinal parasites Ascaris suum and Trichostrongylus colubriformis results in hypophagia that is coupled with an increase in cholecystokinin (CCK) released from I-cells [3,4]; a subset of intestinal epithelial enteroendocrine cells (EECs). Despite only comprising 1% of the epithelium, EECs collectively form the largest mammalian endocrine system. Regulatory peptides and amines are released from EECs in response to luminal nutrients [5] and these peptides signal via vagal afferent fibers to feeding control centers in the brain. These EEC signals in concert with leptin, produced from adipose tissues indicating levels of fat deposits, ultimately control our daily short-term feeding patterns. However, the true biological function and molecular mechanisms that orchestrate the pathways driving hypophagia and weight loss during inflammation have not been addressed. Here, we demonstrate that this two step inflammatory process following T. spiralis infection is mirrored by a biphasic hypophagic response, and mediated by two separate adaptive immune driven mechanisms. We have characterized these two phases using genetically modified mice lacking functional CCK or adaptive immunity and demonstrated that CD4+ T-cells drive I-cell hyperplasia and the resulting CCK is an essential mediator of the initial hypophagia observed during enteritis. Conversely the second phase of hypophagia during skeletal peripheral myositis is CCK-independent but mediated by the anorectic actions of TNFa signaling. Furthermore, we demonstrate for the first time that this The nematode Trichinella spiralis produces a well characterized CD4+ T-cell, Th2 driven transient inflammation in the small January 2013 \| Volume 9 \| Issue 1 \| e1003122 1 Immunoendocrine Cross-Talk Mediates Infection Author Summary The adaptive immune system drives biphasic hypophagia during T. spiralis induced enteritis and peripheral myositis As EEC hyperplasia during inflammation has been previously linked to T-lymphocytes [7,9], the biphasic hypophagia generated by T. spiralis was examined in severe combined immunodeficient (SCID) mice, which lack B and T-cells. SCID mice demonstrated a complete absence of initial hypophagia during T. spiralis induced enteritis and secondary hypophagia during peripheral myositis (Fig. 2A and B). The lack of hypophagia was mirrored by a complete lack of I-cell hyperplasia in parasitized SCID mice (Fig. 2C). This absence of hyperplasia was not seen in all epithelial secretory cells as, concurrent with previous findings [10], indistinguishable goblet cell hyperplasia occurred in infected SCID, adoptively transferred SCID and wild-type animals (Fig. 2D). CD4+ T-cells play a key role in the resolution of T. spiralis infection [11], so to assess if CD4+ T-cells could restore I-cell hyperplasia and hypophagia in SCID mice, CD4+ T-cells (.90% purity; Fig. S2A), were adoptively transferred into SCID recipients before infection. Successful reconstitution was evident from CD4+ splenocytes present post-transfer and via successful worm expul- sion kinetics (Fig. S2B and C).The adoptive transfer of CD4+ T- cells into SCID mice restored I-cell hyperplasia (Fig. 2C) and initial hypophagia during T. spiralis induced enteritis. Recipient mice began to eat less from day 4 p.i., with significant hypophagia at days 6 and 7 (Fig. 2E). This hypophagia was not a direct result of cell transfer alone, as uninfected reconstituted mice displayed no hypophagia (Fig. 2E). Interestingly, the adoptive transfer did not restore the secondary period of hypophagia during the peripheral inflammation induced by T. spiralis (Fig. 2A). Figure 2. Adoptive transfer of CD4+ T-cells to SCID mice restores hypophagia during enteritis. Food intake of naı¨ve and infected wild-type (A), SCID (B) and adoptively transferred SCID mice (E), derived via weighing chow daily. Number of CCK positive I-cells in wild- type, SCID and adoptively transferred SCID mice (C) and number of goblet cells in wild-type SCID and adoptively transferred SCID mice (D); cells/20 VCU accessed via immunohistochemistry and periodic acid- Schiff’s histology staining respectively. Data (n = 4 mice per group) from 2 independent experiments. , P,0.05; , P,0.01; or , P,0.005 between naı¨ve and infected groups, error bars represent SE of means. doi:10.1371/journal.ppat.1003122.g002 Collectively, these data confirm that the biphasic alterations in feeding behavior during T. spiralis induced gastrointestinal and peripheral inflammation is mediated by the adaptive immune system. T. spiralis induced enteritis and myositis induces a biphasic hypophagic response, for which CCK is crucial for the initial period during enteritis Number of CCK positive I-cells in wild- type, SCID and adoptively transferred SCID mice (C) and number of goblet cells in wild-type SCID and adoptively transferred SCID mice (D); cells/20 VCU accessed via immunohistochemistry and periodic acid- Schiff’s histology staining respectively. Data (n = 4 mice per group) from 2 independent experiments. , P,0.05; , P,0.01; or , P,0.005 between naı¨ve and infected groups, error bars represent SE of means. doi:10.1371/journal.ppat.1003122.g002 mice (Fig. 1B). LacZ positive I-cell hyperplasia was indistinguish- able from that of the ‘‘natural’’ I-cell response in wild-type mice (Fig. 1D and E). However, the absence of CCK during this I-cell hyperplasia resulted in the complete absence of initial hypophagia in CCKlacZ mice, despite comparable enteritis (Fig. S1). Even with the absence of CCK and initial hypophagia, CCKlacZ mice still exhibited secondary hypophagia from day 18–19 p.i. and this was comparable to infected wild-type mice (Fig. 1B). The second period of hypophagia occurred in wild-type and CCKlacZ mice despite the resolution of enteritis (Fig. S1) and transpires during the period of larvae encysting within skeletal muscle, representing an extraintestinal inflammatory response to the same biological agent. The lack of I-cell hyperplasia in wild-type mice at day 20 p.i. (Fig. 1E) and the presence of the second phase of hypophagia in CCKlacZ mice (Fig. 1B) confirm this extraintestinal period of hypophagia during myositis as CCK independent. mice (Fig. 1B). LacZ positive I-cell hyperplasia was indistinguish- able from that of the ‘‘natural’’ I-cell response in wild-type mice (Fig. 1D and E). However, the absence of CCK during this I-cell hyperplasia resulted in the complete absence of initial hypophagia in CCKlacZ mice, despite comparable enteritis (Fig. S1). Even with the absence of CCK and initial hypophagia, CCKlacZ mice still exhibited secondary hypophagia from day 18–19 p.i. and this was comparable to infected wild-type mice (Fig. 1B). The second period of hypophagia occurred in wild-type and CCKlacZ mice despite the resolution of enteritis (Fig. S1) and transpires during the period of larvae encysting within skeletal muscle, representing an extraintestinal inflammatory response to the same biological agent. The lack of I-cell hyperplasia in wild-type mice at day 20 p.i. (Fig. 1E) and the presence of the second phase of hypophagia in CCKlacZ mice (Fig. 1B) confirm this extraintestinal period of hypophagia during myositis as CCK independent. Taken together, these data demonstrate a biphasic hypophagia correlating to T. The adaptive immune system drives biphasic hypophagia during T. spiralis induced enteritis and peripheral myositis Furthermore, CD4+ T-cells are identified as the key initiator in I-cell hyperplasia and resulting CCK driven hypopha- gia during T. spiralis induced enteritis. However, the adoptive transfer of functional CD4+ T-cells did not restore the second phase of hypophagia occurring during nurse cell formation- induced myositis. Therefore CD4+ T-cells are not sufficient for this secondary hypophagic period, during T. spiralis induced myositis. T. spiralis induced enteritis and myositis induces a biphasic hypophagic response, for which CCK is crucial for the initial period during enteritis spiralis induced enteritis and peripheral myositis, respectively. Furthermore, increased I-cell function, through the release of CCK, is essential for the initial hypophagia during enteritis, but not the secondary episode during peripheral myositis. T. spiralis induced enteritis and myositis induces a biphasic hypophagic response, for which CCK is crucial for the initial period during enteritis Figure 1. T. spiralis induced enteritis and peripheral myositis produces a CCK dependent and independent bi-phasic hypo- phagia. Food intake of naı¨ve and infected wild-type (A) and CCKlacZ (B) mice, derived via weighing chow daily. Representative CCK (C) and lacZ (D) I-cell staining from day 9 p.i. wild-type and CCKlacZ duodenum respectively. Black arrows indicate I-cells. Black bar = 100 mm. (E) Number of CCK/LacZ positive I-cells/20 VCU in wild-type and CCKlacZ mice as determined from immunohistochemistry or immunofluores- cence respectively. Data (n = 8–10 mice/group) from two independent experiments. , P,0.05; , P,0.01; or , P,0.005 between naı¨ve and infected groups, error bars represent SE of means. doi:10.1371/journal.ppat.1003122.g001 Proximal enteritis induced by T. spiralis has been associated with a period of hypophagia and an increase in CCK and serotonin secreting EECs [7]. Here, mice were examined for alterations in feeding during T. spiralis induced inflammation. Interestingly, a biphasic response in feeding was observed following infection (Fig. 1A). Mice became hypophagic from days 6–10 post infection (p.i.), during the transient period of T. spiralis induced enteritis, and we observed a significant increase in CCK positive I-cells in wild- types (Fig. 1C and E) mirroring hypophagia at days 6 and 9 p.i. Feeding then returned to baseline levels until undergoing a second period of hypophagia from day 18–19 p.i. The secondary period of hypophagia occurs during the period of muscle invasion and peripheral myositis, caused when larvae form the ‘‘nurse cell’’ in which the parasite resides. To further investigate the biological mediators of the hypophagic responses the two phases were mechanistically explored using a panel of genetically modified mouse strains. CCKlacZ mice, which do not express or secrete CCK peptide due to a knock in of a LacZ cassette [8], were infected with T. spiralis and their food intake monitored. Strikingly, the initial period of hypophagia was completely absent in infected CCKlacZ January 2013 \| Volume 9 \| Issue 1 \| e1003122 2 Immunoendocrine Cross-Talk Mediates Infection The secondary period of hypophagia seen during T. spiralis induced peripheral inflammation is caused by the Figure 2. Adoptive transfer of CD4+ T-cells to SCID mice restores hypophagia during enteritis. Food intake of naı¨ve and infected wild-type (A), SCID (B) and adoptively transferred SCID mice (E), derived via weighing chow daily. PLOS Pathogens \| www.plospathogens.org CD4+ T-cell mediated hypophagia results in an augmented protective immune response, through reduction in the pro-inflammatory adipokine leptin Secretory cell hyperplasia during intestinal infection is known to be advantageous during infection. Various goblet and Paneth cell products have been show to have anti-parasitic affects [17,18]. We therefore tested whether I-cell hyperplasia and hypophagia are simply by-products of a parallel switch towards this secretory lineage, or whether I-cell hyperplasia is in itself advantageous during infection. A link between hypophagia, weight loss and immunity is the adipokine leptin; produced mainly by adipose tissue it is a peripheral signal to the body of fat mass deposits but also acts as a pro-inflammatory Th1 cytokine [19]. Therefore reductions in leptin would be anticipated to occur following CCK induced hypophagia and consequent weight loss in this experimental model. Loss of leptin may consequently enhance Th2 immune responses which are protective during nematode infection. CD4+ T-cell mediated I-cell driven hypophagia during enteritis was seen to result in significant weight loss at days 8 and 12 p.i., accompanied by a visible reduction in abdominal fat pads, whereas the brief TNFa driven secondary hypophagia produced no significant alteration in weight at day 20 p.i. (Fig. 4a). This weight loss was correlated with a reduction in serum leptin levels from day 6 p.i. (Fig. 4B). To determine whether alterations in leptin could influence a protective Th2 driven intestinal I immune response, mesenteric lymph node (mLN) cells were polarized towards a Th2 phenotype in the presence or absence of leptin. The addition of leptin resulted in a significant increase in the amount of intracellular pro-inflammatory IFN-y detectable in CD4+ T-cells, as well as a significant reduction in the protective Th2 cytokine IL- 4 (Fig. 4C). To assess if the reduction in leptin during T. spiralis induced enteritis enhances immunity to infection, leptin levels were maintained at basal levels during hypophagia via recombi- nant leptin injection (Fig. 5A). Strikingly, the restoration of basal leptin levels resulted in delayed expulsion of adult worms and a corresponding increase in nurse cell encystation (Fig. 5B). Although no increase in IFN-y levels was seen in re-stimulated mLNs of leptin treated mice, a significant decrease in both Th2 cytokines IL-4 and IL-13 was seen at day 8 p.i (Fig. 5C). A key Th2 driven expulsion mechanism of T. spiralis is mastocytosis and this was seen to be significantly reduced upon the restoration of basal leptin levels analogous to delayed adult worm expulsion (Fig. 5D). CD4+ T-cell mediated hypophagia results in an augmented protective immune response, through reduction in the pro-inflammatory adipokine leptin Taken together this suggests that CD4+ T-cells drive a cascade in which I-cell hyperplasia produces hypophagia and weight loss, lowering pro-inflammatory leptin levels which feed back to influence the protective Th2 immune response, augment- ing mastocytosis and allowing parasite expulsion. Figure 3. Secondary hypophagia during T. spiralis induced peripheral inflammation is absent in p55/p75 2/2 mice. (A) TNFa serum levels during infection in wild-type mice determined via cytometric bead array (B) Number of CCK positive cells/20 VCU, as determined from immunohistochemistry in wild-type and p55/p75 2/2 mice. Food intake of naı¨ve and infected wild-type (C) and p55/p75 2/2 (D) mice, derived via weighing chow daily. Data (n = 7–16 mice per group) are from three independent experiments. , P,0.05; , P,0.01; or , P,0.005 between naı¨ve and infected groups, error bars represent SE of means. doi:10 1371/journal ppat 1003122 g003 The secondary period of hypophagia seen during T. spiralis induced peripheral inflammation is caused by the cachectic cytokine TNFa We next sought to investigate which factors of the adaptive immune response were responsible for the second phase of hypophagia seen during peripheral inflammation induced during the period of nurse cell formation. Both CD4+ and CD8+ T-cells are present during parasite encystation [12] and many apoptotic factors, including TNFa, are detected during nurse cell formation January 2013 \| Volume 9 \| Issue 1 \| e1003122 PLOS Pathogens \| www.plospathogens.org 3 Immunoendocrine Cross-Talk Mediates Infection [13]. Interestingly, TNFa is associated with cachexia in parasite infections [14,15]. Consequently, we examined serum cytokine levels throughout T. spiralis infection. Indeed, TNFa was significantly increased in the serum of infected mice at the time of secondary hypophagia (Fig. 3A), comparable to levels known to directly cause cachexia in mouse infection models [16]. To test the function of increased TNFa during myositis we infected p55/ p752/2 mice, which lack functional TNFa receptors, and assessed if TNFa was responsible for hypophagia during T. spiralis infection. Although initial I-cell hyperplasia and hypophagia during enteritis were present in infected p55/p752/2 mice (Fig. 3B, C and D), remarkably, infected p55/p752/2 mice displayed no period of secondary hypophagia (Fig. 3B and C). Therefore, although the initial CD4+ T-cell and CCK driven hypophagia during enteritis is independent of TNFa, a peripheral peak in TNFa during myositis is functionally responsible for the second phase of hypophagia, via the receptors p55 and/or p75, during T. spiralis induced peripheral inflammation. CD4+ T-cell mediated hypophagia results in an augmented protective immune response, through reduction in the pro-inflammatory adipokine leptin January 2013 \| Volume 9 \| Issue 1 \| e1003122 Discussion , P,0.05; , P,0.01; or , P,0.005 between naı¨ve and infected groups or for indicated comparisons, error bars represent SE of means. doi:10.1371/journal.ppat.1003122.g004 Figure 5. The maintenance of basal leptin levels during T. spiralis infection results in delayed parasite expulsion. (A) Serum leptin levels in PBS and leptin treated mice, determined via ELISA. (B) Adult and larval worm burdens in infected mice. (C) IFN-y, IL-4 and IL-13 cytokine levels from Ag-specific re-stimulation of day 8 p.i. mLN cells from PBS and leptin treated mice, grey lines represent naı¨ve levels; determined via cytometric bead array. (D) Mast cells/20 VCU in PBS and leptin treated mice; accessed via toludine blue staining. Data (n = 9 mice per group) are from two independent experiments. , P,0.05; , P,0.01; or , P,0.005 between naı¨ve and infected groups or for indicated comparisons, error bars represent SE of means. doi:10.1371/journal.ppat.1003122.g005 Figure 5. The maintenance of basal leptin levels during T. spiralis infection results in delayed parasite expulsion. (A) Serum leptin levels in PBS and leptin treated mice, determined via ELISA. (B) Adult and larval worm burdens in infected mice. (C) IFN-y, IL-4 and IL-13 cytokine levels from Ag-specific re-stimulation of day 8 p.i. mLN cells from PBS and leptin treated mice, grey lines represent naı¨ve levels; determined via cytometric bead array. (D) Mast cells/20 VCU in PBS and leptin treated mice; accessed via toludine blue staining. Data (n = 9 mice per group) are from two independent experiments. , P,0.05; , P,0.01; or , P,0.005 between naı¨ve and infected groups or for indicated comparisons, error bars represent SE of means. doi:10.1371/journal.ppat.1003122.g005 Figure 4. Hypophagia and weight loss during T. spiralis induced enteritis reduces the pro-inflammatory adipokine leptin. (A) Change in basal weight during infection. (B) Serum leptin levels during infection, determined via ELISA. (C) Representative CD4+ intracellular IFN-y and IL-4 flow cytometry plots during Th2 polarization in control and leptin treated mLN cells. Data (n = 4–8 mice per group) are from two independent experiments. , P,0.05; , P,0.01; or , P,0.005 between naı¨ve and infected groups or for indicated comparisons, error bars represent SE of means. doi:10.1371/journal.ppat.1003122.g004 Figure 5. The maintenance of basal leptin levels during T. spiralis infection results in delayed parasite expulsion. (A) Serum leptin levels in PBS and leptin treated mice, determined via ELISA. (B) Adult and larval worm burdens in infected mice. Discussion (C) IFN-y, IL-4 and IL-13 cytokine levels from Ag-specific re-stimulation of day 8 p.i. mLN cells from PBS and leptin treated mice, grey lines represent naı¨ve levels; determined via cytometric bead array. (D) Mast cells/20 VCU in PBS and leptin treated mice; accessed via toludine blue staining. Data (n = 9 mice per group) are from two independent experiments. , P,0.05; , P,0.01; or , P,0.005 between naı¨ve and infected groups or for indicated comparisons, error bars represent SE of means. doi:10.1371/journal.ppat.1003122.g005 Figure 4. Hypophagia and weight loss during T. spiralis induced enteritis reduces the pro-inflammatory adipokine leptin. (A) Change in basal weight during infection. (B) Serum leptin levels during infection, determined via ELISA. (C) Representative CD4+ intracellular IFN-y and IL-4 flow cytometry plots during Th2 polarization in control and leptin treated mLN cells. Data (n = 4–8 mice per group) are from two independent experiments. , P,0.05; , P,0.01; or , P,0.005 between naı¨ve and infected groups or for indicated comparisons, error bars represent SE of means. doi:10.1371/journal.ppat.1003122.g004 seems likely a result of the short half-life of loxiglumide as opposed to alternative satiety factors playing an essential role. The previous characterization of CCKlacZ mice demonstrated normal food intake, fat absorption and mass compared to wild-types [21,22]. This coupled with our own observations rules out any underlying defect in feeding of CCKlacZ mice being responsible for the absence of hypophagia during infection. An alternative possibility is that the loss of CCK in brain neurons, rather than gut EECs, underpins the complete absence of hypophagia. However, the persistence of the second phase of hypophagia in CCKlacZ mice and our previous findings involving loxiglumide [7] which does not cross the blood brain barrier, make I-cell hyperplasia and CCK- vagal interactions most likely to mediate gut-induced hypophagia. seems likely a result of the short half-life of loxiglumide as opposed to alternative satiety factors playing an essential role. The previous characterization of CCKlacZ mice demonstrated normal food intake, fat absorption and mass compared to wild-types [21,22]. This coupled with our own observations rules out any underlying defect in feeding of CCKlacZ mice being responsible for the absence of hypophagia during infection. An alternative possibility is that the loss of CCK in brain neurons, rather than gut EECs, underpins the complete absence of hypophagia. Discussion During enteritis food intake is often significantly reduced, perhaps serving to inhibit consumption of contaminated food or to prevent further gut injury. EECs are implicated in this response: elevated I-cell produced CCK and hypophagia have been demonstrated during Ascaris suum and Trichostrongylus colubriformis infection, leading to the hypothesis that CCK was responsible for inflammation induced alterations in feeding [3,4] However, the true biological function and molecular mechanisms that orches- trate the pathways driving hypophagia and weight loss during inflammation have not been addressed. The life cycle of T. spiralis has uniquely allowed us to investigate these questions during both intestinal and peripheral inflammation. Our data demonstrate that these separate inflammatory episodes are mirrored by a biphasic hypophagia driven by two independent immune mediated Figure 3. Secondary hypophagia during T. spiralis induced peripheral inflammation is absent in p55/p75 2/2 mice. (A) TNFa serum levels during infection in wild-type mice determined via cytometric bead array (B) Number of CCK positive cells/20 VCU, as determined from immunohistochemistry in wild-type and p55/p75 2/2 mice. Food intake of naı¨ve and infected wild-type (C) and p55/p75 2/2 (D) mice, derived via weighing chow daily. Data (n = 7–16 mice per group) are from three independent experiments. , P,0.05; , P,0.01; or , P,0.005 between naı¨ve and infected groups, error bars represent SE of means. doi:10.1371/journal.ppat.1003122.g003 January 2013 \| Volume 9 \| Issue 1 \| e1003122 January 2013 \| Volume 9 \| Issue 1 \| e1003122 PLOS Pathogens \| www.plospathogens.org 4 Immunoendocrine Cross-Talk Mediates Infection seems likely a result of the short half-life of loxiglumide as opposed to alternative satiety factors playing an essential role. The previous characterization of CCKlacZ mice demonstrated normal food intake, fat absorption and mass compared to wild-types [21,22]. This coupled with our own observations rules out any underlying defect in feeding of CCKlacZ mice being responsible for the absence of hypophagia during infection. An alternative possibility is that the loss of CCK in brain neurons, rather than gut EECs, Figure 4. Hypophagia and weight loss during T. spiralis induced enteritis reduces the pro-inflammatory adipokine leptin. (A) Change in basal weight during infection. (B) Serum leptin levels during infection, determined via ELISA. (C) Representative CD4+ intracellular IFN-y and IL-4 flow cytometry plots during Th2 polarization in control and leptin treated mLN cells. Data (n = 4–8 mice per group) are from two independent experiments. Discussion However, the persistence of the second phase of hypophagia in CCKlacZ mice and our previous findings involving loxiglumide [7] which does not cross the blood brain barrier, make I-cell hyperplasia and CCK- vagal interactions most likely to mediate gut-induced hypophagia. mechanisms. I-cell hyperplasia and CCK are essential for the initial hypophagia during enteritis, which is orchestrated by CD4+ T-cells. Importantly, we have also identified the second phase of hypophagia during the period of nurse cell formation to be mediated by a separate myositis-induced immune mechanism, fully dependent on the actions of TNFa. T. spiralis infected SCID mice were seen to display I-cell hyperplasia and hypophagia only upon reconstitution of CD4+ T- cells. These data clearly indicate that CCK induced satiety, via vagal afferent fibers signaling to feeding control centres in the brain [5], is an inherent pathway that is utilized by the adaptive immune system to bring about hypophagia and weight loss. This finding corresponds to recent studies showing that CD4+ T-cells restore 5-HT cell hyperplasia in Trichuris muris infected SCID mice [23]. The precise mechanism by which CD4+ T-cells cause EEC hyperplasia during infection remains to be elucidated. EECs have been shown to possess functional TLRs [24] and IL-13 receptors are present on 5-HT cells [9]. However, it has previously been established that during T. spiralis infection in SCID mice NK cells produce ample levels of IL-13 to induce goblet cell hyperplasia [10] yet this IL-13 appears not sufficient to cause EEC hyperplasia. We also detected mRNA for both TNFa receptors p55 and p75 on EECs, yet mice genetically deficient in TNF Furthermore we show for the first time that immune driven weight loss during enteritis results in reduced levels of the Th1 adipokine leptin augmenting a protective Th2 response during infection. Intestinal inflammation is often associated with hypo- phagia and weight loss [1,2] and we have now determined an important immune driven mechanism to explain why this is biologically functional. We have therefore identified a novel molecular pathway and can include I-cell hyperplasia and weight loss as an adaptively driven immune response which, through alterations in leptin, is beneficial during intestinal infection. Our finding that T. spiralis infected mice lacking CCK (CCKlacZ) do not undergo initial hypophagia correlates with our own and other previous findings that a single treatment with the CCK1 receptor antagonist loxiglumide partially restores food intake in parasitized animals [7,20]. PLOS Pathogens \| www.plospathogens.org Immunoendocrine Cross-Talk Mediates Infection Leptin may also directly act on Th2 cytokine production itself as opposed to indirect alterations on Th1 cytokine production [32]. Further study is therefore required to address the leptin-mast cell axis which alters parasite expulsion in our model. As all gut epithelial subtypes are derived from pluripotent crypt stem cells [25], immune cell mediators may alter the transcription factors at the stem cell level leading to altered EEC hyperplasia during infection. Alternatively, analogous alterations in the post- stem cell, neurogenin+, EEC specific progenitor cells could alter EEC proliferation. BrdU labeling studies in experimentally- induced inflammation demonstrated EEC hyperplasia does occur at the level of the stem/progenitor cell rather than fully differentiated epithelial cells [26]. Indeed, the uncoupling of goblet and I cell hyperplasia seen here in infected SCID mice supports the hypothesis that neurogenin+ EEC precursors, rather than the stem cell itself, is targeted by an unknown CD4+ dependent mechanism during T. spiralis driven I-cell hyperplasia. Identifying which factors drive this CD4+ T-cell-stem/progenitor cell interaction is an exciting area for further study. We have identified a novel and specific role for TNFa in hypophagia during T. spiralis induced peripheral inflammation. The absence of the second phase of hypophagia from p55/p75 2/2 mice suggests that the drop in food intake during this extra-enteric inflammatory period is due to the anorexic effects of systemic TNFa or downstream targets. The significant peak of serum TNFa seen in mice correlated with the second period of hypophagia, occurring during nurse cell development and myositis, and strongly supports this notion. Indeed, we observed serum levels comparable to levels known to directly cause cachexia in mouse infection models [16]. Furthermore, TNFa is associated with cachexia in Trypanosoma cruzi infection [14] and during schistosomiasis [15]. There are numerous modes of action by which TNFa could cause anorexia [27]. Although central TNFa levels were not directly monitored, the 20 pg/ml serum TNFa measured during secondary hypophagia is below the levels required to induce anorexia by central administration [28]. It is therefore most likely that TNFa is acting on peripheral afferent nerves, as low level localized cytokine production can trigger afferent nerves without causing an increase in circulating cytokine levels [29]. It is also possible that myalgia and malaise may have contributed to reduced food intake: appetite per se cannot be measured in mice. The observed systemic peak in TNFa occurs during the period of encystation of T. Immunoendocrine Cross-Talk Mediates Infection Immunoendocrine Cross-Talk Mediates Infection receptor signaling demonstrated initial hypophagia and enteritis (Fig. S3A–C) arose independently of its actions. postulated whether I-cell hyperplasia and hypophagia are simply by-products of a parallel switch towards these beneficial secretory lineages or whether I-cell hyperplasia is in itself advantageous in nematode expulsion. Stimulation of the vagus nerve via nutritional release of CCK has also been shown to protect against hemorrhagic shock [31]. Therefore I-cell hyperplasia during nematode infection may represent a previously unidentified anti- inflammatory response. We therefore hypothesized that a reduc- tion in weight as a result of I-cell induced hypophagia may alter the levels of the Th1 adipokine leptin [19]. A reduction in leptin could enhance the protective Th2 immune response to nematode infection. Indeed significant weight loss and reduced leptin levels did occur during T. spiralis induced hypophagia. Recent data on splenocytes demonstrated that leptin alters polarized CD4+ T-cells towards a Th1 phenotype via alterations in proliferation in vitro [32] and we demonstrated parallel results in mLN cells for the first time. Unfortunately CCKlacZ mice have overall reduced basal levels of leptin [33] and were hence unsuitable to study the affect of reduced leptin on intestinal inflammation. We therefore maintained basal leptin levels in infected hypophagic mice and strikingly saw a significant reduction in Th2 cytokines and mastocytosis culminating in delayed worm expulsion. Interestingly mastocytosis was similar in both leptin reconstituted and wild-type mice at day 8 p.i. demonstrating that initially mastocytosis can establish, but without the I-cell driven reduction in Th1 polarizing leptin it is blunted later in infection. These results complement other recent studies in identifying the adipokine leptin as a molecule which can greatly influence the response to infection. Mice lacking the leptin receptor are highly susceptible to infection from protozoa [34], pneumonia [35] and Listeria [36] demon- strating how malnutrition can compromise Th1 driven immunity. However, our data demonstrate that brief alterations in leptin can benefit immunity in terms of Th2 driven resistance to infection. Indeed, a recent study has demonstrated that leptin receptor deficient mice are resistant to experimentally induced Th2- mediated colitis [32]. The precise action of leptin in our studies may be as a direct result of effects on CD4+ T-cell IL-4 production altering mast cell differentiation, proliferation and migration [37] or due to direct effects on mast cells which have recently been shown to express leptin receptors [38]. Immunoendocrine Cross-Talk Mediates Infection spiralis new born larvae in cells of the striated muscle. Encystations are likely to arise from day 4–10 post- infection with rapid growth of the parasite occurring over the following 20 days as terminally differentiated muscle cells re-enter the cell cycle and establish a niche for the parasite [12]. Early non- significant increases in systemic TNFa were seen as early as day 8 post-infection; day 18–21 post-infection may therefore indicate a ‘‘tipping point’’ in peripheral TNFa levels, where significant myositis breaches the threshold required to produce anorexia. TNFa has been shown to be involved in nurse cell formation [13], yet we observed no alteration in nurse cell development in p55/ p752/2 mice that could alternatively explain the observations seen (Fig. S3C–E). The cellular source of TNFa remains to be elucidated. CD4+ and CD8+ T-cells are reported to be present during parasite encystation [12], as are macrophages interestingly peaking during hypophagia [30]. However, as we illustrate here, given the absence of secondary hypophagia in SCID mice reconstituted with CD4+ T-cells, where macrophages are present, the likelihood is that CD8+ T-cells may be the source of cachectic TNFa. Further studies are therefore required to ascertain the cellular source of TNFa which drives the hypophagia during T. spiralis induced myositis. In conclusion, we have identified two separate immune mediated mechanisms of hypophagia during infection induced gastrointestinal and peripheral inflammation, which act via the distinct pathways of I-cell hyperplasia and TNFa cachexia. Furthermore, we demonstrate for the first time an immunoendo- crine feedback loop, in which CD4+ T-cell driven weight loss via CCK reduces leptin levels which impinge on CD4+ T-cell driven effector mechanisms for gastrointestinal infection resolution. Our data elucidate inflammation and weight loss, not just as commonly associated phenomena, but highlights them as a novel immune driven mechanism in parasite expulsion. These data offer potential specific treatment targets to modulate feeding and immune function during inflammatory diseases of the intestine. PLOS Pathogens \| www.plospathogens.org Discussion This partial restoration now January 2013 \| Volume 9 \| Issue 1 \| e1003122 5 January 2013 \| Volume 9 \| Issue 1 \| e1003122 Immunohistochemistry and histology Proximal small intestine was fixed and stained and I-cells were enumerated using, CCK specific, L421 anti-proCCK as previously described [7]. For CCKlacZ detection, transverse 12 mm sections of tissue were cut and fixed in 0.2% glutaraldehyde and stained with X-gal as previously described [8]. Mast or goblet cell sections were stained in toludine blue or Schiff’s reagent, respectively. After mounting, positive cells were enumerated in 20 randomly selected villus crypt units (VCU) and results presented as mean number of positive cells/20 VCU (6 s.e.). Parasites The maintenance, infection and recovery of T. spiralis were carried out as previously described [40]. Food intake and body weight Two experimental groups were compared using Student’s t-test. Three or more groups were compared using the Kruskal-Wallis test, Dunn’s multiple comparison post-test. A p value of #0.05 was considered statistically significant. , P,.05; , P,.01; or , P,.005 for indicated comparisons, error bars represent SE of means. Mice were individually weighed on a daily basis. Food intake per mouse was derived by weighing the chow (B and K, Hull, UK) daily. Kingdom Home Office Scientific Procedures Act (1986) under Department for Environment, Food and Rural Affairs license. In vitro Th2 polarization of mLN cells 6 p 26106/ml mLN cells were stimulated via 5 mg/ml aCD28, 3 mg/ml aCD3 (BD) and polarized via 50 ng/ml IL-4 (Peprotech), 50 mg/ml anti-IFN-c with/without 500 ng/ml recombinant leptin. At 120 hrs 1 mg/ml Brefeldin A/1 mg/ml monensin (Sigma-Aldrich) for IFN-y/IL-4 staining was added for 4 hrs before blocking with anti-FccR (BD). Cells were stained for CD4 (BD) for 30 mins at 4uC before fixing in FACS fix buffer (1% formaldehyde, 0.1% BSA and 0.05% NaN3 in PBS). Cells were permeabilised in 0.1% saponin (Sigma-Aldrich) and stained with biotinylated anti-IFN-c/anti-IL-4 (BD) for 25 mins at RT. Controls were stained with isotype controls (BD). Biotinylated antibodies were detected by streptavidin APC conjugate (Caltag) at 1/200 in saponin for 25 minutes RT. Cells were analyzed on a FACScalibur using Flowjo. Animals Male C57BL/6 and BALB/c mice were obtained from Harlan- Olac Ltd. CCKlacZ mice have a LacZ cassette knocked into the CCK locus on a C57BL/6 background, so homozygote animals are CCK null but faithfully express LacZ in the I cell population [8]. TNFa receptor null p55/p75 2/2 (C57BL/6 background) and severe combined immunodeficient mice (SCID, BALB/c background) were generated as previously described [8,39]. Isolation and in vitro restimulation of mLN cells 6 Isolation and in vitro restimulation of mLN cells mLN cells at 56106 cells/ml in complete media received 50 mg/ ml of T. spiralis antigen (Ag). Supernatants were collected after 24 hrs and cytokines measured using a cytometric bead array kit (BD). Figure S3 Enteropathy and nurse cell formation during T. spiralis infection is not altered in p55/p752/2 mice. (A) Comparison of crypt and villus length in wild-type and p55/ p752/2 mice at naı¨ve, 6 and 21 days p.i., quantified using Image J software. (B) Number of goblet and (C) mast cells/20 VCU in wild-type and p55/p752/2 mice in naı¨ve, 6, 12 and 21 days p.i.; accessed via periodic acid-Schiff’s and toludine blue histology staining respectively. (D) Number of nurse cells visible in field of view in the diaphragm and rectus femoris of infected mice at 21 and 32 days p.i. (E) Cellular infiltration of eosinophils of nurse cells in the diaphragm and rectus femoris of infected mice at days 21 and 32 p.i. (F) Representative haematoxylin and eosin stained images from (E) Black bar = 100 mm. A–D determined via haematoxylin and eosin histological staining. A–F values represent the means 6 SE (n = 4) from 2 independent experiments. , Supporting Information Figure S1 Enteritis in Wild-type and CCKlacZ mice is comparable during T. spiralis infection. (A) Comparison of crypt and villus length in wild-type and CCKlacZ mice at naı¨ve, 6, 12 and 20 days p.i. mice, quantified using Image J software. (B) Number of goblet and (C) mast cells/20 VCU in wild-type and CCKlacZ mice in naı¨ve, 6, 12 and 20 days p.i.; accessed via periodic acid-Schiff’s and toludine blue histology staining respectively. Data (n = 4–8) from 2 independent experiments. , P,0.05; , P,0.01 or *, P,0.005 between naı¨ve and infected groups, error bars represent SE of means. (TIF) Ethics statement Immune mediated secretory cell hyperplasia during intestinal infection is advantageous as goblet and Paneth cell products have been show to have anti-parasitic affects [17,18]. We therefore Mice were housed in specific pathogen free conditions and experiments were carried out in accordance with the United January 2013 \| Volume 9 \| Issue 1 \| e1003122 6 Immunoendocrine Cross-Talk Mediates Infection Immunoendocrine Cross-Talk Mediates Infection Immunoendocrine Cross-Talk Mediates Infection Kingdom Home Office Scientific Procedures Act (1986) under Department for Environment, Food and Rural Affairs license. Serum cytokine detection Serum was obtained from blood at the time of sacrifice via centrifugation at 150006g and cytokines measured using a cytometric bead array kit (BD). Leptin ELISA Mouse leptin ELISA (Linco) was used to detect mouse serum leptin according to manufacturer’s instructions. Adoptive transfer of CD4+ T-cells to SCID mice Adoptive transfer of CD4+ T cells to SCID mice Mesenteric lymph node (mLN) cells were prepared from day 7 p.i. BALB/c mice, in RPMI-1640, supplemented with 10% fetal calf serum, 100 mg/ml penicillin/streptomycin and 1 mM L- glutamine (complete media). CD4+ T-cells were isolated via negative selection using an isolation kit (Miltenyi Biotec). Evaluation of CD4+ purity was via flow cytometry. SCID mice received 46106 cells in P.B.S. via intraperitoneal (i.p.) injection 2 days before infection. Figure S2 Conformation of adoptive transfer of CD4+ T- cells in SCID mice. Flow cytometry plot of (A) MACS purified donor CD3/CD4+ T-cells from day 6 p.i. wild-type mice and (B) representative plot of reconstitution into SCID mice on day 8 recipient splenocytes. Numbers represent percentage of cells in gate of overall lymphocyte gated cells. (C) Worm burdens recovered from wild-type, SCID and SCID adoptively transferred mice at days 6 and 20 p.i. Data (n = 4 mice per group). , P,0.05; , P,0.01 or , P,0.005 for the indicated comparisons, error bars represent SE of means. (TIF) Figure S2 Conformation of adoptive transfer of CD4+ T- cells in SCID mice. Flow cytometry plot of (A) MACS purified donor CD3/CD4+ T-cells from day 6 p.i. wild-type mice and (B) representative plot of reconstitution into SCID mice on day 8 recipient splenocytes. Numbers represent percentage of cells in gate of overall lymphocyte gated cells. (C) Worm burdens recovered from wild-type, SCID and SCID adoptively transferred mice at days 6 and 20 p.i. Data (n = 4 mice per group). , P,0.05; , P,0.01 or , P,0.005 for the indicated comparisons, error bars represent SE of means. References 1. Hartman C, Eliakim R, Shamir R (2009) Nutritional status and nutritional therapy in inflammatory bowel diseases. World Journal of Gastroenterology 15: 2570–2578. of Physiology-Regulatory Integrative and Comparative Physiology 294: R803– R810. 22. 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https://openalex.org/W4234348575	https://bmchealthservres.biomedcentral.com/track/pdf/10.1186/s12913-020-05695-4	English	null	Synergy of policies to strengthen primary care: evidence from a national repeated cross-sectional study	Research Square (Research Square)	2,020	cc-by	8,654	© 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. Jin et al. BMC Health Services Research (2020) 20:865 https://doi.org/10.1186/s12913-020-05695-4 Jin et al. BMC Health Services Research (2020) 20:865 https://doi.org/10.1186/s12913-020-05695-4 Open Access Abstract Background: People bypass primary healthcare (PHC) institutions to seek expensive healthcare at high-level hospitals, leading to escalating medical costs and inefficient use of resources. In 2009, China launched nationwide synergic policies on primary care strengthening, to tackle access to healthcare and financial protection. This study aimed to assess the impact of the two policy areas, health insurance and health workforce, on healthcare seeking behavior. Methods: Drawing on national survey data before (2008) and after (2013) the policies, we linked individual-level data on healthcare-seeking behavior with county-level data on health workforce and health insurance. We constructed a multilevel zero-inflated negative binomial regression to examine the impacts of average reimbursement rate (ARR) of health insurance and the density of registered physicians on outpatient/inpatient visits, and multilevel multinomial logistic regression for the impacts on choice of outpatient/inpatient care providers. Results: Although the increase in health insurance ARR and physician density have positive impacts on individuals’ healthcare use, their impacts might be weakened during 2008 and 2013, and the negative impacts of investment of those in PHC institutions on likelihood of visiting hospitals was larger. The negative impacts of ARR at PHC institutions on likelihood of visiting county-, municipal- and higher-level hospitals in 2013 was 28 percentage points, 66 percentage points and 33 percentage points larger than these in 2008. Conclusions: Primary care strengthening requires synergic policies. Effective mechanisms for coordination across multisectoral actions are necessities for deepening those policies to ensure efficient delivery of healthcare without experiencing financial risks. Keywords: Health insurance coverage, Health workforce availability, Synergic policies, Primary care strengthening Synergy of policies on interrelated health systems function are warranted to strengthen PHC, especially for low- and middle-income countries (LMICs). Health financing and health workforce are two important policy areas. Health- care seeking behavior refers to individuals’ use of health services to meet their health demands, and includes choosing from a range of services and optional healthcare providers [1]. Given that people desire good quality care at a low price, quality and price of health services are two important aspects for improving individuals’ healthcare seeking behaviors. Synergy of policies to strengthen PHC Synergy of policies to strengthen primary care: evidence from a national repeated cross-sectional study Yinzi Jin1,2, Jin Xu2, Weiming Zhu2, Yaoguang Zhang3, Ling Xu3 and Qingyue Meng1,2* Background As Governments strive to progress towards the Sustain- able Development Goals including Universal Health Coverage, concerted efforts are being made to strengthen primary healthcare (PHC) so that people have access to quality health services without experiencing financial risks. * Correspondence: qmeng@bjmu.edu.cn 1School of Public Health, Peking University, 38 Xue Yuan Road, Haidian District, Beijing 100191, China 2China Center for Health Development Studies, Peking University, Box 505, 38 Xue Yuan Road, Haidian District, Beijing 100191, China Full list of author information is available at the end of the article Page 2 of 10 Jin et al. BMC Health Services Research (2020) 20:865 Jin et al. BMC Health Services Research (2020) 20:865 Medical Scheme (NRCMS), Urban Employee-based Medical Insurance (UEBMI), Urban Resident-based Medical Insurance (URBMI), and Urban and Rural Resident-based Medical Schemes (URRMS). Per capita fund for resident-based SHI increased from ¥100 in 2008 to ¥700 in 2018, about 70% from government sub- sides [10]. During the period of 2008 to 2017, people’s demand for health services was rapidly increased, with the outpatient and inpatient services utilization in- creased by 0.9 and 2.5 times, respectively [10]. But the proportion of outpatient and inpatient care provided by PHC institutions decreased by 9.6 and 10.0% points from 2008 to 2017, respectively [10]. Patients continue to by- pass PHC system to seek expensive healthcare at high- level hospitals. As a result, although the proportion of out-of-pocket payments for healthcare decreased, the fi- nancial burden of using healthcare did not fall much, es- pecially for poor households [11]. should focus on the quality and price of health services, guiding patients to choose appropriate healthcare pro- viders for specific health services. Therefore, evaluating the changes of healthcare seeking behavior before and after the policies is of crucial importance to better under- stand the impact of the synergy of the policies—how health financing and health workforce promote the PHC strengthening—are worthy of thinking. Before China’s health system reform in 2009, affected by the economic reforms since the 1970s, the health sys- tem had been once criticized for a massive reduction in financial health protection and substantial increases in out-of-pocket expenses. A large proportion of the popu- lation could not afford the healthcare they needed [2]. In 2009, China launched a nationwide comprehensive health system reform to improve affordable access to quality care [3]. Two major kinds of policies on primary care strengthening have been implemented. The first one is financing policy, related to the price of health ser- vices, with aims to expand healthcare coverage and the benefit package of the social health insurance schemes (SHI) for the population. SHI have set a gradient reim- bursement rate where the rate at PHC institutions is higher than that at higher-level hospitals [4]. p y p As two interrelated policy areas, health insurance coverage and health workforce availability have potential impacts on healthcare seeking behavior [12, 13]. The majority of studies in LMICs have focused on single di- mension of health systems, for example, health financing mechanisms or strengthening of health workforce. Sev- eral studies focusing on strengthening PHC have qualita- tively examined the impact of health systems approach for PHC delivery. However, to our knowledge, no studies have yet quantitively examined the impact of the synergy of policies (combined health system interventions) re- lated to strengthening PHC on healthcare seeking behav- ior. Moreover, there is a value for data on policies at the county level because many health system reform policies are designed and implemented at this local level. To fill this research gap, this study aimed to estimate the im- pacts of the synergy of the policies, including health in- surance coverage and health workforce, on individual’s health seeking behavior, and whether these impacts dif- fered before and after the synergy of policies. Another policy is focused on strengthening the avail- ability of the PHC providers. Lack of qualified health workers is one of the causes for the poor quality of health services. In 2018, 25 and 42% of PHC providers in urban and rural areas, respectively, had less than a junior medical college level of education (the require- ment for a licensed assistant physician) [5]. Therefore, enhancement of the availability of PHC physicians is es- sential for improving quality of PHC. The basic public health service program aims to deliver an essential pub- lic health services package to every Chinese citizen, in which governments subsidize the PHC providers based on the number of covered residents and the perform- ance for service delivery. The national essential drug sys- tem aims to control the over-prescribing drugs, through eliminating mark-ups on drugs dispensed by PHC sys- tem. The local government has increased the budgets and introduced fixed salaries for PHC providers, to com- pensate for loss of income from drug sales [6]. Addition- ally, the local government has issued pay-for- performance scheme for PHC providers, a financial in- centive which links part of income of PHC providers to the quality of their services, to attract qualified PHC providers [7, 8]. Chinese government has invested in health financing and health workforce in an integrated and systemic way, which are profoundly changing ways in which healthcare is financed and delivered [9]. Health insurance reimbursement and health workforce availability From 2008 to 2013, the health insurance ARR increased from 48 to 64%. The ARRs at county hospitals (40% in 2008 and 52% in 2013) were lower than these at higher level hospitals (45% in 2008 and 57% in 2013). The phys- ician density in municipal- and higher-level hospitals in- creased by 1.21 compared with 0.73 in county hospitals and 0.03 at PHC institutions, revealing a widening gap between PHC institutions and hospitals from 2008 to Measures and covariates Healthcare seeking behaviors were measured by the num- ber of outpatient/inpatient visits and the type of health- care providers visited by outpatients/inpatients, including village/community health stations, township/community health centers, county hospitals, and municipal- or higher-level hospitals. PHC institutions refer to village clinics and township health centers in rural areas, and community health stations and community health centers in urban areas. In China, township/community health centers and hospitals offer inpatient services [14]. We measured health workforce density using the num- ber of registered (assistant) doctors per 1000 population at county hospitals and PHC institutions. Health insurance coverage was measured by the SHI average reimburse- ment rate (ARR) of inpatient care at county hospitals and PHC institutions. ARRs were calculated using the mean of actual reimbursement rates for all inpatient services. We used ARR because inpatient reimbursement depends on patient copayment, the official reimbursement rate, and the ceiling. We also considered the variation of services and drug packages included in the SHI, which made the ARR for inpatient services more representative of the practical degree of SHI generosity. Healthcare seeking behaviors of study population Healthcare seeking behaviors of study population Table 1 showed the health needs and healthcare seeking behaviors before and after the synergic policies on pri- mary care strengthening. For health needs, 18.9 and 24.1% of adults reported a sick within 2 weeks, and 24.1 and 33.1% had any chronic disease, respectively in 2008 and 2013. Among those in a need for outpatient care, 39.1% took outpatient care, 27.1% took self-medication, 23.3% continued treatment that took two weeks, and 10.6% took no treatment in 2008; while the proportions of those were 37.2, 14.1, 47.2 and 1.4% in 2013. The ad- missions within 1 year rose from 6.8% in 2008 to 9.0% in 2013. The proportion of patients choosing healthcare in- stitutions within the county (PHC institutions and county hospitals) decreased from 76.9% in 2008 to 72.6% in 2013. Characteristics of samples were shown in Ap- pendix Table 1. Based on Andersen’s model [15–18] and empirical re- search [19–22], we controlled for variables that may act as potential confounders. We divided controlled factors into four components: predisposing factors, enabling fac- tors, health needs, and environmental indicators. Predis- posing factors included age (continuous variable), sex (male/female), marital status (single/married/divorced or widowed), education (no formal education/primary school/junior and high school/junior college and higher- level college), occupation (farmers/unemployed or re- tired/informal employed/formal employed). Enabling factors included income (continuous variable), health in- surance status (NRCMS/UEBMI/URBMI/URRMS/none), distance to the nearest healthcare provider (less than 2 km/2–4 km/4- km and farther). Health needs included sickbed days for illness (continuous variable), presence of chronic diseases (yes/no). Environmental indicators included residence location (rural/urban). Data and sample This study used two nationwide databases and linked individual-level data for the healthcare seeking behav- iors, demographics, and socioeconomic characteristics with county-level data for health workforce and health insurance. Individual-level data were drawn from the China National Health Service Surveys (NHSS), which covered 94 counties with 177,501 respondents before (in 2008) and 156 counties with 273,687 respondents after (in 2013) the policies on primary care strengthening. The NHSS is a nationally representative survey that used four-stage stratified random cluster sampling. County- level data were reported by the health administrative de- partments of the counties sampled by the NHSS in 2008 The financing policy has made remarkable achieve- ments. By the end of 2017, 95% of the population are covered by SHI, including New Rural Cooperative Page 3 of 10 Page 3 of 10 Jin et al. BMC Health Services Research (2020) 20:865 and 2013. Both databases are managed by the National Health Commission (previously the Ministry of Health). In this study, individual- and county-level data were in- terconnected through administrative division codes. the data type of the dependent variables and the number of outpatient visits with extra zeros and over-dispersion, multilevel zero-inflated negative binomial (ML_ZINB) regression was used to investigate the impact of health insurance and health workforce on outpatient/inpatient visits [23, 24]. Multilevel multinomial logistic (MML) re- gression was used to estimate the impact on outpatient/ inpatient choice of healthcare providers, because the dependent variable was categorical random variable [25, 26]. In constructing the model, we assumed that individ- uals maximize their utilities through their decision- making processes [27]. For both of the regressions, two models were fitted: Model 1 regressed each outcome variable on health insurance, health workforce by year; Model 2 added additional regressors of the interaction terms between year and health insurance, year and health workforce. The sign of interaction term in Model 2 could be interpreted as whether the impacts differed before and after health system reform. Impact sizes as the result of the ML_ZINB regressions were presented as incidence rate ratio (IRR), whereas impact sizes of the MML regressions were expressed as relative risk ratio (RRR) [28]. All statistical analyses were performed using Stata 14.0. Health needs Admissions within 1 year (mean%[mean ± SD]) respectively. The rising health insurance ARR and phys- ician density at PHC institutions were positively and sig- nificantly associated with healthcare use. After adding the interaction terms with year, the IRR of interaction terms were lower than 1 (p < 0.001). In other words, compared to 2008, the positive impacts of ARR and physician density on increasing outpatient and inpatient visits dropped significantly in 2013, after controlling for other covariates (Appendix Table 2). Appendix Table 4 shows the results of regressions before and after China’s health system reform, respectively. 2013 (Table 2). Counties with low physician density at PHC institutions had lower proportion of outpatient visits at PHC institutions than those with high physician density both in 2008 and 2013 (Fig. 1). Statistical analysis We compared the healthcare seeking behaviors, and the health insurance reimbursement and health workforce availability between 2008 and 2013. To account for un- measured variations within counties, we applied multi- level random intercept analysis at individual level. Given Jin et al. BMC Health Services Research (2020) 20:865 Page 4 of 10 Table 1 Health needs and healthcare seeking behaviors before and after China’s health system reform 2008 2013 Relative change (%) P value Health needs Prevalence within 2 weeks (mean‰[mean ± SD]) 18.9 (15.1, 22.8) 24.1 (19.9, 28.3) 27.5% < 0.001 Prevalence of chronic diseases (mean‰[mean ± SD]) 24.1 (20.4, 27.8) 33.1 (29.0, 37.2) 37.3% < 0.001 Outpatient healthcare seeking behavior Outpatient visits within 2 weeks (mean%[mean ± SD]) 14.5 (8.4, 20.6) 13.0 (7.9, 18.1) −10.3% 0.105 healthcare-seeking behaviors for people in a need for outpatient care (%) < 0.001 Outpatient care 39.1 37.2 −4.8% Self-medication 27.1 14.1 −48.0% Continued treatment that took two weeks before 23.3 47.2 102.6% No treatment 10.6 1.4 −86.8% The percentage of non-treatment or self-medication due to financial difficulties (%) 24.4 13.6 −44.3% < 0.001 Percentage of outpatient healthcare provider (%) < 0.001 Village/community healthcare stations 49.5 50.2 1.4% Township/community healthcare centers 24.2 22.4 −7.4% County hospitals 17.3 16.9 −2.3% Municipal and higher-level hospitals 8.9 10.5 18.0% Inpatient healthcare seeking behavior Admissions within 1 year (mean%[mean ± SD]) 6.8 (6.5–7.1) 9.0 (8.6–9.4) 32.4% < 0.001 The percentage of people reported a need for admission but did not receive inpatient care (%) 25.1 17.1 −31.9% < 0.001 The percentage of non-hospitalized due to financial difficulties (%) 70.3 43.2 −38.5% < 0.001 Percentage of inpatient healthcare provider (%) < 0.001 Township/community healthcare centers 28.7 21.0 −26.8% County hospitals 48.2 51.6 7.1% Municipal hospitals 11.9 17.9 50.4% Provincial hospitals 8.2 7.3 −11.0% N 177,501 273,687 – ealth needs and healthcare seeking behaviors before and after China’s health system reform Impact of health insurance and health workforce before and after the synergic policies Table 3 shows the results of ML_ZINB regressions. After adjusting for potential confounders, a higher ARR at PHC institutions, physician density at PHC institutions and physician density at county hospitals were associated with a 19% (IRR = 1.19, 95% CI = 1.12–1.27, p < 0.001), 13% (IRR = 1.13, 95% CI = 1.04–1.22, p < 0.001) and 38% (IRR = 1.38, 95% CI = 1.29–1.48, p < 0.001) higher out- patient visits in 2008; while in 2013, the IRRs were 1.04 (95%CI = 0.99–1.10, p < 0.001), 1.11 (95%CI = 1.05–1.18, p < 0.001) and 1.08 (95%CI = 1.02–1.15, p < 0.001), Table 4 shows the RRRs for MML regressions. The ris- ing health insurance ARR and physician density at PHC institutions were negatively and significantly associated with the probability of visiting county and higher-level hospitals. When the ARR at PHC institutions increased by 1 percentage point, the likelihood of inpatients Jin et al. BMC Health Services Research (2020) 20:865 Page 5 of 10 Table 2 Health insurance and health workforce before and after China’s health system reform 2008 2013 Urban Rural Total Urban Rural Total Health insurance health insurance coverage (%) 66.20 91.90 87.10 91.60 97.30 95.20 ARR at PHC institutions, Mean ± SD 0.52 (0.34, 0.71) 0.47 (0.25, 0.69) 0.48 (0.26, 0.71) 0.64 (0.41, 0.86) 0.66 (0.44, 0.89) 0.64 (0.42, 0.86) ARR at county hospitals, Mean ± SD 0.45 (0.34, 0.56) 0.35 (0.24, 0.46) 0.40 (0.27, 0.53) 0.58 (0.49, 0.67) 0.48 (0.40, 0.55) 0.52 (0.44, 0.60) ARR at higher level hospitals, Mean ± SD 0.45 (0.27, 0.63) – 0.45 (0.27, 0.63) 0.57 (0.41, 0.74) – 0.57 (0.41, 0.75) Health workforce Physicians density at PHCs, Mean ± SD 0.46 (0.42–0.50) 0.39 (0.35–0.43) 0.41 (0.36–0.46) 0.56 (0.51–0.61) 0.37 (0.34–0.40) 0.44 (0.40–0.48) Physicians density at county hospitals, Mean ± SD 1.96 (1.89–2.03) 0.55 (0.51–0.59) 0.74 (0.68–0.80) 2.62 (2.49–2.75) 0.80 (0.76–0.84) 1.47 (1.43–1.51) Physicians density at higher level hospitals, Mean ± SD 3.68 (2.56, 4.80) – 3.68 (2.56, 4.80) 4.89 (3.25, 6.53) – 4.89 (3.25, 6.53) N 35 59 94 78 78 156 Table 2 Health insurance and health workforce before and after China’s health system reform N In this study, several key findings were highlighted. First, increasing health insurance ARR and physician density at PHC institutions were associated with more outpatient visits and admissions and more likelihood of visiting PHC institutions. Impact of health insurance and health workforce before and after the synergic policies The results of inter- action terms indicated that the negative impact of ARR at PHC institutions on likelihood of visiting county-, municipal- and higher-level hospitals in 2013 was 28, 66 and 33% larger than these in 2008. Similar patterns were also observed for physician density at PHC institutions and county hospitals (Appendix Table 3). Appendix Table 5 shows the results of regressions among out- patient care in 2008 and 2013, respectively, and Appen- dix Table 6 shows the results of regressions among inpatient care. Impact of health insurance and health workforce before and after the synergic policies Previous studies have found that introduction of PHC providers would lead to a shift of care from specialists to primary care for disease man- agement in different settings [30–34]. In addition, stud- ies in Sweden and Mexico have indicated that government financial investment in PHCs increased the number of visits to PHCs [12, 35]. The majority of stud- ies have focused on single policies (e.g. health insurance or investment in health workforce). However, few have investigated the efforts in a synergic way which contrib- uted to improving use of PHC of a large proportion of the world’s population [36]. Strengthening primary care requires national actions in multiple interrelated health systems policy areas [37]. Increasing the use of PHC may be dependent on improvements in service delivery, including the management of health workforce, as well as in financing. Recognizing these interdependencies makes the task of designing or reforming systems a com- plex one, but is critical for a systemic approach to pri- mary care strengthening. choosing county-, municipal- and higher-level hospitals decreased by 25% (RRR = 0.75, 95% CI = 0.57–0.99, p < 0.001), 45% (RRR = 0.55, 95% CI = 0.37–0.82, p < 0.001) and 46% (RRR = 0.54, 95% CI = 0.36–0.82, p < 0.001), re- spectively, in 2008; while in 2013, the RRRs were 0.54 (95%CI = 0.44–0.66), 0.19 (95%CI = 0.15–0.24), and 0.37 (95%CI = 0.27–0.50), respectively. The results of inter- action terms indicated that the negative impact of ARR at PHC institutions on likelihood of visiting county-, municipal- and higher-level hospitals in 2013 was 28, 66 and 33% larger than these in 2008. Similar patterns were also observed for physician density at PHC institutions and county hospitals (Appendix Table 3). Appendix Table 5 shows the results of regressions among out- patient care in 2008 and 2013, respectively, and Appen- dix Table 6 shows the results of regressions among inpatient care. choosing county-, municipal- and higher-level hospitals decreased by 25% (RRR = 0.75, 95% CI = 0.57–0.99, p < 0.001), 45% (RRR = 0.55, 95% CI = 0.37–0.82, p < 0.001) and 46% (RRR = 0.54, 95% CI = 0.36–0.82, p < 0.001), re- spectively, in 2008; while in 2013, the RRRs were 0.54 (95%CI = 0.44–0.66), 0.19 (95%CI = 0.15–0.24), and 0.37 (95%CI = 0.27–0.50), respectively. Discussion To the best of our knowledge, this is the first study evaluating the impact of health policies on healthcare seeking behaviors, from the perspective of synergic pol- icies to strengthening primary care [29]. Taking advan- tage of the population-based nationally representative survey before and after 2009, we were able to investigate whether the impacts changed with the progress of the health system reform, in which the two policy areas are evolving simultaneously. Our findings can provide impli- cations for further advancing the agenda of deepening the synergic policies on primary care strengthening, by identifying policy entry points to promote PHC systems from a joint vision. Second, compared to 2008, the positive impacts of health insurance ARR and physician density on increas- ing outpatient visits and admissions dropped signifi- cantly in 2013. Diminished marginal return of increasing health inputs in high-resource-density domains implies better strategies that priorities of health resource alloca- tion need to focus on the resource-poor parts such as the PHC institutions [38]. The findings on comparison in healthcare seeking behaviors between 2008 and 2013 Jin et al. BMC Health Services Research (2020) 20:865 Page 6 of 10 Fig. 1 Comparing the proportion of outpatient visits at PHC institutions to physician density at PHC institutions by GDP group, 2008 (A) and 2013 (B) Fig. 1 Comparing the proportion of outpatient visits at PHC institutions to physician density at PHC institutions by GDP group, 2008 (A) and 2013 (B) Fig. 1 Comparing the proportion of outpatient visits at PHC institutions to physician density at PHC institutions by GDP group, 2008 (A) and 2013 (B) hospital admission in China was 16.4%, higher than the average for countries of the Organization for Economic Cooperation and Development, which implies that China needs to examine the appropriateness of inpatient care, including overuse and misuse of medical services. There- fore, China’s health system reform needs to consider re- distributing the existing health resources rather than to continuously increase the health resources, for more ef- fectiveness of financial and service-delivery policy ar- rangements. A previous study showed the encouraging results that the investment in PHC providers showed were consistent with previous studies focusing on China’s health system reform, which indicated that the reform with multipartite policies may make interactional impacts on healthcare use [39]. Nevertheless, these pre- vious studies have not assessed the impact of health sys- tem reform on healthcare seeking behaviors. Discussion Notably, the positive associations with admissions still reminded us with the challenge that a moral hazard situation arises when health insurance coverage is universal, and SHI participants overuse health services especially hospital- based medical services [40–42]. In 2016, the rate of Jin et al. BMC Health Services Research (2020) 20:865 Page 7 of 10 Table 3 Impact of health insurance and health workforce on healthcare use before and after China’s health system reform Self-medication (IRR) Outpatient visits (IRR) Hospital admissions (IRR) Before China’s health system reform After China’s health system reform Before China’s health system reform After China’s health system reform Before China’s health system reform After China’s health system reform ARR at PHC institutions 0.99* (0.99, 1.00) 0.91 (0.90, 0.93) 1.19* (1.12, 1.27) 1.04 (0.99, 1.10) 1.55* (1.28, 1.88) 1.42 * (1.27, 1.59) ARR at county hospitals 0.96* (0.94, 0.98) 0.90* (0.88, 0.92) 1.16* (1.08, 1.23) 1.01 (0.96, 1.06) 1.82* (1.47, 2.25) 1.02 (0.77, 1.36) Physicians density at PHC institutions 0.79* (0.77, 0.81) 0.83 (0.81, 0.85) 1.13* (1.04, 1.22) 1.11* (1.05, 1.18) 1.61 * (1.41, 1.84) 1.00 (0.98, 1.01) Physicians density at county hospitals 0.99 (0.92, 1.06) 0.91* (0.88, 0.95) 1.38* (1.29, 1.48) 1.08* (1.02, 1.15) 1.03 (1.00, 1.05) 0.98 (0.97, 0.99) p < 0.05, p < 0.01, p < 0.001 density at county hospitals was higher than PHC institutions, and had also seen a higher growth, thereby widening the gap of physician density between PHC institutions and hospitals from 2008 to 2013. This unintended result of the reform might lead to the declining use of PHC as a proportion of total health services. The major reason for the unintended re- sults of the reform is the inconsistence of the development of health insurance and health workforce, the two health sys- tem policy areas. Although the SHI has achieved a lot in coverage and service benefit, the quantity of PHC providers is inadequate [9, 45], and the incentive mechanism for PHC providers is weak [8]. largest impact on improving healthcare use [43], so en- suring an adequate availability of PHC providers is one of top priorities to improve the effectiveness of health- care delivery. Our study provided an evidence-based ap- proach for taking steps towards structural adjustment to tackle the sluggish development of existing policy ar- rangements [44]. Discussion There are many complaints about the unavailability of essential drugs on the list of the SHI, that pushes patients to be re- ferred to hospitals and restricts the professional develop- ment of PHC providers [47]. Although the SHI has achieved a lot in coverage and service benefit, the limita- tion of essential drugs covered by the SHI can lead pa- tients bypass PHC institutions to seek health services at high-level hospitals. Second is the financial arrangements for PHC institutions with the delink between revenue and expenditure. The revenues obtained by providing PHC should be turned over to the government financial ac- counts, and the expenditures incurred are financed ac- cording to the standards designed by the government financial department [7]. The delink between revenue and expenditure reduced the financial incentives for PHC pro- viders because their income is fixed and has nothing con- nection with the workload of providing PHC [46]. However, the fee-for-service payment system in hospitals gives hospitals an incentive to attract and retain patients who could otherwise use PHC providers [9]. Third is the salary reform for PHC providers. The percentage of performance-based bonus on the total income is quite low, limiting the financial incentives for providing PHC [8]. The consequent lack of motivation has led to a brain drain to hospitals and out of the health system altogether [48]. In 2017, only 13% of PHC providers had a formal medical education (five years of medical school) in rural and 40% in urban areas [10]. In a word, efforts to cope with the capacity strengthening PHC system have been slow, mostly because of insufficient coordination and frag- mented systems [4, 49]. whole-of-government multisectoral plans, establishing mechanisms for coordination across ministries and other stakeholders, and multi-sectoral mechanism at the stage of monitoring and evaluating enforcement of policies [52]. This study has several limitations. First, the observa- tional nature of our study limited our ability to draw any causal inference from our findings. Rather, the associ- ation found in this study underscored the need for re- search to evaluate the progress of the synergic policies on primary care strengthening from the perspectives of health financing and health workforce. Second, only the 2008 and 2013 round of NHSS were included to evaluate the five-year progress of health system policies. This mid-term impact assessment might limit us to generate policy relevance. Discussion BMC Health Services Research (2020) 20:865 Page 8 of 10 Page 8 of 10 related to the findings of the study. First is the zero-profit medicine policy [6]. Although local governments increase the budgets to balance financial loss of PHC institutions from drugs benefits, this part of financial support is dependent on local government’s financial capacity and cannot make up the loss in most of cases [46]. There are many complaints about the unavailability of essential drugs on the list of the SHI, that pushes patients to be re- ferred to hospitals and restricts the professional develop- ment of PHC providers [47]. Although the SHI has achieved a lot in coverage and service benefit, the limita- tion of essential drugs covered by the SHI can lead pa- tients bypass PHC institutions to seek health services at high-level hospitals. Second is the financial arrangements for PHC institutions with the delink between revenue and expenditure. The revenues obtained by providing PHC should be turned over to the government financial ac- counts, and the expenditures incurred are financed ac- cording to the standards designed by the government financial department [7]. The delink between revenue and expenditure reduced the financial incentives for PHC pro- viders because their income is fixed and has nothing con- nection with the workload of providing PHC [46]. However, the fee-for-service payment system in hospitals gives hospitals an incentive to attract and retain patients who could otherwise use PHC providers [9]. Third is the salary reform for PHC providers. The percentage of performance-based bonus on the total income is quite low, limiting the financial incentives for providing PHC [8]. The consequent lack of motivation has led to a brain drain to hospitals and out of the health system altogether [48]. In 2017, only 13% of PHC providers had a formal medical education (five years of medical school) in rural and 40% in urban areas [10]. In a word, efforts to cope with the capacity strengthening PHC system have been slow, mostly because of insufficient coordination and frag- mented systems [4, 49]. related to the findings of the study. First is the zero-profit medicine policy [6]. Although local governments increase the budgets to balance financial loss of PHC institutions from drugs benefits, this part of financial support is dependent on local government’s financial capacity and cannot make up the loss in most of cases [46]. Discussion Although we did not have data of the latest 2018 round of NHSS which has not been open for analysis, it was reported that healthcare seeking behavior sustained the trend and the health insurance coverage and physician density continued to be improved during 2013 and 2018 [9, 11]. Nonetheless, this interim impact analysis might make our estimates of the associations between health insurance and health workforce and healthcare seeking behavior conservative. Discussion Third, the negative impact of health insurance ARR and physician density at PHC institutions on likelihood of visiting hospitals in 2013 was larger than these in 2008. The results can be explained by the declining use of PHC as a proportion of total health services from 2008 to 2013. Despite the evi- dence of the progress made in strengthening the PHC sys- tem, some challenges remain immense. The physician There are three points on the implementation of policies to explain how the real condition, unlike the policy, are Table 4 Impact of health insurance and health workforce on choice of healthcare providers before and after China’s health system h insurance and health workforce on choice of healthcare providers before and after China’s health system p p y reform Outpatient choice of healthcare providers (reference group is village/community health stations) Before China’s health system reform (RRR) After China’s health system reform (RRR) Township/ community health centers County hospitals Municipal or higher- level hospitals Township/ community health centers County hospitals Municipal or higher- level hospitals ARR at PHC institutions 1.44 (0.96, 2.16) 0.28* (0.18, 0.44) 0.18* (0.10, 0.31) 1.76 * (1.04, 2.74) 1.07 (0.80, 1.44) 0.98 (0.73, 1.32) ARR at county hospitals 1.31 (0.71, 2.39) 1.68 (0.90, 3.15) 2.92*** (1.51, 5.66) 1.44* (1.00, 2.08) 1.72* (1.20, 2.45) 1.87* (1.27, 2.74) Physicians density at PHC institutions 1.23* (1.15, 1.32) 0.43* (0.30, 0.61) 0.76 (0.43, 1.33) 1.34 (0.80, 2.24) 0.95* (0.91, 0.99) 0.98 (0.95, 1.02) Physicians density at county hospitals 1.02 (0.98, 1.06) 1.38* (1.29, 1.48) 1.45 * (1.33, 1.58) 0.91* (0.89, 0.94) 0.98 (0.95, 1.01) 0.99 (0.96, 1.02) Inpatient choice of healthcare providers (reference group is township/community healthcare centers) County hospitals Municipal hospitals Provincial or higher- level hospitals County hospitals Municipal hospitals Provincial or higher- level hospitals ARR at PHC institutions 0.75 (0.57, 0.99) 0.55* (0.37, 0.82) 0.54* (0.36, 0.82) 0.54* (0.44, 0.66) 0.19* (0.15, 0.24) 0.37* (0.27, 0.50) ARR at county hospitals 1.22* (1.06, 1.41) 1.76* (1.27, 2.45) 1.85* (1.34, 2.56) 1.12 (0.91, 1.38) 1.68* (1.39, 2.03) 2.18* (1.74, 2.73) Physicians density at PHC institutions 0.90* (0.84, 0.97) 0.84*** (0.78, 0.91) 0.92* (0.85, 1.00) 0.27* (0.19, 0.36) 0.18* (0.10, 0.30) 0.28* (0.16, 0.48) Physicians density at county hospitals 1.41* (1.29, 1.53) 1.48* (1.34, 1.64) 1.52* (1.37, 1.69) 1.12* (1.07, 1.17) 1.14* (1.09, 1.20) 1.16*** (1.11, 1.22) p < 0.05, p < 0.01, **p < 0.001 Jin et al. Conclusions Primary care strengthening requires synergic policies. Our findings highlighted the role of strengthening PHC on im- proving the effectiveness of financial and service-delivery policy arrangements, and the declining use of PHC as a proportion of total health services could be attributable to the inconsistent development of the two policy areas. Ef- fective mechanisms for coordination across multisectoral actions in an integrated and systemic way are required for deepening those policies to ensure efficient delivery of high-quality healthcare without experiencing financial risks. The implications can guide decision-making on the entry points to reinforce PHC planning, resource alloca- tion, and service delivery in various LMICs. Since quality of care given by PHC providers is still unsatisfactory, patients in real needs choose to bypass the PHC system in favor of hospitals, which resulted in soaring cost of medical care [50]. The synergic policies that are issued to tackle access to healthcare and finan- cial protection have not succeed, even further lower the affordable accessibility of the low-income group. There- fore, further reforms should consider transforming the existing hospital-centered healthcare system to an inte- grated health system based on PHC in a systemic way. A competent health workforce is indisputably import- ant, and a good financing system including effective in- centive mechanisms for PHC providers should continue to focus on aligning incentives for providing quality PHC [51]. Strengthening platforms to design and im- plement more effective multisectoral actions is urgently required. This can include the development of national Funding f h Part of the study is supported by the Beijing Municipal Natural Science Foundation (No. 9204025), and the National Natural Science Foundation of China (No. 71904004). 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Acknowledgements This study is commissioned by National Health Commission and funded by the Center for Health Statistics and Information of National Health Commission. We thank the Center for Health Statistics and Information of the National Health Commission who helped guide this study. This study is commissioned by National Health Commission and funded by the Center for Health Statistics and Information of National Health p 8. Ma X, Wang H, Yang L, et al. Realigning the incentive system for China’s primary healthcare providers. BMJ. 2019;365:l2406. 8. Ma X, Wang H, Yang L, et al. Realigning the incentive system for China’s primary healthcare providers. BMJ. 2019;365:l2406. primary healthcare providers. BMJ. 2019;365:l2406. Commission. We thank the Center for Health Statistics and Information of the National Health Commission who helped guide this study. 9. Meng Q, Mills A, Wang L, et al. What can we learn from China’s health system reform? BMJ. 2019;365:l2349. 9. 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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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