dataesr/scientific-paragraphs-categorization huggingface dataset:
The insert mean size of the libraries was 0. 34 kp for the EV-producing cell samples, and 0. 45 kp for the EV samples (Supplementary Table  S1 ). Sequencing was performed with Illumina, NextSeq500, 75 cycles, single-read, High Output. For each experimental condition, three biological replicates were sequenced. EV-producing cell samples ranged from 9 to 27 million pair-end reads per sample, and EV samples ranged from 30 to 67 million pair-end reads per sample. Reads mapping to the reference genome ranged from 8 to 26 million, and from 0. 38 to 27 million reads for EV-producing cells and EV samples, respectively. Basic statistics of the RNA-Seq data are displayed in Supplementary Table  S1.
RNA coverage was calculated with Bedtools coverage  (Quinlan and Hall, 2010). RNAs with ≥ 90% coverage in at least one EV condition were kept for further indepth analysis. RNA coverage visualization was performed with the Integrative Viewer Software (IGV;  Thorvaldsdóttir et al. , 2013)  on a log scale. Subcellular location prediction was performed with SurfG+  (Barinov et al. , 2009). Clusters of Orthologous Groups (COGs) and KEGG categories were obtained using the eggNOGmapper v2 web tool  (Huerta-cepas et al. , 2017 (Huerta-cepas et al. , , 2019)). Functional enrichment analysis was performed with g:Profiler web-server  (Raudvere et al. , 2019; Reimand et al. , 2019). A maximum value of p 0. 05 was set as a threshold for significative categories. A timepoint clustering study was conducted with the R package maSigPro  (Conesa et al.
RT-qPCR Reverse transcription quantitative PCR (RT-qPCR) was used to validate RNA-seq results. EVs were isolated from the cellfree supernatants of three new independent S. aureus cultures at late-stationary growth phases (12 h) in the absence of vancomycin. EV RNAs were purified as mentioned above. Around 1. 5 μg of RNAs was treated with DNAse I (Amplification Grade, Invitrogen) according to manufacturer's instructions. cDNA synthesis was performed with the high capacity cDNA Reverse Transcription kit (Applied Biosystems). The primers used for quantitative PCR (qPCR) are listed in Supplementary Table  S2  and were designed using eprimer3 software (EMBOSS). qPCR was carried out in a 16 μl volume containing 15 ng cDNA, specific primers (300 nM), and 8 μl IQ ™ SYBR Green Supermix (Bio-Rad). Reactions were run on a CFX96 real-time system (Bio-Rad, France) using the following cycling parameters: DNA polymerase activation and DNA denaturation 95°C for 5 min, 40 cycles of denaturation at 94°C for 15 s, and extension at 60°C for 30 s.
Melting curve analysis was included to check the amplification of single PCR products. Samples setups included biological triplicates and technical duplicates as well as negative controls corresponding to qPCR reactions performed without cDNA (cDNA negative control) and from RT reactions obtained from EV RNAs without reverse transcriptase (RT negative control). Results were analyzed with the GFX Manager software and Ct values were determined. Results with Ct equal or above 40 were considered negative and only experiments with ΔCt ≥ 4 between negative controls and RT samples were considered.
Others, such as spa and RsaC, were differentially abundant between the EVs and the EV-producing cells. To evaluate the influence of the growth phase on EV and EV producing cell RNA composition, a negative binomial-based approach, with the R package maSigPro  (Conesa et al. , 2006)  was applied. Briefly, maSigPro provides a differentially expressed transcript analysis of serial data between experimental groups (e. g. , EV and EV producing cells). maSigPro was applied to the 286 highly covered EV RNAs and identified 91 RNAs with significant temporal profile changes (Padj < 0. 05). RNAs were clustered according to their expression profiles (Supplementary Table  S9 ). Figure  8  shows the six RNA clusters obtained. Three clusters grouped transcripts with a similar expression profile between EV and EV-producing cells: cluster 4 with 8 RNAs (including, e.
FUNDING This work has received financial support from  INRAE (Rennes, France)  and  Institut Agro (Rennes, France). This work was part of the  CARAVEL  project financed by the MICA division from  INRAE. BSRL and VRR were supported by the  International Cooperation Program CAPES/COFECUB  at the  Federal University of Minas Gerais  funded by  CAPES -the Brazilian Federal Agency for the Support and Evaluation of Graduate Education of the Brazilian Ministry of Education  (number  88887. 179897/2018-00and 99999. 000058/2017-03 ,respectively).
ACKNOWLEDGMENTS This work was conducted in the frame of  BactInflam International Associated Laboratory between INRAE (France)  and  UFMG (Brazil). It benefited from the facilities and expertise of the MRic-TEM platform (https://microscopie. univ-rennes1. fr). We are grateful to  Agnes Burel  ( Univ Rennes ,  BIOSIT -UMS 3480 ,  US_S 018 ,  Rennes, France)  for sessions with the microscope. We are also grateful to the genotoul bioinformatics platform  Toulouse Occitanie  ( Bioinfo Genotoul ) for providing computing and storage resources. Sandrine Parayre  ( INRAE, STLO ) and  Sandrine Péron  ( INRAE, STLO ) are warmly acknowledged for the technical assistance during RT-qPCR experiments.
Twenty-eight days after AA, blood was collected in dry tubes to retrieve serum. The animals were sacrificed by intraperitoneal injection of sodium pentobarbital. Aortas were snap frozen in liquid nitrogen. Longitudinal cryosections were cut at 5 μm thickness in optimal cutting temperature (OCT) medium, stained with 4',6-diamidino-2-phenylindole (DAPI) and orcein. Density of nuclei in the media and media surface area were measured in ten fields per aorta at x20 magnification and quantified using a computerized morphometric analysis system (Qwin Software, Leica Microsystems). Immunofluorescence staining of aortic allografts was performed using the following antibodies: polyclonal alpha-smooth muscle actin (alpha-SMA) and IgG, monoclonal CD68 (clone ED-1). Apoptosis was identified by the TUNEL assay.
Detection of circulating DSA was performed in LEW recipients twenty-eight days after AA by adding LEW sera on fresh BN splenocytes mixed with preimmune LEW sera. After incubation with IgG-AF647 and CD45R-FITC, IgG MFI was measured on CD45R negative cells, using a LSRII® flow cytometer and BD FACSDiva® software 6. 0. Statistical analyses were performed using GraphPad Prism 6. 0 (GraphPad Software, San Diego, CA). The differences between groups were evaluated using Mann-Whitney U test and ANOVA, as appropriate. The differences were considered significant when the P value was < 0. 05. These data validate the allogeneic aortic allograft as an accurate model of AMR, in which antibodies binding to donor SMCs are responsible for medial apoptosis and remodeling, as previously described  [4, 5].
INTRODUCTION The LabEx Milieu Interieur (MI) project (www. milieuinterieur. com) is a clinical study aiming to provide the first description of both genetic and environmental determinants of immunologic variance within the general healthy population  (Thomas et al. , 2015). Central to this study, blood samples from healthy volunteers were stimulated by a range of 40 distinct stimuli allowing to characterize the induced immune response (i. e. major secreted cytokines chemokines, transcriptomr etc. see  Duffy et al. , 2014; Urrutia et al. , 2016; Piasecka et al. , 2018). As part of the MI-program skin biopsies were collected from 323 out of 1,000 healthy individuals and primary human fibroblasts were prepared (Genethon, Evry, France). The MI-fibroblasts collection (323 primary fibroblasts) comprises a one-of-a-kind cell collection inasmuch as each derived primary cell isolate is uniquely associated with its corresponding donor data in the LabEx MI genotype-to-phenotype database (e.
Image acquisition and analysis After immunofluorescent labelling of fixed cells, two channel images were acquired in a fully automated and unbiased manner using an automated spinning disk confocal microscope (OPERA QEHS, Perkin Elmer Technologies, UtechS PBI, Institut Pasteur) and a 10× air objective (NA = 0. 4) with the following sequential acquisition settings: (i) 561 nm laser line excitation, filter 600/40 for Cy3 detection, or 488 nm laser line excitation, filter 540/75 for Alexa 488 detection and (ii) 405 nm laser line excitation, filter 450/50 for Hoechst 33342 detection. Forty-seven images per channel, covering roughly the entire surface of each well, were collected for reliable statistical analysis considering potential spatial cell and compound distribution biases. The images and associated metadata were transferred to the Columbus Conductor™ Database (Perkin Elmer Technologies) for storage and further analysis.
Plate was read on the BioPlex 200 (BioRad). Concentration were extrapolated with the Bioplex Manager software (v6. 1) using 5-PL curve-fitting regression algorithms with standards run in duplicate. For data representation, out of ratio values in high concentration (OOR >) were replaced with a value that is twice of the upper limit of quantification (ULOQ), which is measured in the dataset based on the standard curve. Cytokine dosage was performed at the Cytometry and Biomarker UtechS, Institut Pasteur.
To evaluate the delay between HSV-1 infection and the appearance of the VP26-GFP protein in infected cells, we first performed automated imaging microscopy using the IncuCyte Zoom device (Essen Biosciences). The fluorescent signal corresponding to the production of the capsid protein-GFP (VP26), produced after a replication cycle  (Desai and Person, 1998) , appeared only about 9 -10 hours after infection in our experimental conditions. This approach allowed to observe viral replication dynamics by following the fluorescent signal which appearing more rapidly indicated increased susceptibility of cells to infection (data not shown). Our aim was to establish a robust end-point measure that would allow a quantitative assessment of the response to infection. We thus tried a large range of multiplicity of infection (from 0,004 to 10 MOI) and several incubation times after infection to determine the conditions where GFP expression was optimally detected.
de sécurité du médicament (ASNM) on June 22nd, 2012. The study is sponsored by Institut Pasteur (Pasteur ID-RCB Number: 2012-A00238-35) and was conducted as a single centre interventional study without an investigational product. The original protocol was registered under ClinicalTrials. gov (study# NCT01699893). The samples and data used in this study were formally established as the Milieu Interieur biocollection (NCT03905993), with approvals by the Comité de Protection des Personnes -Sud Méditerranée and the Commission nationale de l'informatique et des libertés (CNIL) on April 11, 2018.
Retrieval of information We followed a similar methodology as Azari-Hamidian et al. [49]  for searching the literature. Briefly, published reference documents on medical and veterinary entomology were reviewed to collect information on mosquitoes, diseases, and pathogens  [50] [51] [52] [53]. An online search of scientific papers on mosquito-borne diseases using different search terms was undertaken using online bibliographic databases such as PubMed, Google, and Google Scholar. A combination of the following search terms were used to select publications on mosquito-borne diseases or mosquito species: "mosquito borne pathogens", "mosquito borne diseases", "mosquito borne viruses", "mosquito borne bacteria", "mosquito borne rickettsia", "mosquito fauna'' , mosquito genera including "Aedes", "Culex", "Mansonia", "Anopheles", "arbovirus", "malaria", "filariasis", "avian malaria", "trypanosomiasis", "vectorborne diseases", and "Cameroon".
Funding This work received financial support from the MTN-KFW programme awarded to  N-N SM  and  Wellcome Trust Senior  Fellowship in Public Health and Tropical Medicine [ 202687/Z/16/Z ] awarded to CAN. Part of this study was supported by the  Institut Hospitalo-Universitaire (IHU) Méditerranée Infection , the  National Research Agency  under the programme "nvestissements d'avenir", reference  ANR-10-IAHU-03 , the  Région Provence Alpes Côte d' Azur, and European funding FEDER PRIMI. The funding body did not have any role in the design, collection of data, analysis and interpretation of data, or writing of the manuscript.
Each DNA extract was then amplified in duplicate using real-time PCR assays. All laboratories performed real-time PCR targeting the 'rep529' non-coding DNA element (GenBank accession number AF487550)  [12, 25, 26] : five laboratories used their own laboratory-developed PCR assay and one used a commercialized Toxoplasma-PCR kit (Bio-Evolution, Bussy-Saint-Martin, France). The laboratory-developed PCR assays were performed using a LightCycler 2. 0, LightCycler 480 (Roche, Meylan, France) or StepOne (ThermoFisher Scientific, Montigny-le-Bretonneux, France) apparatus. PCR amplification with the Bio-Evolution kit was performed as recommended by the manufacturer on a Stratagene MX3005 (ThermoFisher Scientific). All six real-time PCR methods were thoroughly optimized and were previously assessed as highly performing methods  [13, 20, 27].
Results In total, five matrices and seven types of spiked samples were examined; 252 samples were DNA-extracted, yielding 648 PCR reactions. All matrices were controlled and found to be Toxoplasma-PCR negative before adding the parasites. All data are reported in S1 Table. In paucicellular samples, for AF and BALF, 100% of the PCR reactions were positive and no increase in Cp was observed over storage duration, at all concentrations tested (Fig  1A ). Thus, at the lowest Toxoplasma concentration, the means of Cp were 27. 82±0. 6 and 28. 30±0. 25 at D0 and D7, respectively, after storage at +4˚C, for AF; and 34. 23±0. 31 and 33. 75±0. 23, respectively, for BALF (Fig  2A ). For the CSF with the lowest T. gondii concentration, the proportion of positive PCR reactions significantly decreased with time and only 50% of reactions were positive at D7 (p<0.
Data analysis was carried out with Torrent Suite Software and variant detection was performed by both germinal and somatic variant caller plugin. The somatic variant caller correctly detected other six ALPS-sFAS patients previously diagnosed in the authors' laboratories. In summary, this approach allows the detection of both germline and somatic mutations related to ALPS by NGS, avoiding the isolation of DNT as the first step. The reads of the somatic variants could be detected even in patients with DNT in the cut off limit. Thus, custom-designed NGS panel testing may be a faster and more reliable method for the diagnosis of new ALPS patients, including those with somatic FAS mutations (ALPS-sFAS).
DNT Immunophenotyping For direct immunofluorescence, peripheral whole blood was incubated using the corresponding monoclonal antibodies as previously described  (14). Cellular acquisition was performed using a Beckman Coulter Navios cytometer (Beckman Coulter, Miami, FL, USA) and data were analyzed with Kaluza 2. 1 software (Beckman Coulter). Eight ALPS-sFAS patients were included in this study showing different DNT percentages (from 4 to 30. 7%) (Table  1 ).
Variant Caller, Annotation and In Silico Prediction Data analysis was carried out with Torrent Suite Software (version 5. 12). Data produced were aligned and mapped to the human GRCh37 (hg19) genome assembly by Torrent Mapping Alignment Program (TMAP). For germline variants, the variant caller plugin with default setting of "Generic PGM Germ Line Low Stringency" (Thermo Fisher Scientific) was performed. Somatic variants were detected by customization of the default setting of somatic variant caller plugin from Torrent Suite Software (TVC, v5. 12. Thermo Fisher Scientific) considering only the exons 7, 8 and 9 of FAS gene. The following parameters were adapted from the somatic variant caller of Torrent Suite server: minimum allele frequency permitted was 0. 01, minimum allele coverage was defined as 3 reads, no minimum coverage on either strand was defined but those variants with strand bias above 0.
85 and strand bias pvalue below 0. 05 was filtered out. Variant annotation was performed with Ion Reporter Software (version 5. 10). The software Integrative Genomics Viewer (IGV)  (26)  was used to inspect the aligned reads (bam files) of each sample in order to check the consistency of nucleotide calls. VarSome  (27)  was used for in silico pathogenicity prediction and for a comprehensive interpretation of the variants. The somatic FAS mutations were detected with this approach in seven out eight patients. It is important to consider that in the patient 8 (P8) which we could not be able to detect the somatic mutation, he had the DNT in the limit for our consideration as an ALPS patient (required criterium). In P1 and P4, the mutations were detected even with slightly high DNT.
9% (case of P7). Variant annotation and pathologic interpretations for new variants were performed with Ion Reporter Software and VarSome, respectively (Table  3 ). Somatic mutations previously described in P2-P7  (13, 24, 25)  and germline variants were excluded from variant annotation and in silico analyses. Somatic variant of P1 (c. 651+2T>C) was predicted as pathogenic and was previously reported as disease causing in ALPS-FAS patients  (10, 28). The somatic variant detected in both P1 and P4 (c. 587A>G) was not found in Clinvar and was classified as benign (twenty-one benign versus no pathogenic scores), but it was not detected in Sanger sequencing from their isolated DNT (data not shown). The somatic variant found in C5 (c. 675T>C) is probably a benign synonymous variant that not predicted an aberrant splicing (27, 29) (Table  3 ).
Thus, targeted gene panel sequencing is the optimal option to allow very deep coverage in a cost-effective way. Increasingly, new approaches are being developed for the exploration of somatic disease-causing mutations, being more frequent than expected. High-throughput NGS is presented as an important tool for the discovery of new diseases caused by postzygotic mosaicism  (34) (35) (36) (37) (38). Hence, there is a role for customized targeted NGS approach in some circumstances looking for somatic variants when germline variant calls remain noncontributory and might help correlate clinical and cellular phenotypes with genomic datasets. In an attempt to facilitate the molecular diagnosis of ALPS-sFAS patients, we describe clinical, immunologic and molecular findings of a new ALPS-sFAS patient diagnosed through NGS targeted gene panel.
FUNDING This work was supported by grants from  Fondo de Investigación Sanitaria  ( FIS-PI16/2053 ) to LA. The project has been co-financed with FEDER funds. ML-N was co-financed by " Fondo Social Europeo, Programa Operativo de empleo juvenil (YEI) ". The study was also supported by the  Institut National de la Santé et de la Recherche Médicale  (INSERM) and by government grants managed by the  Agence National de la Recherche  as part of the " Investment for the Future" program  ( ANR-10-IAHU-01  and  ANR-18-RHUS-0010 ), the  Ligue Contre le Cancer-Comité de Paris, Fondation ARC pour la recherche sur le CANCER , the  Centre de Référence Déficits Immunitaires Héréditaires (CEREDIH) , the  Agence National de la Recherche  ( ANR-14-CE14-0026-01  " Lumugene ";  ANR-18-CE17-0001  " Action ").
10 5 PBMCs per condition were suspended in X-vivo15 medium (Westburg) and activated as previously described  (22). Cell samples were analyzed using a 4-laser LSR Fortessa flow cytometer (BD Biosciences) using Diva software and analyzed with FlowJo software v10. 6. 1 (FlowJo, LLC). Antibodies and panels used for flow cytometry analysis are listed in supplementary Table  1.
In vitro ADCC assays of human PBMCs Cryopreserved isolated PBMCs from healthy volunteers and cancer patients were provided by ImmunXperts (Gosselies, Belgium), and ConversantBio (Huntsville, Alabama). PBMCs were thawed and resuspended in complete RPMI medium supplemented with 10% FBS + 50U/mL Pen/Strep (Westburg). 2. 5x10 5 cells were distributed per well and incubated at 37°C, 5% CO 2 for 20 h with either anti-TIGIT mAb, its corresponding IgG isotype control (BioXcell), or Rituximab at a final concentration of 6. 6 nM. Cells were then stained for surface markers as indicated in Supplementary Table  1. After washing, cells were fixed and, immediately before acquisition, AccuCheck Counting beads were added for absolute quantification following manufacturer's specifications. Data were acquired as described for flow cytometry.
Flow cytometry on dissociated CT26 murine tumors Two days after the second antibody treatment and 10 days post-tumor inoculation, CT26 tumor bearing mice were sacrificed and eight tumors per group were collected in ice-cold PBS and prepared for flow cytometric analysis. Surface staining was performed according to Table  1. Cells were fixed overnight at 4°C, treated again with Fc block (anti-CD16/CD32, eBioscience) for 15 min at RT, followed by intracellular staining with FoxP3-APC (supp Table  1 ) in permeabilization buffer for 30 min at 4°C. Cells were washed and resuspended in FACS buffer prior to acquisition. For ex vivo stimulation, cells were treated as previously described  (22)  and according to supplementary Table  1. Data were acquired on a MacsQuant flow cytometer (Miltenyi Biotech) and analyzed with FlowJo TM V10.
Statistical Analyses Statistical analyses are described in the figure legends. For tumor growth curves, data were fitted with a linear mixed model using SAS JMP® version 13 after log transformation of tumor volumes. Fixed effects included time and treatment. Polynomial functions of time were included based on significance of the polynomial term in sequential tests. The necessity to include random effects was assessed by a Wald test. Good model fit was assessed by plotting the fitted versus observed tumor growth curves and by residual analysis. A treatment difference in tumor growth was tested by a joint hypothesis test for the interaction terms of time and treatment  (25).
TIGIT is expressed by specific cancer cells and represents a target for direct cytotoxicity TIGIT has been described to be specifically expressed in immune cells  (1). Following investigation of public databases for TIGIT gene expression in cancers, we identified a high proportion of heme malignancies among samples with high expression level (Supp Fig. 6 ). To determine if this observation was due to strong TIGIT expression by immune cells in these blood cancers or to expression on tumor cells themselves, we acquired fresh samples from patients with Sezary syndrome (SS), a particular form of cutaneous T cell lymphoma (CTCL) with circulating clonal CD4 + tumor cells. To differentiate the normal and tumor CD4 + T cell population in these samples, we first determined the TCR-V rearrangement corresponding to the tumor clone.
Accordingly, during the course of Etigilimab development, an anti-TIGIT mAb under clinical evaluation, it was demonstrated that preferred Treg depletion is correlated with improved antitumor therapeutic activity of isotypes engaging activatory FcR, and peripheral Treg depletion was observed in patients during phase 1 trial  (41). Treg depletion may be explained by the better affinity of the surrogate anti-TIGIT mAb used in our study as compared to clone 10A7  (3, 40). Overall, these results demonstrate the better clinical potential of anti-TIGIT mAbs with high affinity for activatory FcR over FcR-disabled isotypes while limiting the possible depletion of effector T cell populations. To further understand the TIGIT expression in cancer, we compared several gene expression databases for TIGIT expression in different cancer and found the highest level of TIGIT in heme malignancies, particularly in T cell lymphoma.