dataesr/scientific-paragraphs-categorization huggingface dataset:
2C  and D ). A slight increase in the helix propensity was observed for the N-terminus, whereas a slight decrease was observed for the C-terminus. For all seven peptides the minimal inhibitory concentration preventing all growth (MIC 100 ) was determined as described earlier. 51 The assay was performed at least twice for each peptide, with four replicas each time. Overall, the data is very consistent as shown by the averages and standard deviations depicted in Fig. 2  Helix propensity of magainin 2 and the effect of terminal modifications. Secondary structure propensities determined using assigned chemical shifts and the ncSPC tool 62 range from pure sheet (À1) to pure helix (+1). Secondary structural propensity for magainin 2a (A) in TFE/buffer (1 : 3 v/v) and (B) in aqueous buffer. Fig. 3.
Data analysis For each well, the ratio between the absorbance at 570 nm and the absorbance at 600 nm was calculated. The average value for the blank medium was then subtracted from all wells, before normalizing against the average values from wells containing bacteria but no peptide. The lowest peptide concentration where no reduction of resazurin was observed is considered the minimal inhibition concentration (MIC 100 ). Average values with standard deviations were calculated based on at least two experiments with four replicas each. For 1 : 1 peptide mixtures, the expected MIC 100 value was calculated under the assumption of the peptide activities being additive (no synergism or antagonism) from eqn (  1.
Acknowledgements We gratefully acknowledge  Delphine Hatey  and  Bruno Vincent  for helping with peptide synthesis and solution NMR spectroscopy, respectively. The discussions with  Martin Hof  and  Mariana Amaro  are much appreciated. The nancial contributions of the  Agence Nationale de la Recherche  (projects MemPepSyn  14-CE34-0001-01 ,  Biosupramol 17-CE18-0033-3 , Naturalarsenal  19-AMRB-0004-02  and the  LabEx Chemistry of Complex Systems   10-LABX-0026_CSC ), the  University of Strasbourg , the  CNRS , the  Région Alsace  and the  RTRA International Center of Frontier Research in Chemistry are gratefully.
Ethics The study protocol and informed consent forms were reviewed and approved by the National Ethics Committee of the Central African Republic and the Clinical Research Committee of the Institut Pasteur in Paris. The trial was registered prior to the enrolment of the first participant under the ClinicalTrials. gov Identifier, NCT01746199. All pregnant women provided written, informed consent before participating in the study. An independent Data Safety Monitoring Board (DSMB), set up prior to the beginning of the trial, regularly reviewed and monitored the safety data collected. This trial was funded by the Fondation de France and conducted by the Institut Pasteur de Bangui and the Institut Pasteur de Paris. All treatment received during pregnancy in relation to the pregnancy and malaria were undertaken by the study (including caesarian sections).
Data collected: The following data were collected from pregnant women at inclusion: socio-demographic characteristics, clinical data, obstetric history, and gestational age estimated either by the date of the last menstrual period (LMP) or by uterine fundal height if LMP was unknown. CD4 and blood cell counts were determined. Malaria diagnosis using RDT, TBS and Polymerase Chain Reaction (PCR) was performed. During follow-up, clinical examination was performed every month. The total number of doses of SP-IPTp delivered during pregnancy was documented. Adverse effects were recorded according to their severity stage (low, moderate or severe) and relation to SP-IPTp or CTX. CD4 count was determined and any suspected malaria episode was investigated by laboratory testing (RDT, thick blood smear and PCR).
Medians and interquartile ranges (IQR) and proportions were used to describe the distributions of continuous and categorical variables, respectively. Distributions of variables were compared between the two arms using the Student test or Mann-Whitney U test for continuous variables, Chi-Squared or Fisher exact test for categorical variables. Data were recorded in an Access database and analyzed using Stata 15 software (StataCorp LP, College Station, TX, USA). Statistical significance was achieved for p values < 5%.
Acknowledgments We are grateful to all the women and children who participated in this trial, the investigators and medical teams who valiantly executed this clinical trial in difficult conditions. Financial support: This study was supported by the  Fondation de France. Disclaimer: The funding institution was not involved in the study design, data analysis, data interpretation or the writing of the report. We thank the  Foundation Total  for providing insecticide-treated bednets for all pregnant women. Authors'addresses:  Muriel Vray ,  Laura Tondeur  and  Lenaig Le Fouler ,  Unité d'Epidémiologie des Maladies Emergentes, Institut Pasteur, Paris, France ,  E-Mails: Muriel. Vray@pasteur. fr,laura. tondeur@pasteur. fr,lenaig. le-.
ACKNOWLEDGMENTS We thank  Guiying Lin ,  Xiaomin Chang ,  Jin Li ,  Jianmei Li ,  Xinzhu Chen ,  Yuqi Yang ,  Jianglong Guo , and  Xuejun Hong  for the help in some of the experiments and for the genetic screen. The expert technical contribution of  Luhong Lin ,  Yongxin Liao ,  Fengyi Zhang ,  Yanzhen He  for the screen is gratefully acknowledged. We are indebted to  Prof. Ni  and his collaborators ( Tsinghua University ) for giving us access to his resource of  Trip RNAi lines (Tsinghua Fly Center ) and also the  Vienna Drosophila Resource Center  (VDRC, www. vdrc. at)and  Bloomington Drosophila Stock Center  ( NIH P40OD018537 ) for their resource. We thank  Anne Beauvais  and  Jean-Paul Latge  for the gift of the RFP-labeled A. fumigatus wild-type strain,  Erjun Ling  for the gift of the PPO1 antibody.
3i ) showing the multilineage potential of the Krt17+ cells as previously demonstrated in vivo (Fig. 1f ). In silico approach reveals anal TZ differentiation path. Next, we have used computational models to predict the hypothetical trajectory or pseudotime  25  that could stand from our single cell mRNA sequencing datasets. We have examined the differentiation trajectory that could exist between the anal TZ clusters, the anal canal cluster and the rectum cluster. Differentiation trajectory reconstruction on the four remaining clusters using the slingshot R package  26, 27  suggests three trajectories (Fig. 4a ). However, only two trajectories suggest a differentiation trajectory. This is illustrated by pseudotime values, which contain information about ranking and distance of every cell within a given trajectory (Fig.
8e  and Fig. 2g ). More interestingly, an additional cluster, absent in unwound analysis (Fig. 2 ), was identified as "TZ hybrid state" cells. This cluster is enriched for TZ marker Krt17 and glandular markers Muc13 and Krt8. Then, pseudotime analysis was performed on the post-wound single cell RNAseq dataset (Fig. 6b ). We found three major trajectories representing a hierarchical relationship between TZ cells to anal canal/suprabasal cells and wounded epithelial cells. The third trajectory stands for TZ cells transitioning to the "TZ hybrid state" to finally go towards the rectum which support our previous data, where TZ cells displayed multipotency when subjected to injury (Fig. 5 ). In contrast to the normal condition (Fig. 4 ), the rectal trajectory in the wounded tissue (pseudotime 3 Fig.
First, we have confirmed by immunostaining that the AP-1 transcription factor JunB was enriched at the anal TZ (Fig. 6d, e ), but not the anal canal or the rectum confirming the scRNAseq analysis. Moreover, in a wounded situation we showed that JunB is also expressed in cells participating in both chemical and mechanical wound repair (Fig. 6f  and Ext. Data Fig. 7c ). We further showed using shRNA in the organoid culture, that JunB is essential for TZ cells ability to differentiate properly. Two shRNA JunB (sh2 and sh3), which have the most efficient JunB downregulation, showed alteration in differentiation markers Krt10 and loricrin by immunofluorescence and western blot analysis on FACS sorted RFP+ organoids (Fig. 6i, j ). These data suggest that JunB may participate into the wound repair by promoting differentiation of the anal TZ cells, which may undergo further plasticity program to give rise to a fully functional glandular cell type.
Total number of cells was counted using DAPI staining in stratified and glandular epithelia. The number of GFP+ cells was counted using Zen software and photoshop CC2019 and the percentage of GFP cells was calculated according to total number of cells (reported as 100%). Immunostaining and histology. The anorectal tissue was fixed in 4% paraformaldehyde overnight at 4 °C. The sample was then washed three times in PBS 1× and put in sucrose 30% overnight at 4 °C. Lastly, the tissue was put in two volumes of sucrose 30% and one volume of OCT (Tissue-Tek, Sakura, Torrance, CA) for few hours and embedded in OCT compound. For some antibodies, such as Krt6 and nectin-4, the anorectal tissue was fixed in methanol for 30 min at -20 °C instead of the paraformaldehyde and process in 30% sucrose, as previously described.
The list of primary and secondary antibodies is in the Supplementary Table  1. Fluorescent images were acquired using a Confocal microscope ZEISS LSM 880. All images were analyzed using ZEN software and Photoshop. Histologies were performed by the IPC/CRCM Experimental Pathology platform (ICEP), and analyzed by two independent pathologists at the Paoli Calmettes Institute. EdU staining. Mice were injected intraperitoneally with 0. 25 mg EdU (Abcam, ab146186) for 4 h and tissue was processed as described in the previous sections. The Click-iT EdU AlexaFluor 647 Imaging kit, (Invitrogen C10640) was used to detect proliferative cells according to the manufacturer instructions. Oil Red O staining. Slides were dip in 60% isopropanol one time quickly then stained in working Oil Red O solution (30 ml of stock solution mixed with 20 ml of distilled water) for 15 min at RT.
At least 100 slides with 9 µm-thick sections were screened for each mouse and no GFP+ crypts were found. d Quantification of the percentage of GFP positive cells, representing the anal TZ Krt17+ derived cells, found in the glandular epithelium at 1week, 2 weeks and 4 weeks post-wound (n = 3 mice quantified for each time point). (Source data are provided as a source data file). Two-tailed paired t-test; error bars, mean ± SEM *p = 0. 0115, **p ≤ 0. 0100, ***p < 0. 0001. e Krt17+ anal TZ cells give rise to differentiated enteroendocrine cells (Chromogranine A+, inset 3-fold zoom) and goblet cells (alcian blue+, inset 2. 5-fold zoom) (n = 3 independent experiments from three mice). Scale bars are 50 μm. ACD Inc. ) were used in the RNAscope fluorescent assay according to the manufacturer's protocol (Catalog Number 320293).
Cell sorting was performed using a FACS Aria II equipment (BD Bioscience) and FACS diva software. Organoid culture. After FACS isolation, 4000-10,000 cells were cultured in 25 µl Matrigel (Corning, 356231) on 48-well plate. Three hundred microliter of medium (Supplemental Table  2 ) supplemented with 10 μM Y-27632 (only first 5 days to avoid anoïkis) were added to wells and the plate was incubated at 37 °C, 5% CO 2. The medium was changed every 3-4 days. Upon outgrowth, organoids were passaged every 10-15 days either by mechanical dissociation by gentle pipetting or by enzymatic dissociation with TrypLE express enzyme (Gibco TM , 12605010). Medium was supplemented with 10 μM Y-27632 the first 3 days. Imaging organoids. Brightfield images, GFP and RFP expressions were monitored using the Leica M205 FA stereomicroscope.
See Supplemental Table  1  for complete reference list of the antibodies used. Full scan blots are provided in the Supplementary information. Membranes were then washed three times with TBS 0. 1% Tween for 10 min. Fluorescent secondary antibodies or HRP-coupled secondary antibodies listed in the supplementary Table  1  were used at 1/1000 in 5% non-fat milk or 5% BSA. Fluorescents images were acquired using Biorad Chemidoc imager and HRP immunoblots were developed using standard ECL Prime (Amersham, RPN 2236). Real-time PCR. Total RNA was isolated, using a Qiagen Rneasy Micro Kit, and used to produce cDNA using the Maxima first strand cDNA synthesis kit. Reverse transcription reactions were diluted to 10 ng/μl and 1-2 μl of cDNA was used to perform Real time PCR using the CFX96 real-time PCR System, CFX Manager Software and the Ssofast EvaGreen Supermix reagent.
All reactions were run in triplicate and analyzed using the ΔΔCT method with relative expression normalized to GAPDH. (See Supplemental Table  3  for all primer sequences). Fig. 6  Post-wound single cell RNA sequencing reveals a TZ population enriched in the transcription factor JunB. a Main cell populations visualized on UMAP from scRNA-seq of anal epithelial cells 1 week of post-EDTA wound b Structure of the differentiation trajectory reconstruction using the slingshot R package suggests three trajectories. Pseudotime values show an elongated distribution on each trajectory suggesting that there are more intermediate phenotypes and therefore a differentiation process. c Hierarchical relationship between clusters 4, 5, and 6 and expression of key genes expressed in TZ hybrid state cells. d In tissue, JunB (white) is specifically expressed by transition zone cells and is absent in Krt8 (red) glandular epithelium (n = 3 independent experiments from three mice).
i, j JunB knockdown impairs TZ cells differentiation properties. i Western Blot analysis showing JunB downregulation in TZ organoids infected with sh2 and sh3 JunB compared to control shRNA and sh1JunB infections (n = 6 independent experiments from six biologically independent samples). Differentiation marker Krt10 expression is decreased following JunB downregulation in organoids infected with sh2 and sh3 JunB (n = 2 independent experiments from two biologically independent samples). GAPDH was used as a loading control. (Source data are provided as a source data file). j Immunofluorescence confirms the downregulation of Krt10 in sh2 and sh3 JunB organoids (n = 5 independent experiments from 5 independent samples). The defect in differentiation is also detected using antibody against Loricrin (n = 5 independent experiments from five independent samples).
Scale bars are 50 µm (d, e) (TZ organoid) (f, j), and 20 µm (e) (TZ rectum). Single cells RNA sequencing. After tissue dissection and dissociation, FACS purified suspended cells were partitioned into nanoliter-scale Gel Bead-In-Emulsions (GEMs) with the Chromium Single Cell Controller (10× Genomics) (performed by HalioDx company and TGML Marseille Luminy platform). After cell encapsulation and barcoding, library preparation followed the standard scRNAseq protocol comprising reverse transcription, amplification, and indexing (10× Genomics). Sequencing was performed using a NextSeq Illumina device (Illumina). Illumina bcl files were basecalled, demultiplexed and aligned to the mouse mm10 genome using the cellranger software (version 3. 1. 0, 10× Genomics). All downstream analyses were done with R/Bioconductor packages, R version 4.
0. 3 (2020-10-10) [https://cran. r-project. org/; http://www. bioconductor. org/]. Raw counts were imported into R and single cell data was analyzed with the 'Seurat' package v. 3. 2. 2  21. After filtering for library size (between 200 and 5000 features per cell) and mitochondrial gene expression (less than 25%), pre-processing was performed using Seurat functions for counts normalization (NormalizeData using the LogNormalize method), scaling (ScaleData using the 2000 most variable features), dimension reduction with principal component analysis (PCA) (RunPCA with default parameters), construction of a shared nearest neighbor (SNN) graph (FindNeighbors using ten dimensions of reduction as input) and clustering (FindClustering with a resolution of 0. 2). Data was adjusted for cell cycle using the CellCycleScoring function and regressing for S and G2/M phases scores.
After visualization with Uniform Manifold Approximation and Projection (UMAP) dimensional reduction technique [https://arxiv. org/abs/1802. 03426], contaminating cell types were filtered out (i. e. , anal glands, hair follicle, and fibroblasts) followed by re-scaling of each dataset. An additional filtering based on the expression of Lgr5, Lhx2, Tbx1, was necessary in the normal sample to remove hair follicle cells that were contaminating the basal cluster. After filtering of bad quality and contaminating cells, 939 cells from the normal sample and 4147 cells from the wounded sample were used for all downstream analyses. Markers of each cluster were identified using the wilcox test option of the FindAllMarkers function, with a bonferroni p value adjustment. The same criteria for filtering, dimension reduction, clustering, visualization, adjusting for cell cycle, and pseudotime were used to analyze the normal and the wounded sample.
Cell-filtering parameters are nFea-ture_RNA > 200, nFeature_RNA < 5000, percent. mt < 25%. Other parameters included ScaleData (x, vars. to. regress = c("S. Score", "G2M. Score")), RunPCA(x) # default parameters, FindNeighbors(dims = 1:10), FindClusters(resolution = 0. 2), RunUMAP(dims = 1:10). Pseudotime analysis. We used the dynverse R package to compare across pseudotime algorithms and choose the one best suited for our dataset  27. Final trajectory (pseudotime) analyses were performed using the slingshot R package  26. To this end, clustering information was extracted from Seurat objects and passed directly to slingshot's main function, using the "Basal" cell cluster as the starting cluster. The same "granularity" parameters (i. e. , Omega = 20) were used for both conditions, normal and wounded, to assure comparability.
Immunostainings were performed the same day with the same antibody solution and identical imaging settings were applied, when comparing different conditions. Images were acquired with a Confocal microscope ZEISS LSM 880 and were analyzed with ImageJ. A threshold was applied to all images to remove the background before measuring the integrated density, while keeping the same ROI for each images (see Supplemental methods for details). For EdU quantification, eight areas per mice and per region were manually counted using Zen software and photoshop CC2019. Krt17 expression was used to separate TZ from anal canal and rectum. At least eight different areas of TZ and anal canal and 16 different crypts from nine mice were quantified (n = 3 per condition). Percentage of EdU+ cells was calculated according to total number of DAPI cells (reported as 100%).
Acknowledgements Processing of the slides for histology was performed by  Emilie Agavnian  in the experimental histopathology (ICEP) core facility at the  Institut Paoli Calmettes. All organoid cultures were done in the 3D-Hub-O platform at CRCM. We thank  Manon Richaud  and  Françoise Mallet  in the Flow cytometry facility,  Daniel Isnardon  and  Magda Rodriguez  (especially for the RNAscope quantification) in the microscopic core facility at the  CRCM ,  Charlyne Gard  for the scRNA seq experiment at  Aix Marseille Univ, INSERM, TAGC, TGML, Marseille, France. Ghislain Bidaut in the Cibi platform  for the initial scRNAseq analysis,  David Owens  for the K17CreERT2 mouse lines and  Marc Lopez  for the anti-nectin-4 monoclonal antibody. Jean-Christophe Orsoni  and  Arnaud Capel  in the mouse facility at the  CRCM.
Samuel Granjeaud  for helping with statistics. This study was supported by  ANR  grant # ANR20-CE13-0009-01  (G. G. ) and  ANR-10-INBS-0009-10  (platform TAGC), partly supported by research funding from the  Canceropôle Provence-Alpes-Côte d'Azur, Institut National du Cancer and Région Sud , grants from the  Excellence Initiative of Aix-Marseille University A * Midex , " Investissement d'avenir" (CapoStromEx)  and  Inserm Plan Cancer AAP  single cell (G. G. and H. H. V). L. M. is a recipient of the  French ministerial research fellowship  and the  Ligue National Contre le Cancer  fellowship.
Methods This study was conducted using data from the ASAS-PerSpA dataset, which has been described elsewhere  [20]. In brief, ASAS-PerSpA was a multicentre observational study with a cross-sectional design in which a total of 24 countries participated. Its main aim was to investigate clinical peripheral rheumatologic features in consecutively included SpA patients and evaluate the validity of existing outcome measures of peripheral rheumatologic features.
Patients In the ASAS-PerSpA study, patients with a diagnosis of SpA (n ¼ 4465) were included between July 2018 and February 2020, representing 24 countries in four geographical regions. The study was approved by the ethical committees in all countries (complete list available in Supplementary Data S1, available at Rheumatology online) and written informed consent was obtained from participants prior to inclusion. For this analysis, only patients with a definite diagnosis of axSpA were included, which was defined as axSpA and either r-axSpA or non-radiographic axSpA (nr-axSpA) as a disease subgroup.
The precise pathophysiological mechanisms underlying axSpA remain unclear, but as different types of HLA-B27 are found in different parts of the world (e. g. HLA-B*27:05 in Europe and HLA-B*27:04 in Asia) and the association between HLA-B27 and axSpA varies between races  [19] , one might have expected to find more variation in age at onset and its association with HLA-B27 across geographical regions. Race was unavailable in the ASAS-PerSpA dataset, yet we expect the majority of the patients included in each geographical region to identify with its most prominent race, hence a clear effect of race would have been seen in the data. Additionally, many other factors are thought to have an influence on the occurrence of axSpA, such as other genetic factors and differences in the human microbiome and environmental factors, such as smoking  [5, 25] , which makes the relative consistency in age at onset all the more intriguing.
A potential limitation of this study is the fact that data were collected cross-sectionally based on patient records and patient-reported information, which has resulted in some missing data, especially regarding HLA-B27 status, as this was not specifically analysed for this study. Nonetheless, all geographical regions contained both patients with HLA-B27-positive andnegative disease and patients whose HLA-B27 status was unknown were not different than those with nonmissing data (data not shown).
Acknowledgements This study was conducted under the umbrella of the ASAS. We would like to acknowledge all the patients and investigators who participated in this research. We would like to thank the  ASAS-PerSpA Steering Committee members, ClinInfo  and the pharmaceutical companies supporting this initiative. Funding: No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this article. The  ASAS-PerSpA study  was conducted under the umbrella of the ASAS with unrestricted grants from  AbbVie ,  Pfizer ,  Eli Lilly ,  Novartis ,  UCB ,  Janssen  and  Merck. The funders did not have any 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.