dataesr/scientific-paragraphs-categorization huggingface dataset:
05; **P ≤ 0. 01; ***P ≤ 0. 001; NS, non-significant). To confirm that Itsn2-Y554 phosphorylation correlated with CD3 chain phosphorylations during TCR stimulation with a cognate antigen, we generated an independent phosphoproteome data set from OT-I CD8 + T cells that express a TCR specific for the ovalbumin (OVA)-derived SIINFEKL (N4) peptide bound to H-2Kb MHC-I molecules. OT-I T cells were stimulated with pMHC-I tetramers laden with N4 peptide for 15, 30, 120, 300, and 600 s. Globally, analysis of this data set showed results very comparable to those obtained with CD4 + T cells  (Fig EV4, Dataset EV3). In brief, 59% of the phosphosites identified and 88% of the phosphosites regulated by TCR stimulation in the CD4 + T cells were also detected in OT-I CD8 + T cells (Fig EV4A  -C ). Moreover, phosphorylation kinetics of phosphosites regulated by TCR stimulation were remarkably conserved across both data sets (Fig  EV4B  and C ).
Discussion Pioneering phosphoproteomic studies have identified molecular events induced by receptor ligation  (Nguyen et al, 2009; Brockmeyer et al, 2011; Humphrey et al, 2015; Navarro et al, 2011). LymphoAtlas complements these studies by focusing for the first time on early phosphorylation events triggered by receptor ligation in primary cells. This study provides a time-resolved, quantitative atlas of protein phosphorylations before and during the first 10 min of TCR stimulation. We quantified and determined the relative changes in phosphorylation on 7,180 sites (unique sites plus combination of sites quantified together on the same peptides) corresponding to more than 2,000 proteins. A number of these phosphosites (1,777 out of 7,180) were not reported in previous studies, thus highlighting the deep coverage of this data set.
LymphoAtlas is a unique resource to explore the phosphorylation events underlying TCR signaling initiation in primary T cells. To facilitate the exploration of this data set by the scientific community, a Web application that interactively displays phosphorylation kinetics of selected sites from the LymphoAtlas database is available at https://bmm-lab. github. io/LymphoAtlas/ (see also  Appendix Fig S2). By quantitatively capturing changes in phosphorylation dynamics, LymphoAtlas provides a rationale to the identification of therapeutic targets to advance immunotherapies based on T-cell reprogramming. Finally, the approach developed here lays the foundation for further systematic explorations of dysregulated signaling networks underlying diseases or resulting from molecular perturbations or gene inactivation in genetically engineered mice.
Flow cytometry Stained cells were analyzed using an LSRII system (BD Biosciences). Data were analyzed with the Diva software (BD Biosciences). Cell viability was evaluated using SYTOX Blue (Life Technologies). The following antibodies were used: anti-CD5 (53-7. 3), anti-CD4 (RM4-5), anti-CD8a (53-6. 7), anti-TCRb (H57-597), anti-CD44 (IM7), and anti-CD69 (H1. 2F3), all from BD Biosciences. T-cell isolation and short-term expansion CD4 + or Cas9-EGFP OT-I CD8 + T cells were purified from pooled lymph nodes and spleens with a Dynabeads Untouched Mouse CD4 + or CD8 + T Cell Kit (Life Technologies); cell purity was 95%. CD4 + -purified T cells were expanded for 48 h with plate-bound anti-CD3 (145-2C11, 5 lg/ml) and soluble anti-CD28 (37-51; 1 lg/ ml both from EXBIO). Then, T cells were harvested and grown in the presence of IL-2 (5-10 U/ml) for 48 h prior to stimulation for phosphoproteomic analysis.
Samples were loaded onto a C18 precolumn (300 lm inner diameter × 5 mm, Dionex) at 20 ll/min in 5% acetonitrile, 0. 05% TFA. After 5 min of desalting, the precolumn was switched online with a 75 lm inner diameter × 50 cm C18 column (in-house packed with Reprosil C18-AQ Pur 3 lm resin, Dr. Maisch; Proxeon Biosystems, Odense, Denmark) equilibrated in 95% solvent A (0. 2% formic acid) and 5% solvent B (80% acetonitrile, 0. 2% formic acid). The pre-enriched peptides and TiO 2enriched phosphopeptide samples were eluted at 300 nl/min flow rate using a 240 min gradient of solvent B (4-12% in 44 min, 12-30% in 184 min, 30-40% in 12 min). The pY-enriched peptides were eluted at 300 nl/min flow rate using a 110-min gradient of solvent B (5% to 25% in 80 min, 25% to 50% in 30 min). The mass spectrometer was operated in data-dependent acquisition mode with the XCalibur software.
Protein identification and quantification Raw MS files were analyzed by MaxQuant version 1. 5. 2. 8. Data were searched with the Andromeda search engine against mouse entries of the SwissProt protein database (UniProtKB/Swiss-Prot Knowledgebase release2015_05, Mouse taxonomy, 16,699 entries), the Biognosys iRT peptides, and a list of potential contaminant sequences provided in MaxQuant 1. 5. 2. 8. The search included methionine oxidation, protein N-terminal acetylation, and serine, threonine, and tyrosine phosphorylation as variable modifications, and carbamidomethylation of cysteines as a fixed modification. Specificity of trypsin digestion was set for cleavage after lysine or arginine, and three missed cleavages were allowed. The precursor mass tolerance was set to 20 ppm for the first search and 4.
5 ppm for the main Andromeda database search, and the mass tolerance in MS/MS mode was set to 25 mmu. Validation was performed through a false discovery rate set to 1% at protein and peptide-spectrum match (PSM) level determined by target-decoy search in MaxQuant (with additional filters for peptide validation: minimum length of 6 amino acids and a minimum Andromeda score of 40 for modified peptides). MaxQuant processing of files from phosphorylated peptides was performed independently for each enrichment step (TiO 2 , pY-IP) and each biological series of experiment (replicates 1-4), with the "match between runs" option of MaxQuant enabled for label-free relative quantification of peptide ions across stimulation time points and injection replicates. The minimal ratio count was set to 1 for calculation of LFQ intensities.
Experimental design and statistical rationale Antibody-based TCR stimulation of primary mouse CD4 + T cells was repeated four independent times with distinct pools of mice. For each condition (experiment, time point after stimulation, sample type), several MS injection replicates were performed, leading to 206 raw files considered for statistical analysis. The detailed description of each analysis (raw file name, sample type, biological replicate number, MS technical replicate number, analytical conditions) is available in the "Sample names" sheet of the Dataset EV1. N4based TCR stimulation of OT1 CD8 + T cells was repeated three independent times with distinct pools of mice. For each condition (experiment, time point after stimulation, sample type), three MS injection replicates were performed, leading to 108 raw files considered for statistical analysis.
The detailed description of each analysis (raw file name, sample type, biological replicate number, MS technical replicate number, analytical conditions) is available in the "Sample names" sheet of the Dataset EV3. The quantitative proteomic analysis was performed using the statistical package R (R Development Core Team, 2012; http://www. R-project. org/) and R scripts related to the analysis can be found online: The antibodybased stimulation of CD4 + T cells can be found on https://github. c om/mlocardpaulet/TCR_ABStim2018; the N4 stimulation of OT1 CD8 + T cells on https://zenodo. org with the https://doi. org/10. 5281/zenodo. 3556731; and the source code of the shiny-web application on https://github. com/mlocardpaulet/LymphoAtlas_App. Phosphorylation site relative quantification was performed independently on the TiO 2 and the phosphotyrosine IP samples with the intensities of the "Phospho (STY).
At this stage, only the phosphosites with a localization score ≥ 75% in a minimum of one replicate were kept, and the phosphoserines and phosphothreonines were removed from the phosphotyrosine IP samples. Conversely, the phosphotyrosines quantified in the pY-IP were removed from the TiO 2 data set. Moreover, for statistical analysis, phosphosites with high numbers of missing values were not considered: Only phosphorylation sites that were detected in at least 3 conditions (time points), and presenting in each of them a minimum of two quantification values from independent replicate experiments, were retained. For those, kinetics were normalized between biological replicates before being subjected to an ANOVA with Tukey's correction. To avoid biases coming from the random draw of replacement values, this statistical process was performed 200 independent times and phosphosites were considered statistically significant if they presented a p-value of the ANOVA ≤ 0.
Statistics and computational methods In all experiments, data are presented as mean AE SEM unless stated otherwise. The number of biological replicates is defined in the figure legends. For flow cytometry experiments and cell proliferation analyses, statistical significances were calculated using two-sided paired Welch Student's t-test. t-SNE analysis was performed on means of intensities of phosphosites that were calculated between biological replicates and scaled by the maximal value observed over the course of TCR stimulation. Determination of the clusters was performed with the ClusterX function with default parameters from the R package ClusterX (https://github. com/JinmiaoChenLab/Cluste rX) based on Rodriguez et al's algorithm  (Rodriguez & Laio, 2014). Within the t-SNE plot, P-value corresponding to the local enrichment of an annotation term (UniProt keyword or kinase-substrate relationship) in the neighborhood of an annotated site was calculated by comparing, using an hypergeometric test, annotations between the subset of sites that fell within a maximum distance of 4 (Euclidean distance calculated with t-SNE coordinates) and the set of all TCRregulated sites.
Barcoding, labeling, and acquisition To stop stimulation, cells were fixed with 2% PFA for 15 min at room temperature. Fixed cells were washed and barcoded using palladium isotopes (Cell-ID TM 20-Plex Pd Barcoding Kit from Fluidigm, CA, USA). 2. 5 × 10 6 barcoded cells were then stained with metal-conjugated antibodies directed against surface antigens, washed, and permeabilized with cold methanol (95%) for 20 min. After permeabilization, cells were washed and stained with metal-conjugated antibodies directed against intracellular antigens before acquiring data on a Helios mass cytometer (Fluidigm, CA, USA). Antibodies used for surface and intracellular staining are listed in Appendix Table  S1.
Data availability The mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium (http://proteomecentral. proteomexc hange. org) via the PRIDE partner repository with the data set identifiers PXD014225 (CD4 + T cells stimulated with anti-CD3/anti-CD4 antibodies) and PXD016583 (OT-I CD8 + T cells stimulated with MHC-I tetramers loaded with N4 agonist peptide). Expanded View for this article is available online.
Acknowledgements We thank  N. Jarmuzynski ,  E. Maturin , and  S. Durand  for their technical help, as well as  Y. Couté ,  C. Schaeffer ,  AM. Hesse ,  C. Ramus ,  A. Hovasse ,  S. Ferré , and  E. Same  for their assistance in the optimization of phosphoproteomic methods. This work was supported by  CNRS ,  INSERM , the  Agence Nationale de la Recherche  (LymphoScan project to R. R), the  European Research Council (ERC)  under  FP7 Program  (grant agreement no. 322465  (INTEGRATE) to B. M. ), and the  European Union 's  Horizon 2020 Research and Innovation Program  (grant agreement no. 787300  (BASILIC), to B. M. ), the  MSDAVENIR Fund  (to B. M. ), the  Investissement d'Avenir program  of the  French Ministry of Research ProFI  ( Proteomics French Infrastructure ,  ANR-10-INBS-08  to O. B.
X-ray crystallography Crystallization screening was carried out by the sitting-drop vapor-diffusion method with a Mosquito (TTP Labtech) automated crystallization system. All crystals were grown at 18˚C using the hangingdrop vapor-diffusion method. Crystals of 15-20 mg/ml LtgA were grown at 18˚C and appeared within 2-3 days. LtgA was crystallized in a 1:1 (v/v) ratio against a well solution of 33% (w/v) PEG 6000 and 100 mM HEPES, pH 7. 5. Crystals were rectangular in shape and grew to approximately 200-300 mm in length. The data set was collected at the Soleil Synchrotron (Beamline Proxima-1) (Supplementary file 2). Phasing by molecular replacement was performed using Phenix  (Adams et al. , 2010). Building was performed using Coot  (Emsley and Cowtan, 2004) , and restrained refinement was carried out using a combination of Phenix and the ccp4 software suite  (Adams et al.
Accession numbers Coordinates and structural data have been submitted to the Protein Data Bank under the accession code 6H5F. protection of animals used for scientific purposes. The laboratory at the Institut Pasteur has the administrative authorization for animal experimentation (Permit Number 75-1554) and the protocol was approved by the Institut Pasteur Review Board that is part of the Regional Committee of Ethics of Animal Experiments of Paris Region. All the invasive procedures were performed under anesthesia and all possible efforts were made to minimize animal suffering.
(d) LtgA plus anhydro product (1,6-anhydro-chitotriose) (PDB ID: 5OIJ). The online version of this article includes the following figure supplement(s) for figure 1: Figure supplement 1. Conservation of alpha helix 30 amongst diverse lytic transglycosylases. Figure supplement 2. Binding of LtgA to the Peptidoglycan.
Acknowledgements We acknowledge the synchrotron beamline staff (PROXIMA-1 at SOLEIL and X06DA at  SLS ) for their assistance. We are extremely grateful to  Frederick Saul ,  Patrick Weber  and  Marco Bellinzoni  for their constant helpful guidance, advice and assistance. We thank  Dr. Antoine Forget  for the advice on and help with figure presentations. AHW was supported by an  EMBO  long-term fellowship ( ALTF 732-2010 ) and an  Institut Carnot-Pasteur Maladies Infectious fellowship. This work was supported by an  ERC  starting grant (PGN from SHAPE to VIR 202283) and a  Fondation pour la recherche medicale (FRM)  grant  Programme d'Urgence  ( DBF20160635726 ) to IGB. This study received funding from the  French Government  0 s  Investissement d 0 Avenir program ,  Laboratoire d 0 Excellence 'Integrative Biology of Emerging Infectious Diseases ' (grant no.
ANR-10-LABX-62-IBEID ). IS was supported by the  Institut Carnot Pasteur Microbes and Santegiven  to the  Pasteur-Paris University PhD program  and the  'Fin de these de  science' number  FDT201805005258  granted by  "Fondation pour la recherche medicale (FRM). The  UtechS Photonic BioImaging (Imagopole) ,  C2RT ,  Institut Pasteur  was supported by the  French National Research Agency (France BioImaging ;  ANR-10-INSB-04 ; Investments for the Future).
Design of covNHR miniproteins We designed covNHR miniproteins harbouring only two of the four sub-pockets of the NHR crevice using the crystallographic structure of the complex between C34 and covNHR-VQ (PDB: 6R2G) as template. This covNHR variant binds C34 with sub-pM affinity  34. Three different miniproteins were initially designed, one called covNHR-N containing the two N-terminal pockets (NTP and MP), a second one named as covNHR-M encompassing the two central pockets (MP and HP), and a third protein, designated covNHR-C, with the two C-terminal pockets (HP and CTP). The molecules were built by clipping the NHR helices and connecting the new ends with short loops. The loops were manually built establishing helix capping interactions and maintaining favorable backbone psi and phi angles.
Protein and peptide samples The NHR and CHR gp41 sequences used in this work are described in Figure  1. The reference gp41 sequence was taken from the full gp160 precursor glycoprotein of the HIV-1 BRU isolate (Swiss-Prot entry sp|P03377|ENV_HV1BR). The covNHR miniproteins were computationally designed using SwissPDBviewer  54  and YASARA software  55. The DNA encoding the protein sequences were synthesized and cloned into pET303 expresion vectors (Thermofisher Scientific, Waltham, USA). To facilitate purification by Ni-Sepharose affinity chromatography, the protein sequences were histidine tagged at the C-terminus with the sequence GGGGSHHHHHH. The covNHR proteins were produced and purified following the protocol previously described  33. Synthetic CHR peptides, both N-acetylated and Camidated, were acquired from Genecust (Luxembourg), with a purity > 95%.
This work has been funded by the Spanish Ministry of Economy and Competitiveness  (grant:  BIO2016-76640-R ),  ANRS  and the  Vaccine Research Institute  for the  Investissements d'Avenir program  to CM and by the  European Fund for Research and Development  from the  European Union. SJ acknowledges a short-term fellowship from  Boehringer-Ingelheim Fonds. MCM was a recipient of a.
Serial sections were collected at ×20 magnification to reconstruct each whole-hippocampal image software (ZEN2. 1, Zeiss). The images were collected as Z-series of 18 optical slices to obtain a sufficient resolution to perform the morphological analysis of microglial cells. For each mouse, 3 representative stacks of images of the hippocampus were recorded. Positive cells for Iba-1 were counted (50-100 cells) and their morphology analyzed in each area e. g. the cornu ammonis (CA)1/CA2, CA3 and the dentate gyrus (DG). Image analysis and processing were performed with the software Image J -Fiji  [24]  using the "concentric circles" plugin. For the Sholl analysis, the intersection number per radian was defined each 5 μm from the center of each cell (n = 3 mice per treatment with 50-100 microglia analyzed per mouse).
This antibody recognizes only the pro-form of mouse IL-1β and does not detect the cleaved and secreted mature IL-1β form. Cells were then washed and re-suspended in lysing solution (BD FACS™ Lysing Solution) before the acquisition. Data were acquired with a flow cytometer (BD FACSCanto II) and analyzed with FlowJo v7. 6. 5 software (Tree Star, Ashland, OR). Very low SSC and very low FSC were excluded to strictly define the populations of interest. IL-1β pro-form staining was measured using geometric mean fluorescence intensity (GMFI). For the analysis, live single cells were pre-gated. Then, CD11b + /CD45 low cells were gated as microglia, while CD11b + /CD45 high cells or CD11b -/CD45 high cells were gated as infiltrating macrophage or lymphocyte cells, respectively. FMO controls were also included to define populations of Fixable Viability Dye cells and CD45, CD11b, and IL-1β-expressing cells.
Relative fold change in vehicle group (grey bar) and in rmIL-33 group (black bar) were quantified versus sham group (S; white bar). Minocycline-treated mice were also analyzed at 48 h post-surgery (dotted bar). IL-33 injection delayed the resolution of inflammation highlighted by an increase of inflammatory markers at 48 h administration. A partial reduction of this effect was observed under minocycline exposure, especially for IL-1b and Ifng mRNA. Data are represented as mean ± SEM (n = 4-6). Statistical comparisons were made using Kruskal-Wallis followed by Dunn's multiple comparison test for each group vs. Sham. *P ≤ 0. 05, **P ≤ 0. 01, ***P ≤ 0. 001. In addition, a comparison between rmIL-33 (48 h) and rmIL-33 + mino (48 h) groups was performed using the Mann-Whitney test. #P ≤ 0. 05, ##P ≤ 0.
b, c, d Histograms showing the number of Iba1 + cells by mm 2 quantified in each area (CA1/CA2 in b, CA3 in c, and DG in d). Quantification of Iba1 + cells was analyzed from 3-5 sections per mouse (n = 3-4 for each group). In rmIL-33-injected mice, the number of microglia was exacerbated in all hippocampal areas. Interestingly, under minocycline treatment, these increases were drastically limited. Data are represented as mean ± SEM. Statistical comparisons were analyzed using Kruskal-Wallis and uncorrected Dunn's test to generate P values for each paired comparison (each group vs. Sham without minocycline). *P ≤ 0. 05 Fig. 4  Effects of rmIL-33 treatment on microglial morphology in hippocampal areas. a Representative high magnification of Iba1 + microglia in sham, vehicle-or rmIL-33-treated mice (Scale bar = 10 μm).
b, c Sholl analysis was performed after Iba1 immunohistochemistry on the hippocampal section from 48 h post-surgery mice. b To evaluate the ramification complexity of microglial cells, the number of process intersections per radius was reported graphically in the curve. c Bar graphs show the cumulated number of intersections at distances up to 25 μm from the soma. The analyses were performed for each hippocampal area and for each group without or with minocycline treatment. In all hippocampal areas, Sholl analysis showed that microglia maintained hyper-ramified state following IL-33 injection, although vehicle treatment induced an intermediate-ramified state, as compared to sham. The minocycline pre-treatment impaired IL-33 effect on microglial morphology. Data are represented as mean ± SEM.
1%). Habituation in a novel environment was analyzed in intrasession (a) and in intersession 24 h and 48 h post-surgery (b). mRNA expression of pro-inflammatory markers (Nos2 in c, Il1b in d, Tnfa in e, and Ifng in f) or anti-inflammatory markers (Arg1 in g, Chil3 in h, Il10 in i, and Igf1 in j) was quantified by RT-qPCR normalized against 18S RNA at 48 h post-administration in WT/sham (WT-S), IL-1αβ KO/sham, IL-1αβ KO/vehicle and IL-1αβ KO/rmIL-33-treated groups (n = 4-6). Relative fold change in groups was quantified versus WT/sham group (S; white bar). Data are shown as mean ± SEM. The habituation analysis (a, b), n = 8-10 per group corresponding to a pool of 2 independent experiments. Two-way ANOVA followed by a Sidak post hoc test was used to analyze the distance traveled among the two groups (IL-1αβ KO injected with vehicle or rmIL-33).
Patients And Methods Patients were enrolled if they were aged of 18 years or older, with a diagnosis of TAK (according to the ACR criteria and/or Ishikawa criteria modi ed by Sharma) established between October 2014 and July 2017. All patients were treatment-naive or free from any immunosuppressive therapy (DMARDs and biological-targeted therapies) for at least 4 months. If previously initiated, steroids must have been started within the month preceding inclusion. The trial is registered on ClinicalTrials (NCT02101333), and could be nd at https://clinicaltrials. gov/ct2/show/NCT02101333? cond=takayasu&draw=2&rank=8; registration date 02/04/2014, registration number NCT02101333). All patients gave written informed consent and also consent for publication. The trial conformed the ethical guidelines of the Declaration of Helsinki and was approved by the Institutional Review Boards of the Ethic committee of "Cochin Hospital University" (CPP Ile de France, Cochin Hospital, 02/02/2014)(N° ID-RCB 2017-AO3380-53).
Importantly, 31% of the proteins present in the Enriched Flemmingsome (150 proteins) were already demonstrated to be localized to the furrow, bridge or midbody during cytokinesis and/or to be involved in cytokinesis (Supplementary Table  1 , TAB2). This demonstrates the strength of our proteomic study, as well as its potential for identifying new candidates (339) important for cytokinesis. The Enriched Flemmingsome thus represents a useful resource for the cytokinesis community and we created a dedicated website (https://flemmingsome. pasteur. cloud/) with continuous updated literature on each hit, as a reference database. Following up on the Enriched Flemmingsome, we found that ALIX, syntenin and syndecan--4 are indeed highly enriched at the midbody, then at the abscission site (Fig. 2 ).