dataesr/scientific-paragraphs-categorization huggingface dataset:
Gene expression. Total RNA from mouse microdissected lung compartments, whole lung, BAL, or cultured cells was extracted with Nucleospin RNA kits (Macherey-Nagel, Duren, Germany). RNA was reverse-transcribed with the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). A preamplification of cDNA from microdissected compartments was performed before real-time PCR using the PreAmp kit (Applied Biosystems). cDNA was amplified using SYBR Green or TaqMan assays. Relative quantification was performed as described. 23 croarrays. Lung total RNA (0. 5-2 mg) was processed on Affymetrix GeneChip Mouse Genome 430 2. 0 Array (Santa Clara, CA) or Applied Biosystems Mouse Whole Genome Arrays (Foster City, CA). Microarrays were analyzed respectively using the Robust Multichip Average method and the Bioconductor Linear Models for Microarray Data package and using the NeONORM method as described.
29, 30 IAME-compliant microarray data are available at http://mace. ihes. fr (3133411542) and www. ncbi. nlm. nih. gov/gds (GSE45369). Laser microdissection. Whole lungs were fixed, embedded in OCT compound (Sakura, Nagano, Japan) and frozen as described. 31 ections (15-20 mm) were stained with cresyl violet as manufacturer procedure (Ambion, Austin, TX). Using the LMD AS microscope (Leica, Wetzlar, Germany), 410 samples of epithelial layer of bronchi or alveolar regions per experiment were cut out and collected into RNA lysis buffer. Cell Culture. The normal human bronchial epithelial cell BEAS-2B was grown in complete epithelial cell culture medium (Promocell GmbH, Heidelberg, Germany). The human bronchial epithelial cell 16HBE was grown in Dulbecco's modified Eagle's medium (DMEM) with 5% fetal calf serum (FCS).
ACKNOWLEDGEMENTS We thank  Dr Christophe Carnoy  for critical reading of the manuscript,  Dr Edward Miao  for plasmids encoding NLRC4-deficient flagellins, and  Dr Philippe Gosset  for  BEAS-2B  and  16HBE  cells. L. V. M. , D. C. , D. F. , S. C. , J. T. and J. C. S. were funded by  INSERM ,  Institut Pasteur de Lille, Region Nord Pas de Calais  ( ARCir Europe  n1R07028EE and PhD fellowship),  Univ Lille Nord de France ,  Agence Nationale de Recherche sur le SIDA  ( n12008/ 058 ),  INSERM-CONICET  joint project ( n1174 ),  Reseau National des Ge  ´nopoles (n1203), and  European Community  ( STREP SavinMucoPath INCO-CT-2006-032296 ). AGB was funded by the  Agence Nationale pour la Recherche  ( Physio2007 n1013-01 ) and the  Genopole Evry. DISCLOSURE The authors declared no conflict of interest.
Ehrlichia sp. UFMG-EV trp36 gene evolved from a highly divergent clade within E. canis To study the evolution of trp36 gene we used a combination of phylogenetic and evolutionary analysis (see Additional file 1 for detailed description of materials and methods). The gene trp36 has been widely used to study the genetic diversity of E. canis strains  [7] [8] [9] [10]. We performed maximum likelihood and neighbor joining phylogenetic analyses with trp36 nucleotide sequences available in GenBank (Additional file 1) to study the evolution of Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV trp36 in relation to E. canis trp36. The phylogenetic analysis showed that Ehrlichia sp. UFMG-EV and Ehrlichia sp. UFMT-BV trp36 are separated but clustered together with E. canis strains from South Africa, Taiwan and Brazil (Figure  1 ).
Acknowledgments This research was partially supported by the  EU   FP7   ANTIGONE  project number  278976. JJV was sponsored by project  CZ. 1. 07/2. 3. 00/30. 0032 ,co-financed by the  European Social Fund  and the state budget of the Czech Republic. ACC was supported by a grant from the  Ministère de l'Education Supérieure et de la Recherche of France.
Ethical aspect This study is based on data collected retrospectively at two different times among the same population of gendarmes. A major outbreak of CHIKV in Reunion Island ended in June 2006. In that context, it was decided that a study on the prevalence and consequences of infection among gendarmes would be conducted, because CHIKV was considered to be a possible work-related disease. All the gendarmes on duty in Reunion Island were offered a serological test for CHIKV and self-report questionnaires by their military physician. Testing was considered to be part of routine care, but patients were informed about the study by a written notice attached to the selfquestionnaire and the military physician received their oral consent before inclusion. Only those who accepted the blood test and questionnaire were included.
Statistical analysis To identify the prognostic factors associated with persistent arthralgia or arthritis 30 months after infection (median duration of follow-up in 2008), we first analyzed the entire 2008 study sample using a multinomial logistic regression with "no rheumatic disorders" as the reference category. To implement the regression model, all the predictive variables were first assessed individually using univariate regression models. Then, variables with a significant p-value < 0. 20 were included in the multivariate regression model, and a backward stepwise procedure was used to keep as predictors the variables with p-value <0. 05. These regressions were performed using STATA software, version 9. The CHIKV acute stage symptoms were largely shared by CHIKV + patients and absent in CHIKV-patients.
For these analyses, XL-Stat 9 and R 2. 15. 1 softwares were used. The MCA displays the individuals and variable categories as labeled points in a multiple dimensional space. It decomposes all the information represented by variables and individuals into multiple factorial axes, such that each successive factorial axis captures a part of the total information in decreasing order  [17]. Thus, the first two factorial axes capture the maximum amount of information available in the data. With the help of these two factorial axes, the graphic display of variable categories can reveal the structural relation between variables. In the graph, the more distant a variable category is from the origin, the higher its contribution is (i. e. the contribution of categories near the origin is small), but outliers situated too far from the origin are no longer informative.
In the study, sex categories were thus excluded as there were only 3 females, which led to a distortion of the MCA graph (the category "women" representing an outlier that was too far from the origin). Most interestingly, the interpretation of data associations can easily be read on the MCA graph: the closer the variable categories are on the graph, the stronger they are associated. Here, the categories of self-perceived recovery at 30 months and rheumatic disorders were superimposed on the MCA graph to observe which symptoms were closer to (i. e. were associated with) self-perceived recovery, arthralgia and arthritis. The strength of these associations was tested in binary analyses using the Chi 2 test. One property of MCA is the possibility of providing a severity scale from 0 (corresponding to the theoretically least severe case) to 100 (corresponding to the theoretically most severe case) for each factorial axis  [18].
Fluorescent labelling of lung cell suspensions was undertaken upon re-suspension in HCF medium (Hanks medium for flow cytometry) with 3% decomplemented fetal bovine serum (FBS, Invitrogen). Fc receptors were blocked with anti-CD16/32 (clone 2. 4G2, BD Biosciences). Cells were stained using antibodies specific for CD4 (clone RM4-5) and CD8 (clone 53-6. 7) obtained from BD Biosciences. Samples were fixed in a 1. 25% paraformaldehyde solution in PBS for at least 1 h, acquired on a FACSCalibur (BD Biosciences) and analyzed using the CellQuest software (BD Biosciences). Analysis of cell populations was undertaken with appropriate gating according to side and forward scatters to exclude dead cells as well as silica particles. CD4+ T cells were separated and isolated by magnetic cell separation system (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) by positive selection with anti-CD4 magnetic beads.
This work was funded by the '' Marshall'' Programme d'excellence Diane convention , by the  Fonds de la Recherche Scientifique Medicale (FRSM) , by  Actions de Recherche Concertees, Communautefrancaise de Belgique, Direction de la Recherche Scientifique  ( ARC 09/14-021 ), by the  Fondation Contre le Cancer , by the  Fonds de la Recherche Scientifique (FNRS ;  Project PDR T. 0119. 1314633768 ), by the  European Commission  under  FP7-HEALTH-F4-2008  (Contract no. 202047 ), and by  Agence Nationale de securitesanitaire de  l'alimentation, de l'environnement et du travail (ANSES, France). F. H. is a Research Associate with the  FNRS, Belgium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Gene Data Expression Analyses Cluster analysis. GenBank accession number -GSE 32229. Log 2 signals were extracted after interslide quantile normalization and performed with R (Bioconductor). Probe sets showing minimal variation across all samples were filtered out, only retaining those 3217 with a coefficient of variation over 0. 1, and using them to cluster the samples through the hierarchical agglomerative algorithm. Average linkage and Euclidian distance were used as linkage and similarity methods, respectively. Supervised classification was carried out using Cluster and TreeView software. Detection of differentially expressed genes. Statistical analyses used the R and Bioconductor software. To identify over-or under-expressed genes, we used the empirical Bayesian method for differential expression calculation  [26]  implemented in the Linear Models for Microarray Analysis (LIMMA) package.
Immunohistochemistry and immunofluorescence Immunohistochemistry was performed as described previously  [20]  using antibodies directed against phospho-histone 3 (Upstate), PCNA and GRP78/Bip (Abcam), cleaved caspase-3 (Cell Signaling), Muc2, and ATF6a (Santa Cruz Biotechnology, Tebu-bio), phospho-eIF2a (Cell Signaling), Foxp3 (eBiosciences), and Muc4  [25]. Immunofluorescence studies were performed using antibodies directed against KDEL (Enzo Life Sciences), GRP78/Bip and active caspase 3 (Cell Signaling), and Muc2 (Santacruz), and then labeled with the appropriate secondary antibody (Life Technologies). Nuclei were stained using TO-PRO-3 iodide. Fluorescence was detected by confocal laser scanning microscopy (CLSM-510-META, Zeiss). All images were acquired using the Zeiss LSM Image Browser software.
Flow cytometry analysis Cell suspensions were incubated with PE-, FITC-, APC-, or PerCP-conjugated mAbs against mouse CD3, CD4, CD8, CD11c, CD19, CD44 high , CD62 l u w , NK1, CD45Ly. 1, and CD45Ly. 2 (BD Biosciences) at optimal concentrations for 20 minutes at 4uC. Antibodies for the intracellular staining of Foxp3+ Tr eg cells were from eBioscience. Labeled cells were analyzed using a BD-LSR II device and CELLQuest software (BD Biosciences).
Acknowledgments The authors thank  Pr. Jean-Fre  ´deric Colombel for helpful discussions and contribution to manuscript editing,  Drs. Laurence Bougne  `res-Vermont and  Matthieu Collin  from  INSERM-Transfert ,  Matthieu Coutet  from  INSERM-Transfert Initiative , and  Pr. Renato Monteiro  ( INSERM UMR-S699 ) for helpful discussions,  Olivier Thibaudeau  ( IFR02 ) and  Sylvie Mosnier  ( Beaujon Hospital ) for helpful technical assistance, and  Association Francois Aupetit (AFA), and labex ''Inflamex ''. Electron microscopy was performed by  Alain Grodet  in the setting of the  Atelier de Microscopie ElecTronique (AMET)   IFR02/CRI.
This work was supported by grants from : Institut National de la Santeet de la Recherche Medicale (INSERM) ,  INSERM-Transfert (CoPoC 's grant to XT and EOD),  Agence Nationale de la Recherche  ( ANR Emergence  to EOD),  Association Francois Aupetit (AFA)  (to XT and EOD),  Assistance Publique-Hopitaux de Paris (AP-HP)  (to EOD:  Interface  Fellowship),  University Denis Diderot Paris 7, and labex ''Inflamex '' (to EOD). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Peroxidase activity was revealed using an ECL (enhanced chemiluminescence) or ECL advanced kit. The luminescence was captured with a Fuji intelligent dark box camera (LAS 3000, Fujifilm, Dusseldorf, Germany). Quantifications were performed with the Multigauge V3. 0 software (Fujifilm). Immunofluorescence. For LAMP-1 detection, cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0. 2% Triton-X100. For 8-oxo-G detection, cells were fixed in 4% paraformaldehyde in PBS and postfixed in 70, 95 and 99% methanol for 30 min at -20 °C and then rehydrated by 95 and 70% methanol at -20 °C for 3 min and three washes in PBS. The primary antibodies used were anti-LAMP-1 (Transduction Labs, Erembodegem, Belgium; L76620) and anti-8-oxo-G (AB5830 Chemicon International, Frederiksberg, Denmark).
Flow cytometric analyses were performed using a Beckman Coulter (Pasadena, CA, USA) Epics XL-MCL, a BD LSR Fortessa (Becton Dickinson, Erembodegem, Belgium) or a BD FACSCanto II (Becton Dickinson). 8-oxo-G detection was performed exactly as above for microscopic analysis. Aggresome detection was performed using the ProteoStat Aggresome detection kit (Enzo life Sciences) according to the manufacturer's instructions. Collected data were exported to the WinMDI 2. 9 (J. Trotter, Salk Institute for Biological Studies, La Jolla, CA, USA) or FlowJo (Ashland, OR, USA) softwares for detailed analysis. Sorting of NHEKs was performed on a BD FACS Aria (Becton Dickinson) or on a Coulter FACS Altra (Beckman Coulter). Selected subpopulations with the ad hoc forward scatter factor and/or fluorescent staining intensity values were electrostatically sorted in air, collected in complete culture medium and put again in culture.
Acknowledgements. This work was supported by the  Centre National de la Recherche Scientifique , the  Université Lille 1 , the  Université Lille 2 , the  Ligue contre le Cancer , the  Association pour la Recherche sur le Cancer , the  Institut Pasteur de Lille  and the  SIRIC OncoLille (Grant INCa -DGOS-Inserm 6041 ). ED had a fellowship from the  Institut Pasteur de Lille  and the  Région Nord/Pas-de-Calais. JN has a fellowship from the  Université Lille 2  and from the  Association pour la Recherche sur le Cancer. NM had fellowships from the  Centre National de la Recherche Scientifique  and the  Région Nord/Pas-de-Calais , the  Ligue contre le Cancer  and the  Lille 1 University. We thank the  Bioimaging Center Lille-Nord de France (Campus Calmette) ,  Antonino Bongiovanni  and  Hélène Bauderlique  for imagery and cytometry facilities.
The data sets for the 1. 5 and 2. 0 A ˚resolution structures were refined using HKL-2000  (Otwinowski & Minor, 1997) , while HKL-3000  (Minor et al. , 2006)  was used for the 1. 6 A resolution data. Molecular replacement with Phaser  (McCoy, 2007)  in the PHENIX  (Adams et al. , 2010; Terwilliger et al. , 2008)  suite was used to solve the phase problem using EndA from S. pneumoniae as the search model (PDB entry 3owv;  Moon et al. , 2011). A partially refined model of this structure (space group P1) was then used as a search model in molecular replacement against the 1. 5 A ˚resolution data set. The same R free reflections were used for each of the high-resolution data sets. The 2. 0 A ˚resolution Mg 2+ -bound GBS_NucA data set was solved by molecular replacement (space group P6 3 ) using the structure from the 1.
Despite the low overall sequence and structural conservation, the motifs of these nucleases aligned well, as did the histidine residue thought to serve as the general base and the asparagine chelating the divalent metal in the active site (Fig. 3a ) Structural superposition of GBS_NucA (H148A) with the most closely related EndA (PDB entry 3owv;  Moon et al. , 2010)  reveals that the two nucleases have a nearly identical structure (r. m. s. d. of 1. 1 A ˚over 192 C atoms; Figs. 3b, 3c  and 3d ) and electrostatic surface potential (Figs. 4g, 4h, 4j  and 4k ). The most notable difference is in the conformation of the N-termini: in GBS_NucA the N-terminus is comprised of a short coil that is somewhat variable in structure, leading to an 11-residue -helix (A, Gln48-Ile58). The N-terminus of EndA, by comparison, has a longer coil which begins on the 'front' face of the enzyme and travels across a hydrophobic 'shoulder' of the enzyme to the 'back' face (Fig.
3d ). There it forms two smaller -helices (A, Gln51-Val57; B, Asp60-Gln65) residing in a position similar to A in GBS_NucA (Figs. 4a  and 4b ). Although the reasons for this structural difference are unclear, the variation could be related to the localization of each enzyme: NucA is secreted into the extracellular medium, while EndA can either be secreted or membrane-associated as a component of the competence complex  (Lacks & Neuberger, 1975; Puyet et al. , 1990). Other small structural variations can be observed in external loop regions, which might be influenced by differences in both sequence and crystal-packing interactions (Fig. 3c ). GBS_NucA aligns less well with the structure of Spd1 (r. m. s. d. of 1. 9 A ˚over 141 C atoms using PDB entry 2xgr;  Korczynska et al. , 2012; Figs. 3e, 3f and 3g).
Biochemical characterization of NucA surface residues: effects on substrate binding and catalysis Thus far, there are no published structures of EndA, Spd1 or other similar metal-finger nucleases in complex with a bound DNA or RNA substrate. Similar attempts to cocrystallize such a complex with GBS_NucA have likewise failed (unpublished data; see Supporting Information). However, X-ray crystal structures exist for the -Me periplasmic nuclease VVN from Vibrio vulnificus in complex with duplex DNA [PDB entries 1oup  (Li et al. , 2003)   c ) GBS_NucA (H148A) surface residues chosen for mutagenesis with likelihood of involvement in catalysis (red) or DNA substrate binding (cyan or purple). (Wang et al. , 2007) ]. VVN is unreleated to the Streptococcus sp. -Me nucleases and exhibits no sequence conservation.
Sequence interruptions of the VVN nuclease sequence are shown as repeated dots, with the residue numbers below. (b, c, d) Structural superposition of GBS_NucA (H148A) (blue) and EndA (H160A) (PDB entry 3owv; Moon et al. , 2011; orange), emphasizing the 'front' (b), 'back' (c) and 'side' (d) faces of the enzymes. (e, f, g) Structural superposition of GBS_NucA (H148A) (blue) and Spd1 (PDB entry 2xgr (Korczynska et al. , 2012; green), emphasizing the 'front' (e), 'back' (f) and 'side' (g) faces of the enzymes. Dashed circles highlight regions of structural dissimilarity.
FLUORESCENCE MICROSCOPY The moDCs and dDCs (10 5 cells per assay) were cultured on glass slides for 18 h. After fixation in 3% paraformaldehyde for 15 min, they were permeabilized by 0. 1% TritonX-100 for 2 min and then incubated for 30 min with bodipy phallacidin (Invitrogen) to label filamentous actin (F-actin). Cell nuclei were labeled with DAPI (Invitrogen) for 10 min and slides were mounted on Mowiol (Invitrogen). Pictures were taken using a confocal microscope DMI16000 (Leica, Nanterre, France) and analyzed using Image J software (National Institute of Health, USA). In some experiments, moDCs and dDCs were incubated with C. burnetii and B. abortus for 18 h. C. burnetii and B. abortus organisms were revealed using human and bovine specific Abs, respectively. Secondary Abs consisted of anti-human and -bovine Abs coupled with 555 Alexa fluor.
MICROARRAYS Total RNA of moDCs and dDCs (2 × 10 6 cells per well) was extracted using the RNeasy minikit (Qiagen, Courtaboeuf, France) and DNAse treatment  (Gorvel et al. , 2014). The expression of modulated genes was analyzed using 4X44k Human Whole Genome microarrays (Agilent Technologies, Les Ulis, France) and three biological replicates per experimental condition, as recently described  (Ben Amara et al. , 2010). Sample labeling and hybridization were performed using One-Color Microarray-Based Gene Expression Analysis. Slides were scanned at a 5 μm resolution with a G2505C DNA microarray scanner (Agilent Technologies, Les Ulis, France). Image analysis and intraarray signal corrections were performed using Agilent Feature Extractor Software A. 9. 1. 3. Microarray data analysis was performed using the R (v.
2. 15) and Bioconductor libraries, as recently described  (Moal et al. , 2013). In brief, raw data were filtered and normalized using the Agi4x44PreProcess library. Unsupervised and supervised analyses were carried out using hierarchical clustering, principal component analysis (PCA) (made4 library,  Culhane et al. , 2005) , and Significance Analysis of Microarray (SAM) algorithm (siggenes library). Genes were considered to be differentially expressed when absolute fold change (FC) was above 2. 0. Functional enrichment analysis was performed on selected genes with DAVID tools  (Dennis et al. , 2003) , using the Gene Ontology (GO)  (Ashburner et al. , 2000) , and Kyoto Encyclopedia of Genes and Genomes (KEGG)  (Okuda et al. , 2008)  pathways. Functional pathways were designed using the Cytoscape and Inkscape softwares.
The acquisition pulse sequence used was a 90°pulse sequence with water presaturation and 10 s recycle time. Typically, the accumulation of 2,048 scans was used. Spectra were referenced using the 3-(trimethylsilyl) 3, 3, 2, 2-tetradeuteropropionic acid Na salt (d4-TSPA, Sigma-Aldrich, USA) signal at 0 ppm. FID signals were processed using Topspin 2. 1 Ò software. A line broadening of 0. 3 Hz was applied. Peakfit Ò software was used for spectral deconvolution to access chemical shift, coupling constants and peak areas.
LTA structural elucidation Using chemical shifts previously assigned  (Jang et al. 2011; Morath et al. 2001) , data described in Table  1  were used for LTA structure elucidation. The average chain length of the poly (glycerol-phosphate) backbone and degree of D-alanine and N-acetylglucosamine substitutions were quantified from the results of Peakfit Ò software deconvolution of LTA. The total amount of glycerol was determined from the area of the region between 3. 8 and 4. 2 ppm. The area ratio of glycerol and a-methylene group of the membrane anchor between 2. 2 and 2. 5 ppm identified the different average chain length. The area ratio of substitute alanine and total amount of glycerol gave the proportion of glycerol esterified by alanine. The area ratio of a-D-N-acetyl-glucosamine H 4 (d = 3.
RNA extraction, reverse transcription, and quantitative PCR analysis Total RNA was extracted from cell populations by TRIzol (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from 0. 5-1 μg of total RNA using the Superscript II reverse transcriptase (Invitrogen). Fifty-five nanograms of cDNA were used as a template and mixed with 10 μL of 2× Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) and primers (Supplementary Table  S1 ) in a reaction volume of 20 μL. Cycling conditions were 10 minutes at 95°C, 60 cycles of 15 seconds at 95°C, and 1 minute at 60°C. Each sample was run in duplicate on ABI PRISM 7900HT Sequence Detection System (PE Biosystems). Results were analyzed using the comparative ΔΔCt method: data are presented as the fold difference in gene expression normalized to the housekeeping gene β2-microglobulin and relative to a relevant reference sample.
Results hDLL4 upregulates MSI-1 transcript levels in CRC cells by a NOTCH2/3-mediated mechanism We preliminarily established conditions for ligand-mediated stimulation of NOTCH signaling in CRC cells. To this end, MICOL-14 tum cells transfected with the CSL-luc plasmid were stimulated by hDLL4 and NOTCH activity measured by in vitro luciferase reporter assay 72 h later. An 8-fold increase in luciferase activity was observed in hDLL4-stimulated cells, compared with basal levels (Supplementary Fig. S1A ). Importantly, these effects were blocked by anti-NOTCH2/3 but not by anti-NOTCH1 mAb (Supplementary Fig. S1A ). Because MICOL-14 tum cells express NOTCH1-3 but lack NOTCH4 receptors  (13) , we could conclude that DLL4 stimulation triggers NOTCH signaling mainly through NOTCH2 and/or NOTCH3. These data were also supported by measurement of endogenous components of the NOTCH pathway by qRT-PCR.