dataesr/scientific-paragraphs-categorization huggingface dataset:
Acknowledgements We are indebted to  Christophe Lamaze ,  Cedric Blouin  and  Laurence Abrami  for DHHC clones,  Erwann Loret  for anti-Tat antibodies,  Corinne Lionne ,  Laurent Chaloin , and  Gudrun Aldrian  for help with Tat HPLC-purification,  Simon Lachambre  and  Fanny Martini  for pioneer phagocytosis experiments,  Jean-François Guichou  for expert assistance during ITC experiments,  Nicole Bec  and  Sarah Cianferani  for MALDI-TOF/TOF analysis and  Sara Salinas  for providing primary neurons. This work was funded by  ANRS ,  Sidaction  and the  FRM (Equipe FRM   20161136701 ) to B. B. and by  ANR  and  FRM  to N. V.
Trib1-ROSA Mb1Cre mice were obtained by crossing Trib1-ROSA KI mice with Mb1Cre Mice (24) that will allow Trib1 overexpression specifically in B cells from the pro-B cell stage. The presence of the Mb1Cre transgene was assessed by a PCR performed on tail DNA, using the following primers: Forward 5′ ACCTCTGATGAAGTCAGGAAGAAC-3′ and Reverse 5′-GCAGATGTCCTTCACTCTGATTCT-3′. All animal Hs99999903_m1, GAPDH: Hs99999905_m1, TRIB1: Hs00179769_m1). Each sample was amplified in triplicate in a StepOnePlus real-time PCR machine (Applied Biosystems). Relative expression levels were calculated with the StepOne v2. 1 software (Applied Biosystems), using the comparative cycle threshold method, and normalized to the endogenous control Hprt1, Gapdh and/or Beta-actin.
After a washing step, cells were fixed with 100 µL of fixation buffer from Fixation/Permeabilization kit (eBioscience) during 20 min at room temperature, in the dark. Cells were then permeabilized with the permeabilization buffer from Fixation/Permeabilization kit (eBioscience) and incubated for 30 min at room temperature in the dark with anti-mouse IgG1 (PE, Southern Biotech) and washed before acquisition by a cytometer. For all stainings, cells were analyzed using the FACS Calibur (BD Biosciences). The data were analyzed with FlowJo software (Treestar).
B cell Phenotype in Mice Overexpressing Trib1 in B cells We next assessed the development of B cells in primary and secondary lymphoid organs in Trib1-ROSA Mb1Cre mice, by flow cytometry analysis. The proportions of the different subsets of B cells in BM, spleen, and LNs were not different between Trib1-ROSA Mb1Cre and Trib1-ROSA control mice, showing that Trib1 overexpression in B cells does not have any impact on their development (Table  1 ). We detected in the spleen and LNs two distinct populations of GFP positive B cells in Trib1-ROSA Mb1Cre mice (Figure  2D ). However, the proportions of MZ, T1, T2, and follicular B cells were not different between splenic GFP + and GFP high populations. Of note, as expected, Trib1 overexpression in B cells did not impact neither T cell development (Table  S1  in Supplementary Material), nor the expression of activation markers CD44 and CD69 after stimulation with anti-CD3 and anti-CD28 antibodies for 72 h in vitro (data not shown).
B-cell-specific Trib1 Overexpressing Mice Do not Develop any sign of lupus Trib1-ROSA Mb1Cre mice were analyzed to evaluate the development of lupus symptoms. They do not develop any proteinuria, even at old age (18 months) (data not shown). We quantified the basal level of serum anti-dsDNA IgM autoantibodies, one of the hallmarks of SLE disease, by ELISA. At a young age (3-month-old), Trib1-ROSA Mb1Cre and Trib1-ROSA produce the same amount of anti-dsDNA IgM, whereas at 6 months, Trib1-ROSA Mb1Cre mice produce even less autoantibodies than control mice (Figure  4A ). At 3 months, as mice from C57BL/6 genetic background do not produce high titers of autoantibodies, we boosted the production of anti-dsDNA autoantibodies by an injection of LPS. The injection of LPS mimics a bacterial infection and induces a polyclonal activation of B cells, including autoreactive B cells producing natural autoantibodies.
DiscUssiOn We identified an overexpression of TRIB1 in human SLE B cells during clinically inactive disease by transcriptome analysis  (10). This overexpression was confirmed in a second cohort of SLE quiescent patients, by real-time qRT-PCR. This led us to study the Mass spectrometry analysis was performed on immunoprecipitation product (ntotal = 5). In each subgroup defining their main function, the proteins are ranked according to the number of experiments in which they were found as Trib1 partners (column « n ») and their confidence score. (Accession numbers are coming from Swissprot Database). Our results showed that this was not the case. Altogether, these characteristics made this new mouse model suitable for this study. Trib1-ROSA-Mb1 mice did not display any symptom of lupus disease: activation status of B cells is normal, and we did not detect any feature of renal disease.
FUnDing This work was supported by grants from Strasbourg University (UdS), Centre National de la Recherche Scientifique (CNRS), Arthritis Courtin foundation, by the Agence Nationale de la Recherche (ANR-13-JSV3-0007-01, LEDsGO; and ANR-11-EQPX-022), and by EU-funded (ERDF) project INTERREG V "RARENET. " LS, JR-L, and DB were supported by the Ministère de la Recherche et de la Technologie. JR-L was supported by Arthritis Courtin foundation. VD was supported by Initiative of Excellence (IdEx), Strasbourg University, France.
All Amplification experiments were repeated at least thrice to check reproducibility. Purified PCR products were sequenced in both directions on an ABI 3500 sequencer analyzer (Applied Biosystems, CA, USA) by using the secondary PCR primers and the BigDye Terminator v3. 1 Cycle Sequencing Kit (Applied Biosystems, CA, USA). The obtained sequences were inspected using the 4 peaks software (https://nucleobytes. com/4peaks/index. html), edited with the BioEdit sequence alignment editor (version 7. 2. 5), and analyzed for DNA database search and comparisons using the BLAST server (www. ncbi. nlm. nih. gov/ BLAST). Genotypes were named using the established gp60 genotype nomenclature  [31].
For each transition, the collision energy was experimentally optimized by testing 7 values (step of ±3 V) centred on the reference value. The reference value was calculated using the equation given by the supplier. The isolation width for both Q1 and Q3 was set to 0. 7 m/z unit. Mass data collected during LC-SRM were processed with the Skyline open-source software package 3. 6. 1  48. Area intensities of each precursor were manually checked. Time-lapse video microscopy. Diluted precultures (1/1000) of Candida albicans were incubated for 1 hour at 37 °C without agitation in glass bottom μ-Dish 35mm,high (Ibidi) previously treated with poly-L-lysine. The rhodamine-labelled peptides (Rho-L-Ctl or Rho-D-Ctl) were then added to the culture at a concentration of 10X MIC and incubated for 30 min at 37 °C under agitation.
To perform time-lapse video microscopy, the peptides were removed and replaced by Sabouraud medium. Yeasts were then plated at 37 °C, 5% CO 2 , on a Nikon Eclipse Ti inverted microscope equipped with an Andor Zyla sCMOS camera and a 60X objective. To prevent evaporation, a layer of mineral oil (Sigma) was added on top of the media prior imaging. Images (fluorescence and phase contrast) were captured every 10 min for 20 hours using Nikon NIS-Elements AR software, and processed with ImageJ.
B cell progenitors (B220 + ) were divided into immature (CD43 + ) and mature (CD43 -) cells on the basis of CD43 expression. Data quantified in the panels on the right show the percentage of live cells in the indicated gates. (D) Thymus from WT and Itsn2 -/-littermates were analysed by flow cytometry. Gating strategy is shown on the left. T cell progenitors (CD3 + ) were divided into double negative (CD4 -CD8 -), double positive (CD4 + CD8 + ), CD4 single positive (CD4 + CD8 -) and CD4 single positive (CD4 -CD8 + ) cells. Data quantified in the panels on the right show the percentage of CD3 + cells in the indicated gates. (E-H) Spleens from WT and Itsn2 -/-were analysed by flow cytometry. (E) Identification and quantification of B cells (B220 + TCRb À ) and T cells (B220 -TCRb + ). (F) T cells were subdivided into CD4 (CD4 + CD8 -) and CD8 (CD4 -CD8 + ) T cells and quantified.
1 BM and 50% WT or Itsn2 -/-BM, immunised with NP-KLH in Alum, and spleens of immunised animals were analysed by flow cytometry at day 13. CD45. 1 + and CD45. 2 + NP-specific GC B cells (NP + GL-7 + CD95 + , E) and Tfh (CD4 + CXCR5 + PD-1 + , F) are shown. Graphs on the right represent the CD45. 2/CD45. 1 ratio calculated from the percentages of cells in the indicated gates. (G) 80-20% mixed chimeras in mMT recipients were infected with 10 4 PFU of Vaccinia virus via intra-footpad injections, and popliteal lymph nodes were analysed by flow cytometry at d7. GC (GL-7 + CD95 + , G) and Tfh cells (GL-7 + CD95 + , H) are shown. Data are representative of 2-3 independent experiments with at least 4 mice in each group. Student's t-test, ns p>0. 05, *p<0. 05, *p<0. 01, ***p<0. 0001. DOI: https://doi.
Flow cytometry Single cell suspensions were prepared from spleen, lymph nodes, bone marrow or peritoneal lavage. After blocking Fc receptors using anti CD16/32 antibodies, cells were stained with the appropriate combination of the following antibodies: B220 (RA3-6B2), TCRb (H57-597), CD21 (7G6), CD4 (GK1. 5), CD8 (53-6. 7), CD3 (145-2 C11), CD23 (B3B4), CD24 (M1/69), CD43 (S7), Ly-51 (6C3), B and T cell activation antigen (GL7), CD95 (Jo2), CD138 (281. 2), IgG1 (A85. 1), CD5 (53-7. 3), CD11b (M1/ 70), CD19 (1D3), IgM (1B4B1 or II/41), IgD (11-26), CXCR4 (2B11), CD86 (GL1), CD84 (mCD84. 7), ICOSL (HK5. 3), SLAM (TC15-T2F12. 2), Ly108 (330-A5), CXCR5 (2G8), PD-1 (543), CD45. 1 (A20), CD45. 2 (104), MHCII (25-9-17), CD40 (3/23), CD69 (H1-2F3). Data was acquired on LSR Fortessa (BD Bioscience) and analyzed with FlowJo (Treestar, Ashland, Oregon).
1% Triton for 1 min and stained with Alexa Fluor 488 phalloidin (Molecular Probes). TIRF images were acquired with an EMCCD camera (iXon3 897, Andor, Northern Ireland) coupled to a TIRF microscopy system (Cell R; Olympus, Japan) with 488 nm, 561 nm and 640 nm lasers (Olympus). Images were recorded with Cell R software (Olympus) and analyzed with ImageJ software (NIH). Lymph nodes and spleen were embedded in OCT and frozen in cold isopentane. 10 mm wide frozen sections were cut with a cryostat. Sections were fixed in 4% paraformaldehyde, blocked with PBS containing 1% BSA and 10% goat serum (IF blocking buffer). Staining was performed in IF blocking buffer with a combination of the following antibodies: GL7-AF647, B220 PB (RA3-6B2), TCRb FITC or PE (H57-597), anti-k PE (187. 1), F4/80 APC (BM8).
Confocal imaging was performed with a Zeiss LSM 780 microscope, using a 20x objective (Plan-Appochromat 20X/0. 8NA) for spleen sections or a 40x objective (Plan-Appochromat 20X/1. 3NA, oil immersion) for LN sections. Tiled images were stitched using the ZEN software (Zeiss), and analysed in Imaris (Bitplane) or ImageJ (NIH). For multiphoton microscopy, explanted popliteal and inguinal lymph nodes were prepared as described  (Carrasco and Batista, 2007) , and imaged with an upright multiphoton microscope (Olympus), a 25X, NA 1. 05 water immersion objective, and a pulsed Ti:sapphire laser (Spectra Physics MaiTai HP DeepSee) tuned to 820 nm. Emission wavelengths were detected through band-pass filters of 420-500 nm (CTV), 515-560 nm (CFSE) and 590-650 nm (SNARF-1). Multidimensional movies were analyzed with Imaris.
Experimental data and statistical analysis Sample sizes were chosen on the basis of published work in which similar phenotypical characterization and similar defects were reported. Cohort randomization or 'blinding' of investigators to sample identity was not done in this study. In some experiments, the data for each study group were compared with Student's t-test or (when applicable) with a paired t-test and P values were calculated. Normal distribution of samples was assumed on the basis of published studies with analysis similar to ours. All statistical analysis was performed using the Prism software  (Graphpad Inc, San Diego, California). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Acknowledgements We thank the biological resource unit for animal husbandry (The  Francis Crick Institute) , the flow cytometry unit (The  Francis Crick Institute ), and the peptide chemistry unit for synthesis of Eapeptide (The  Francis Crick Institute ). The Itsn2 -/-strain was generated by the trans- NIH Knock-Out Mouse Project (KOMP)  and obtained from the  KOMP Repository  (www. komp. org). We thank  Daniel Lingwood  for kindly sharing with us the Hemagglutinin trimer and Flu vaccination protocol. We thank  Beatriz Montaner  and  Kathrin Kirsch  for help with managing animal colonies. We thank  Selina J Keppler  and all the members of the  Lymphocyte Interaction Laboratory  for scientific discussions and critical reading of the manuscript. We thank  Jan Sondenkamp  ( The Francis Crick Institute ) for imaging the ELISPOT plates.
Despite extensive optimization, crystals that diffracted to higher resolution were not obtained. While equally poor diffraction was initially obtained with CTL0886 C crystals, reductive methylation of surface exposed lysines  (Walter et al. , 2006)  enabled the growth of cubic crystals that diffracted synchrotron X-rays to 1. 90 Å resolution (Table  S2 ). The structure of CTL0886 C was determined using selenomethionine-labeled recombinant protein (also dimethylated) by Se-SAD and refined against the native highresolution dataset. The final model is comprised of a single polypeptide in the asymmetric unit, 1 sulfate anion and 98 water molecules. The CTL0886 C structure is comprised of 13 α-helices spanning two separate domains (Figure  2A ). Domain 1 consists of helices α1-7 arranged in a 7 helix bundle (residues 353-517).
Structural superposition of CTL0886 C domain 1 and geranylgeranyl transferase (GGTase) from Enterococcus faecalis (PDB ID: 3P8L,  Wallrapp et al. , 2013)  indicates these proteins align with an RMSD of 2. 52 Å across 110/164 Cα atoms (Figure  2B ) despite sharing only 5% sequence identity. GGTases transfer geranylgeranyl (GG), a 20-carbon lipophilic chain, to C-terminal cysteine residues of specific proteins  (Vandermoten et al. , 2009). The most striking similarity between these two structures is the highly conserved spatial organization of each α-helix within the bundle. CTL0886 C domain 1 is, however, lacking several α-helices typically found within the PTS family, including those involved in dimerization and extended regions typically found within the active site of PTS family enzymes (e.
DISCUSSION Our results converge to identify CT622 as a secreted protein that plays multiple and crucial roles in the initiation and support of the C. trachomatis growth cycle. We first demonstrated that CT622 translocation occurs through a T3S-dependent mechanism. Gong et al. also came to this conclusion but based on the use of a drug that was later shown not to be a specific inhibitor of T3S, therefore our FIGURE 5 | Knocking out CT622 expression impairs inclusion growth. (A) Dot plot distribution of the inclusion area in cells infected with WT, AS9, or AS9 complCT622 bacteria for 24 h before fixation and DNA staining with Hoechst. Random fields were taken and the area of all inclusions in that field were measured using Zen software. Gray bar, median. The star indicates a significant difference (Mann-Whitney-Wilcoxon test).
G Zhong (University of Texas). Mouse Goat secondary antibodies were conjugated to Alexa488 (Molecular Probes), to Cy3 or Cy5 (Amersham Biosciences). Hoechst 33342 was from Molecular Probes. Images were acquired on an Axio observer Z1 microscope equipped with an ApoTome module (Zeiss, Germany) and a 63× Apochromat lens. Images were taken with an ORCA-flash4. OLT camera (Hamamatsu, Japan) using the software Zen.
05% tween-20 TBS (TTBS) and probed for 1 h at room temperature with HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies diluted 1:1,000 in mTBS. Finally, blots were washed with TTBS followed by TBS, incubated with chemiluminescent substrate (Millipore), and imaged using a Bio-Rad Chemidoc MP with Image Lab software.
, 2014). Briefly, cells were washed with PBS and gently trypsinized. After a washing step in PBS cells were fixed in PFA 2% in PBS and bacteria were stained using anit-Hsp60 antibody (Affinity BioReagents, MA3-023) followed with Alexa488-conjugated secondary antibodies. Infection rates were measured using a Gallios flow cytometer (Beckton Coulter). The titration assay performed on the first round of infection allowed to correct for variations between experiments, and the data were expressed as inclusion forming units (IFU) obtained at 48 hpi for an input of 10∧3 IFU.
Mass Spectrometry Analysis, Database Search, and Protein Identification Digested peptides were analyzed by nano LC-MS/MS using an EASY-nLC 1000 (Thermo Fisher Scientific) coupled to a Q Exactive Orbitrap mass spectrometer. For input samples, about 1 µg of each sample (dissolved in 0. 1% FA) were loaded and separated at 250 nl/min on a home-made C 18 50 cm capillary column picotip silica emitter tip (75 µm diameter filled with 1. 9 µm Reprosil-Pur Basic C 18 -HD resin, (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany)) equilibrated in solvent A (0. 1% FA). The peptides were eluted using a two slopes gradient of solvent B (0. 1% FA in ACN) from 2 to 5% in 5 min, 5 to 30% in 150 min, 30 to 60% in 70 min at 250 nL/min flow rate (total length of the chromatographic run was 240 min including high ACN level steps).
For pull down samples, half of the sample was injected on a 30 cm long column and peptides were separated with a 120 min long gradient. The Q Exactive (Thermo Fisher Scientific, Bremen) was operated in data-dependent acquisition mode with the XCalibur software 2. 2 (Thermo Fisher Scientific, Bremen). Survey scan MS were acquired in the Orbitrap on the 300-1,700 m/z range with the resolution set to a value of 70,000 at m/z = 400 in profile mode (AGC target at 1 E 6). The five most intense ions per survey scan were selected for HCD fragmentation (NCE 27), and the resulting fragments were analyzed in the Orbitrap at 17,500 of resolution (m/z 400). Isolation of parent ion was fixed at 1. 6 m/z and underfill ratio at 1%. Dynamic exclusion was employed within 45 s. Data were searched using MaxQuant (1.
4. 1. 2 version) (with the Andromeda search engine) against the Human database from  SwissProt and TrEMBL (2014. 01. 14, 88 ,500 entries including 39,715 from swiss prot), and the C. trachomatis database from  Uniprot (2014. 10. 20, 889 entries). The following search parameters were applied: carbamidomethylation of cysteines was set as a fixed modification, oxidation of methionine, and protein N-terminal acetylation were set as variable modifications. The mass tolerances in MS and MS/MS were set to 5 ppm for each, respectively. Maximum peptide charge was set to 7 and 5 amino acids were required as minimum peptide length. Peptides and proteins identified with an FDR lower than 0. 1% were considered as valid identification. Label free analysis was done by using the "match between run" feature of MaxQuant (3 min time window).
Experimental phase information was obtained for the CTL0886 C structure by Se-SAD using AutoSol within the Phenix suite  (Adams et al. , 2002 (Adams et al. , , 2010)) , which identified nine unique selenium atoms in the asymmetric unit. Phenix Autobuild correctly traced 148/299 Cα atoms (Map-model CC = 0. 58, R work /R free = 41. 32/46. 48) within a single CTL0886 C polypeptide. Anomalous phases were combined with the complete 1. 90 Å native diffraction dataset using CAD  (Collaborative Computational Project, 1994). Subsequently, Phenix. Autobuild was then used to trace 241/299 of the expected amino acids from the combined experimental maps with R work /R free = 23. 05/26. 88. Structure refinement was carried out using Phenix. One round of individual coordinates and isotropic atomic displacement factor refinement was conducted, and the refined model was used to calculate both 2F o -F c and F o -F c difference maps.
ACKNOWLEDGMENTS We are very grateful to  Dr. C. Parsot  for initially pointing to the labile N-terminal portion of CT622 as a putative chaperone binding site, and for reagents and advice, and to  Sara Campos  for technical help. We acknowledge the contribution of the Recombinant Proteins in Prokaryotic Cells platform. We thank  Thibault Chaze  ( Proteomics platform ) for analysis of the pull-down experiment. We are thankful to  T. Hackstadt  ( Rocky Mountain Laboratories, USA ) and  G. Zhong  ( University of Texas, USA ) for anti-TarP and anti-CT813 antibodies respectively. This work was supported by an  ERC   Starting Grant  ( NUChLEAR N • 282046 ), the  Institut Pasteur  and the  Centre National de la Recherche Scientifique. MB and associated research was supported by  NIGMS   P30 GM110761.
A schematic workflow of the study is shown in Fig. 1. By studying genome-wide data of the nine animals, we detected 424 NS-SNPs possibly associated with PVL. We further genotyped these 424 candidate SNPs in the 42 Mauritian cynomolgus macaques experimentally infected with SIV (including the nine animals subjected to whole genome sequencing). For that purpose, we elaborated a customized sequence capture system. The sequences surrounding the SNPs of interest were captured from the 42 animal genomic DNAs and sequenced by a multiplexed approach by using the Illumina technique. The genes containing the variants most probably associated with PVL were analyzed.
The relatedness of animals is a consequence of the founder effect and isolation of Mauritian macaque population as evoked in the introduction (for more details see Supplementary note S1). The study of nine complete genome sequences led us to detect 424 candidate SNPs potentially associated with the PVL, out of which 297 were NS variants located in 188 functional genes. The list of these 188 macaque genes in which associated variants were detected, was compared with the list of genes which have been reported in the literature to be associated with HIV1 infection or replication. Among the 188 macaque genes 26 genes are present in the NCBI database. The list of these genes is given in Supplementary Table  S2. A high number of SNPs associated with PVL were found in the class II region of the MHC on macaque chromosome (chr.
) 4. Among these SNPs, only one lay in the macaque C4H6orf1 gene homologous to the human C6orf1 gene which is known to be associated in humans with the replication of the HIV (by reference to the NCBI database). Interestingly, it has been reported that knockdown of C6orf1 (chr. 6 open reading frame 1) by siRNA inhibits HIV-1 replication in HeLa-derived TZM-bl cells. The genotype of 42 animals did not confirm the association of the all SNPs lying in the MHC class II region. Outside the MHC, probabilities less than 0. 01 (Fisher's exact test) were confirmed for only 5 NS-SNPs which were submitted to MLR analysis. In case of NS-SNPs we detected inside the MYH13 (Myosin, Heavy Chain 13) and MYH8 (Myosin, Heavy Chain 8) genes, it has been reported that HIV-1 protease cleaves the human myosin heavy chains 8 and 13 in vitro.
Complete genome sequencing. For six animals, the library construction, sequencing, and initial quality check were performed at Beijing Genomics Institute (Shenzhen, China) (for details see: Osada et al. 5  ). As for the three remaining animals the library construction, sequencing, and initial quality check was performed at the CNAG using standard Illumina protocols (Barcelona, Spain). Calling of variants and search for their functional impact. All raw sequence reads were mapped to the draft genome of the cynomolgus macaque macFas5 reference sequence using BWA-MEM 0. 7. 5a-r405 with default parameters. Picard tools (https://github. com/broadinstitute/picard) were used to mark and remove PCR duplicates and to aggregate alignments and generate a single BAM file for each individual. Whole-genome alignments were deposited to the European Nucleotide Archive  19  under the accession number PRJNA (deposit in databank under submission).