dataesr/scientific-paragraphs-categorization huggingface dataset:
Acknowledgments This work has been carried out with financial support from the  Spanish Ministerio de Ciencia y Tecnología (MCYT)  under Research Project No. FIS2011-25161 , and  Junta de Andalucía  under research projects  P10-FQM-5735  and  P09-FQM-4584. It was also partially funded by the  French Government program Investissements d'Avenir (LABEX INTERACTIFS , reference  ANR-11-LABX-0017-01 ) and by a grant from the french district PoitouCharentes.
Deregulation of signaling pathways controlled by protein phosphorylation underlies the pathogenesis of hematological malignancies; however, the extent to which deregulated phosphorylation may be involved in B-cell non-Hodgkin lymphoma (B-NHL) pathogenesis is largely unknown. To identify phosphorylation events important in B-NHLs, we performed mass spectrometryebased, label-free, semiquantitative phosphoproteomic profiling of 11 cell lines derived from three B-NHL categories: Burkitt lymphoma, follicular lymphoma, and mantle-cell lymphoma. In all, 6579 unique phosphopeptides, corresponding to 1701 unique phosphorylated proteins, were identified and quantified. The data are available via ProteomeXchange with identifier PXD000658. Hierarchical clustering highlighted distinct phosphoproteomic signatures associated with each lymphoma subtype.
amjpathol. org -The American Journal of Pathology 35%). Dynamic exclusion was set to two MS/MS acquisitions, followed by exclusion of the same precursor ion for 2 minutes. Maximum ion injection times were set to 300 milliseconds for MS and 100 milliseconds for MS/MS. Automatic gain control was set to 1 Â e 6 for MS and 5000 for MS/MS. Charge state screening was enabled to discard þ1 and unassigned charge states. Technical duplicate data for each of the MOAC elutions (ammonium hydroxide and pyrrolidine) and triplicate data for the phosphotyrosine immunoprecipitation samples were acquired.
Bioinformatics Analysis RAW files were converted to mzXML using msconvert  16  and searched against the Swissprot Human protein database (http:// www. uniprot. org) appended with common proteomics contaminants and reverse sequences as decoys. Searches were performed with X!Tandem version 2010. 10. 01. 1 (http://www. thegpm. org/TANDEM) using the k-score plug-in. 17, 18 For all searches, the following search parameters were used: parent monoisotopic mass error of 50 ppm and fragment ion error of 0. 8 Da, allowing for up to two missed tryptic cleavages. Variable modifications were oxidation of methionine, carbamidomethylation of cysteine, and phosphorylation of serine, threonine, and tyrosine. The search results were then postprocessed using PeptideProphet (http://peptideprophet. sourceforge.
net) and ProteinProphet (http://proteinprophet. sourceforge. net). 19e21  Spectral counts were obtained for each cell line using ABACUS. 22 Tyrosine-enrichment data were processed through ABACUS separately from the serine and threonine data. ABACUS results were filtered to only retain proteins with a ProteinProphet probability of >0. 7. Only phosphorylated peptides with a probability of >0. 8 were considered for spectral counting. These parameters resulted in a protein false-discovery rate (FDR) of 0. 1 for the serine and threonine data. For the tyrosine enrichment, these ABACUS parameters resulted in a protein FDR of 0. 0045. This ABACUS output was used for all subsequent analyses to quantify the relative abundance of phosphorylated peptides/proteins. Phospho-site localization was performed with an inhouse reimplementation of the Ascore algorithm, as described by Beausoleil et al.
The minimum motif occurrence for Motif-X was set to 20, and only motifs with P < 10 À5 were considered. Hierarchical clustering of the spectral count data were performed separately for each enrichment type (tyrosine versus serine/threonine). Apart from this, the procedure was performed in the same manner on both data sets, as follows. Only proteins with a total spectral count higher than nine in at least one of the cell lines used in this study were retained. After filtering, all protein spectral counts were normalized first to the total spectral counts within each cell line, and then to the total across all cell lines. These normalized values were log2 transformed and underwent hierarchical clustering with a multiscale bootstrap resampling for 10,000 iterations using the pvclust package in R (http://www.
The mean efficiency of p-Ser/p-Thr peptide enrichment with MOAC was 83. 4% (SD, 6. 5%), and the mean efficiency of p-Tyr peptide enrichment in pY-IP was 54% (SD, 14. 7%). The reproducibility between the biological triplicates ranged from 0. 65 to 0. 85 (Supplemental Table  S2 ). In total, 868,578 MS/MS spectra were assigned to 6640 unique phosphorylated peptides. The data have been deposited to the ProteomeXchange Consortium (http:// proteomecentral. proteomexchange. org) via the PRIDE (PRoteomics IDEntifications database) partner repository  27  with the data set identifier PXD000658. MS/MS analysis of peptides obtained from the MOAC enrichment led to identification of p-Ser peptides (4752 unique peptides) and/or p-Thr peptides (1292 unique peptides) and p-Tyr peptides (233 unique peptides) with a peptide FDR at 0.
BCR Signaling Pathway Is Phosphorylated in GC-Derived B-NHLs To investigate phosphorylated signaling pathways in the three B-NHL subtypes, we interrogated our data set using the functional annotation tool of DAVID software version 6. 7 (http://david. abcc. ncifcrf. gov/summary. jsp). 33, 34 The logarithm-transformed P values of the top 10 Kyoto Encyclopedia of Genes and Genomes pathways identified in BL, FL, or MCL are plotted in Figure  3. Pathways identified with p-Ser/p-Thr proteins in BL, FL, and MCL indicated that the three B-NHLs shared common phosphorylation of specific pathways, such as the spliceosome, ErbB signaling pathway, ubiquitin-mediated proteolysis, and the mammalian target of rapamycin pathway (Figure  3A ). This analysis also underlined the differential phosphorylation of specific pathways, such as the BCR signaling pathway and the insulin signaling pathway in BL and FL cell lines compared with the MCL cell lines.
Immunoblotting, IRS-1 immunoprecipitation and analysis of cellular PIP 3 Proteins from cell lysates (30 mg) were separated by SDS-PAGE and the abundance/activation status of specific proteins assessed by immunoblotting  [25]  using primary antibodies to proteins of interest followed by exposure to an appropriate peroxidaseconjugated IgG for 1h at room temperature. Immunoreactive bands were detected by enhanced chemiluminescence on Kodak X-OMAT film and quantified using Image J software (http:// rsbweb. nih. gov/ij/). Analysis of p85-PI3K association with IRS1 and cellular PIP 3 content were respectively assessed by coimmunoprecipitation with IRS1 and use of a sensitive time-resolved fluorescence resonance energy transfer (FRET)-based assay as described previously  [28, 29].
AdpA, also known as BldH, has been identified as a conserved major transcriptional regulator involved in the formation of aerial mycelia in various Streptomyces species  [3] [4] [5] [6]. AdpA is a member of the family of AraC/ XylS regulator proteins that contain a C-terminal domain with two helix-turn-helix DNA-binding motifs; these features are strictly conserved in all Streptomyces AdpAs in the StrepDB database  [7]. The N-terminal domain of AdpA is responsible for its dimerization and regulation  [8, 9]. Protein/DNA interaction experiments identified the following consensus AdpA-binding site in S. griseus: 5′-TGGCSNGWWY-3′ (with S: G or C; W: A or T; Y: T or C; N: any nucleotide)  [10]. AdpA was discovered and has mostly been studied in S. griseus, in which it was first shown to activate expression of about thirty genes directly.
Statistical analysis of array data R software  [32]  was used for normalization and differential analysis. A Loess normalization  [33]  was performed on a slide-by-slide basis (BioConductor package marray;  [34] ). A paired t-test was used for differential analysis. Variance estimates for each gene were computed under the hypothesis of homoscedasticity, together with the Benjamini and Yekutieli P-value adjustment method  [35]. Only genes with a significant (P-value < 0. 05) fold change (Fc) were taken into consideration. Empty and flagged spots were excluded, and only genes with no missing values were analysed. A few genes which displayed excessive variation were analysed using the Vmixt method from the VarMixt package  [36]. We defined our cut-off for microarray data acquisition as Fc <0.
625 or Fc > 1. 6 with P-value < 0. 05. The genome of S. lividans 1326 was sequenced only recently  [24] , so we used the StrepDB database  [7] , and in some cases a basic local alignment search tool (Blast), to identify S. lividans orthologs (SLI gene number) of S. coelicolor genes. We also used the protein classification scheme for the S. coelicolor genome available on the Welcome Trust Sanger Institute database  [37].
qRT-PCR analysis Oligonucleotide pairs specific for cchA (SLI0459), cchB (SLI0458), SLI0755, SLI6586, ramR (SLI7029), hyaS (SLI7885) and hrdB (SLI6088, MG16-17) (Additional file 1: Table  S1 ) were designed using the BEACON Designer software (Premier BioSoft). RNA samples were extracted from cultures in YEME liquid medium at OD 450nm values of about 0. 3, 0. 8, 1. 5, 1. 9 and 2. 3 (time points A, B, C, D and T, respectively). Aliquots of 20 μg of RNA were treated twice with 2 Units of DNase I with the TURBO DNA-free reagent (Ambion) for 30 min at 37°C. Reverse transcription and quantitative real-time PCR were performed as previously described  [25]. PCRs involved a hybridization step of 55°C, except for ramR, SLI0755 and cchB where a temperature of 58°C was used. Each assay was performed in triplicate and repeated with at least two independent RNA samples.
Microarray experiments were performed on RNA samples extracted at time T. b. Change in gene expression S. lividans adpA mutant compared to the wild-type at each time point of growth. RNA was extracted from S. lividans wild-type 1326 and adpA mutant cells cultivated in liquid YEME medium after various times of growth (OD 450nm of 0. 3, 0. 8, 1. 5, 1. 9 and 2. 3, respectively, at time points A, B, C, D and T). Relative amounts of SLI0755, SLI6586, hyaS, cchA, cchB, ramR PCR product were measured by qRT-PCR. At each time point of growth, gene expression levels were normalized using hrdB as an internal reference and are indicated in this figure as the n-fold change in adpA mutant compared to the wild type. Results are expressed as means and standard deviations of at least three replicates. Data are representative of at least two independent experiments for each strain at each growth time.
Note that a different scale is used for hyaS. differences between the expression level of tested genes and that of a reference gene. A P-value < 0. 05 was considered significant. In silico analysis and electrophoretic mobility shift assays (EMSA) Several AdpA-binding site sequences, identified in S. griseus by DNase I footprinting experiments  [10, 13, 18, 23] , were used with the PREDetector software (version 1. 2. 3. 0) [39]  to generate a S. griseus matrix  [25]. This matrix was used with the S. coelicolor genome sequence (the S. lividans genome sequence was not available during the course of this study and is still not available on PREDetector software) to identify putative AdpA-binding sites upstream from S. lividans AdpA-dependent genes (scores > 3). The StrepDB database  [7]  and Blast were used to identify S.
Theses genes encode proteins of several different classes according to the Welcome Trust Sanger Institute S. coelicolor genome database  [37] : 72 of the genes are involved in metabolism of small molecules, including seven playing a role in electron transport (e. g. SLI0755-SLI0754, cydAB operons) (Table  1 ); 18 encode proteins involved in secondary metabolism, for example the cchA-cchF gene cluster (SLI0459-0454) involved in coelichelin biosynthesis  [43]  and the SLI0339-0359 cluster encoding the putative deoxysugar synthase/glycosyltransferase. Deletion of adpA in S. lividans also affected the expression of 32 genes involved in regulation including ramR (SLI7029), wblA (SLI3822), bldN (SLI3667), hrdD (SLI3556) and cutRS (SLI6134-35)  [1, 6]. Sixty-two genes involved in the cell envelope  [37]  were differentially expressed in the adpA mutant; they include hyaS (SLI7885)  [44] , chpE, chpH  [1] , SLI6586 and SLI6587 which were strongly down-regulated in the adpA mutant (Table  1 ).
For cchA/cchB and SLI0755/SLI0756, larger amounts of AdpA were necessary for near complete displacement of the DNA probe to a Gene expression in the S. lividans adpA mutant was compared to that in the wild-type, using S. coelicolor microarrays. Table  1  shows a selected subset of the genes (see Additional file 2: Table  S2  for the complete list). The genes presented here were further studied or are discussed in the text because of their role in Streptomyces primary or secondary metabolism  [1, 6, 17]. b Gene names for S. coelicolor (SCO) and S. lividans (SLI) and annotated function are from the StrepDB database  [7]. c S. coelicolor microarrays were used for transcriptome analysis of the S. lividans adpA mutant (the complete microarray data set is presented in Additional file 2: Table  S2 ).
The S. lividans genome sequence was recently made available  [24]  and SLI ortholog gene numbers were identified as SCO gene orthologs with StrepDB database  [7]. The expression of genes shown in bold was analysed by qRT-PCR. Intergenic DNA regions between genes labelled with asterisks were analyzed by EMSA (Figure  2 ). A SCO7658-orthologous sequence (98% nucleotide identity according to BLAST) was detected in S. lividans, downstream from hyaS, but it was not annotated as a S. lividans coding DNA sequence (CDS). However our microarray data suggest that this sequence is indeed a CDS or alternatively that the S. lividans hyaS CDS is longer than annotated. d SCO genes and their S. griseus orthologs studied and described under another name found on StrepDB database  [7]  or see "References". e Fold change (Fc) in gene expression in the S.
lividans adpA mutant with respect to the parental strain with P-value < 0. 05, as calculated by Student's t-test applying the Benjamini and Hochberg multiple testing correction. ± indicates average Fc of some gene operons (see Additional file 2: Table  S2  for details). f From a protein classification scheme for the S. coelicolor genome available from the Welcome Trust Sanger Institute database  [37] : macromolecule metabolism (m. m. ), small molecule metabolism (s. m. ). protein-DNA complex. In a competition EMSA performed on SLI6586/6587 with an excess of the corresponding unlabelled probe, AdpA-binding to the labelled probe decreased (data not shown). We also tested a hyaS promoter in which one (highest score) of the three putative AdpAbinding sites was mutated (at position -134 to -129, see Additional file 3: Figure  S1a ): the affinity of AdpA for this promoter region was reduced and one protein-DNA complex disappeared (Additional file 3: Figure  S1b ).
These results suggest that one dimer of AdpA binds the adjacent sites -129 and -123 of S. lividans hyaS promoter and another dimer binds the -100 site resulting in the formation of the two DNA-AdpA complexes depicted in Figure  2. These EMSA experiments demonstrated that S. lividans AdpA directly binds to five intergenic regions and confirmed the in silico prediction presented in Table  2. S. In silico analysis of the S. coelicolor genome using PREDetector software (version 1. 2. 3. 0, the S. lividans database was not available at the time this analysis was performed)  [39]  to analyse orthologs of S. lividans AdpA-dependent genes. The S. coelicolor AdpA-binding sites identified were checked for their conservation and location using the S. lividans genome StrepDB database  [7]  (see legend c).
b Genes are named according to the StrepDB database  [7]. *binding sites located between S. coelicolor genes transcribed in the opposite orientation. c Putative S. coelicolor AdpA-binding sites were found in silico with PREDetector  [39] ; # putative site located in the upstream from the CDS of cchB. The site location given corresponds to the position of first nucleotide most distant from the translation start point of the first gene named. The positions of some sites are not the same for the S. lividans orthologs as indicated in brackets (S. lividans StrepDB database  [7] ). ~putative sites are in the CDS of SLI6587. Predicted CDS diverge between SLI6586 and SLI6587 locus and their orthologs SCO6197 and SCO6198, resulting in a smaller intergenic region in S. lividans. lividans AdpA directly regulates at least the six AdpAdependent genes listed above and identified by microarrays and qRT-PCR analysis.
Acknowledgements We thank  T. Msadek ,  S. Dubrac ,  E. Johnson  and  J. -L. Pernodet  for helpful discussion and critical reading of the manuscript, and  O. Poupel  for assistance with qRT-PCR analysis. We are grateful to  G. Bucca  for her advice and help with microarray handling. We thank  Alex Edelman & Associates  for correcting the manuscript. This work was supported by research funds from the  Institut Pasteur  and  Centre National de Recherche Scientifique. A. Guyet was the recipient of fellowships from the  Ministère de l'Education Nationale, de la Recherche et de la Technologie , the  Pasteur-Weizmann foundation  and the ERA-IB European grant. AG thanks  BBSRC  and  R. Daniel  for his constant support during the preparation of this manuscript.
Despite this, no replicative virion could be detected at the tumor injection site 4 days after the last VSV dose neither for the WT nor the various mutants (data not shown). WT VSV and the M M51R mutant are actively being developed for clinical oncolytic applications. For obvious safety reasons, viral replication has to be restricted both in space and were injected subcutaneously with B16 cells and infected with a single 5 × 10 8 PFU intratumoral dose of either VSV WT or the mutants on day 7, harvested 15 minutes after injection (day 0) and on indicated days postinfection. Virus titers were determined using a standard plaque assay. Data are the mean ± SEM of three tumors. (b) C57Bl/6 mice (n = 9/group) were injected subcutaneously with B16 cells and infected locally at the tumor site with 5.
0 × 10 8 PFU of WT or mutant VSV on day 7. On day 8, tumors were harvested, homogenized, and supernatants were analyzed for chemokine expression levels using a multiplex assay. Data show the combination of three independent experiments. (c) C57Bl/6 mice (n = 6) were injected subcutaneously with B16 cells and infected locally at the tumor site with 5. 0 × 10 8 PFU of WT or mutant VSV on day 7, 9, and 11. Sera from the indicated days after infection were tested for total neutralizing immunoglobulins and IgGs using a plaque reduction assay. Indicated data points represent serum dilutions that reduced plaque formation by 50%. Data represent the mean ± SEM of three independent experiments. *P < 0. 05, **P < 0. 01. time, which however limits virus availability for oncolysis. For VSV, this is likely achieved through the induction of the innate antiviral immune response within infected tumor cells and through the rapid generation of neutralizing antibodies.
DISCUSSION Oncolytic virotherapy has proven to be a promising cancer treatment in several successful clinical studies. 8, 22, 23 VSV, one of the most studied prototypic oncolytic viruses, is now in clinical trials for liver cancer treatment in the United States (NCT01628640). However, a number of questions remain unanswered about the mechanisms that contribute to virotherapy efficacy. One such question is the relative contribution of the immune response versus the intrinsic oncolytic activity of a particular virus to the treatment outcome. To address this question and assess if a virus mutational status impacts the antitumoral immune response, we sought to determine the ability of WT, M, and G VSV mutants  We found that the in vitro replication potential of the various VSV strains studied did not correlate with their respective capacity to persist in tumor tissue in vivo.
Samples were acquired on a FACS Fortessa (BD Biosciences, San Diego, CA) and analyzed using the Flowjo software (Tree Star, Ashland, OR). Anti-CD45 PE/CF-594, anti-CD25 APC, and anti-NKp46 Alexa 700 were purchased from BD Biosciences. Anti-PD-1 fluorescein isothiocyanate (FITC), anti-CD4 APC/Cy7, anti-CD4 FITC, anti-CD8 phycoerythrin (PE)/Cy7, anti-CD62L Alexa 700, anti-CD44 PercP/Cy5. 5, anti-B220 allophycocyanin (APC)/Cy7, anti-CD11b Pacific Blue, anti-Gr1 PE/Cy5, anti-CD11c FITC, anti-F4/80 APC, anti-PD-L1 Brilliant violet 421, and anti-CD86 PE were purchased from BioLegend (San Diego, CA). Purified anti-VSV-G from the monoclonal VSV-Indianaspecific IgG-secreting hybridoma VI10  47  (kindly obtained from Dr R. M. Zinkernagel) was coupled to Alexa fluor 647 fluorochrome using the Alexa Fluor 647 Protein Labeling Kit (Life Technologies, Burlington, ON).
Cells were stained for surface markers, then fixed and permeabilized for intracellular staining. IFN-γ PE and TNF-α APC were obtained from BioLegend and granzyme B eFluor 450 was purchased from eBiosciences (San Diego, CA). In vitro infections. B16gp33, HepG2, L929, and Vero cells were infected at a multiplicity of infection of 10 or mock infected. At 24-hour postinfection, cells were trypsinized, harvested, washed, and resuspended in FACS buffer. Cells were labeled with anti-PD-1 FITC, anti-PD-L1 brilliant violet 421, and anti-H2 PE (BioLegend) and analyzed by flow cytometry using a FACS Fortessa instrument (BD Biosciences) and the FlowJo software (Tree Star). Tumor surface molecules staining. Mice were injected into the flank with 5 × 10 5 B16gp33 cells in 100 µl PBS. A single VSV dose was given intratumorally 7 days after inoculation and tumors were harvested 24 hours later.
Tumors were then dissociated to achieve single-cell suspensions using a nylon 100 μm cell strainer (BD Falcon), washed, and resuspended in FACS buffer. Cells were labeled with anti-CD45 PE/CF594, anti-PD-1 FITC, anti-PD-L1 brilliant violet 421, and anti-H-2 PE (BioLegend) and analyzed by flow cytometry using a FACS Fortessa instrument (BD Biosciences) and the FlowJo software (Tree Star).
MHC-I recognition assay. Mice were injected into the flank with 5 × 10 5 B16gp33 cells in 100 µl PBS. A single VSV dose was given intratumorally 7 days after inoculation and tumors were harvested 24 hours later. Tumors were then dissociated to achieve single-cell suspensions using a nylon 100 μm cell strainer (BD Falcon), washed and coincubated with carboxyfluorescein diacetate succinimidyl ester-labeled P14 transgenic splenocytes (gp33-41-specific T cells) at a 1:1 ratio in the presence of monensin A (20 μg/ml) and APC-coupled anti-CD107a antibody for 5 hours. Cells were stained for surface markers and analyzed by flow cytometry using a FACS Fortessa instrument (BD Biosciences) and the FlowJo software (Tree Star).
(c) C57Bl/6 mice (n = 6) were injected subcutaneously with B16 cells and infected locally at the tumor site with 5. 0 × 10 8 PFU of WT or mutant VSV on day 7, 9, and 11. Sera from the indicated days after infection were tested for total neutralizing immunoglobulins and IgGs using a plaque reduction assay. Indicated data points represent serum dilutions that reduced plaque formation by 50%. Data represent the mean ± SEM of three independent experiments. *P < 0. 05, **P < 0. 01.
ACKNOWLEDGMENTS This work was supported by the Jeanne and J. -Louis Lévesque Chair in Immunovirology from the  J. -Louis Lévesque Foundation  and from a grant from the  Canadian Institutes of Health Research  ( MOP-89797 ). V. J. and M. -P. L. acknowledge studentship support from the  Fondation Armand-Frappier. P. L. holds a  Fonds de recherche du Québec-Santé postdoctoral  award and a  Thomas F. Nealon, III   Postdoctoral Research Fellowship  from the  American Liver foundation.