dataesr/scientific-paragraphs-categorization huggingface dataset:
Activity and inhibition assays Human and smHDAC8 activity testing was carried out with the HDAC8 Fluorimetric Drug Discovery Kit (Fluor de Lys(R)-HDAC8, BML-KI178) from Enzo Life Sciences according to the manufacturer's instructions with a substrate concentration of 50 mM. Fluorescence was measured in a plate reader (BMG Polarstar) with excitation at l = 390 nm and emission at l = 460 nm. Inhibition data for human HDACs 1, 3 and 6 were obtained according to published procedures with Z(Ac)Lys-AMC as the substrate and trypsin as the developing agent  [67]. HDAC1, 3 and 6 were purchased from BPS Bioscience.
Acknowledgments We are grateful to  Alastair McEwen  ( IGBMC ) for his kind assistance during X-ray data collections. We wish to thank members of the  ESRF-EMBL  joint structural biology groups and the SOLEIL synchrotron for the use of their beamline facilities and for help during data collection. We also thank  Nicolas Bertheaume  ( CIIL, Lille ) for his help with the TUNEL assays.
This project and the authors of this manuscript were financed by the  European Community  grant  SEtTReND  ( FP7-Health contract no. 241865 ). This work was also supported by institutional funds from the  Centre National de la Recherche Scientifique (CNRS) , the  Institut National de la Santeet de la Recherche Medicale (INSERM) , the  Universitede Lille 2 , the  Universitede Strasbourg (UDS) , the  French Infrastructure for Integrated Structural Biology (FRISBI) , and from contracts  CNPq  ( 306879/2009-3 ,  480576/2010-6  and  573839/2008-5 ) and  FAPEMIG  ( PPM-00439-10 ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Alternatively, B. trematum has only been isolated from humans and is found to be associated with ear and wound infections  (6). B. hinzii strain ATCC 57130 was isolated from an immunocompromised person, and B. trematum strain CCUG 13902 was isolated from a human leg wound. The sequencing data for both these strains were obtained from PCR-free random fragment libraries sequenced on the MiSeq (Illumina, Hayward, CA) platform using indexed paired-end 250nucleotide (nt) v2 chemistry and resulted in ~700-fold coverage for each genome. The nonindexed read length was 250 nt, with 84. 4% of the postfilter paired-end reads having Q30 or greater. The sequence reads were subsampled (~2. 2 M reads) and assembled into contigs using Velvet  (7)   The genomes were analyzed with the RAST server  (8). A total of 4,586 coding sequences were predicted for B.
This may suggest a closer relationship between the two bird-associated species (B. avium and B. hinzii) and B. petrii (isolated from the environment) in comparison with the other Bordetella species. Nucleotide sequence accession numbers. These whole-genome shotgun projects have been deposited in GenBank under accession no. AWNM00000000 (B. hinzii) and no. AWNL00000000 (B. trematum). The versions described in this paper are the first versions, accession no. AWNM01000000 and AWNL01000000, respectively.
ACKNOWLEDGMENTS This work was funded by an operating grant from the  Canadian Institutes of Health Research  (grant no. MOP-102706 ) and a  Program International de Collaboration Scientifique (Franco-Canadien)  grant from the  CNRS. N. R. S. was a recipient of a  Natural Sciences and Engineering Research Council of Canada  scholarship. We thank  James C. Richards  from  NRC, Ottawa , for providing us with bacteria after our dear colleague  Malcolm B. Perry  passed away.
5T33MM cells are considered 5T33MM+ and CD11b-. Appropriate isotype controls were used. All samples were acquired on a FACS Canto and analysed with FACS Diva software (BD biosciences). For human iNKT analyses, an antibody mixture composed of anti-CD3-FITC (clone sk7, BD biosciences)/anti-CD16CD56-PE (clone 3G8, My31, Analis, Ghent, Belgium)/anti-CD45-Texas Red (clone J33, Analis)/anti-CD19-PC5 (clone J3-119, Analis) was used to determine the amount of T lymphocytes on peripheral blood cells. In a second tube, anti-CD3-PC5 (clone ucht1, Analis), anti-TCRVa24-FITC (clone 6B11, BD biosciences) and anti-TCRVb11-PE (clone C21, Analis,) was used to gate iNKT cells.
Unstimulated iNKTs secreted a background level of up to 3006251 pg/mL IFN-c during the course of the disease. These data indicate that a drop in cytokine secretion from the iNKTs could be preceded by a decline in iNKT number. To confirm that this is an actual decline in iNKT number and not the result of internalization of the Va14 receptors, we performed RT-PCR of the Va14 receptor of healthy and terminally diseased murine liver iNKTs (Figure  3C ). If the receptor would be internalized, mRNA levels would still be high. One of the 5T33MM samples was used as reference and the other values were calculated relative to this. We could find that the mRNA expression of Va14 correlated to the number of iNKTs measured by FACS with a clear drop in iNKT number in terminally diseased 5T33MM mice. Only very slight IL-4 production (662 pg/mL) was observed when a-GalCer was administered to the culture (data not shown) indicating that liver iNKTs are skewed to a Th1 profile and can therefore be used as an immunotherapeutic tool in MM when activated by adjuvants such as a-GalCer.
Acknowledgments We thank  Angelo Willems ,  Carine Seynaeve  and  Marie Joos de ter Beerst  for their expert technical assistance. We would also like to thank  Dr. Claude Preudhomme ,  Dr. Christophe Roumier ,  Pierre-Marie Degand ,  Laurence Wicqart  and  Francis Pryfer  of the tumourbank of  Lille  ( Centre de Biologie-Pathologie, Laboratoire d' Hematologie, 2 avenue Oscar Lambret, CHRU ,  59 037 LILLE Cedex ).
France French researchers have been very active for more than 10 years in developing MLVA for the genotyping of pathogenic bacteria and fungi of global health interest (concerning humans, animals and plants) or which may pose a bioterrorist threat. These developments have included the setting up of new assays and of tools accessible on the Internet to facilitate the development of such assays  [8]. Of particular interest are online databases presenting MLVA typing data, including the first one, made public in 2002  [8] , the development and commercialisation of typing kits and the provision of typing services. MLVA is currently in the phase of entering routine practice in a number of reference laboratories and a market seems to be emerging in France. MLVA is primarily used in France for six bacterial species of high medical interest.
The MLVA used for Mycobacterium tuberculosis  [9]  is now well-known worldwide as mycobacterial interspersed repetitive units-variable-number tandem repeat (MIRU-VNTR), owing to the efforts of a company (Genoscreen, Lille, France) in Institut Pasteur Lille and to the importance of this pathogen. This assay has also served as a pilot for the development of large-scale MLVA typing and associated databases. More recently, MLVA has been developed for Staphylococcus aureus, Legionella pneumophila and Pseudomonas aeruginosa, with the production of fully automated assays and of typing kits by the Centre Européen d'Expertise et de Recherche sur les Agents Microbiens (CEERAM) at La Chapelle sur Erdre. In the L. pneumophila assay, 12 loci are coamplified in a single multiplex PCR  [10]. Alternatively, the assays can be set up locally, with no need to buy kits, since all the necessary information is published  [10] [11] [12].
MLVA is also in routine use for Streptococcus pneumoniae, with more than 1,000 genotypes publicly accessible from the the Robert Picqué Military Hospital in Bordeaux  [13]  and for Acinetobacter baumanii  [14]. An MLVA assay for Streptococcus agalactiae has also been developed in France and additional MLVA assays are currently being developed by the Agence nationale de sécurité sanitaire de l'alimentation (ANSES) for zoonotic agents and by the Centre de coopération internationale en recherche agronomique pour le développement (CIRAD) for plant pathogens. MLVA assays, which are now used worldwide, have also been developed for major bioterrorist agents, including Yersinia pestis and Bacillus anthracis  [15] , as well as minor agents, such as Brucella spp. [16] , together with associated online databases.
The third version was released in 2007 and a fourth, which will be able to manage a variety of sequencebased assays in addition to MLVA, is currently under development. The second database  [13] , developed by the Robert Picqué Military Hospital, was released in 2007. Importantly these two websites allow external users to create their own database, with user-defined species, set of loci, etc. , independently of the hosting institution. The resulting databases can be shared within a community or even made publicly accessible. The other two MLVA databases were developed by the Institut Pasteur in Paris  [18]  and Guadeloupe  [19] ; the latter is dedicated to M. tuberculosis. A list of websites hosting MLVA genotyping databases for a number of pathogens is maintained at the genomes and polymorphisms website  [8].
At CSRSV, a MLVA-15 research project has been developed for C. botulinum in collaboration with laboratories of the other countries participating in the European Biodefence Laboratory Network (EBLN). Strains were provided mainly by the CNRB and also by other EBLN institutions. This MLVA scheme improved the discriminatory power compared with the previous MLVA-10 scheme for C. botulinum  [39]. The analysis was extended to B and F toxin serotype strains, in addition to A serotype strains: five newly characterised MLVA loci were added to the previous 10-MLVA scheme and new groups were described. To date, MLVA data have been obtained for about 300 international C. botulinum strains, whereas profiles from 79 strains across Europe have been published  [40].
In addition, the national reference laboratory for tuberculosis, located within RIVM, uses the MIRU typing assay (24-loci MLVA) for M. tuberculosis. The MLVA schemes developed at RIVM and a typing tool for these pathogens are maintained at RIVM  [43]. The typing tool allows interrogation of a MLVA-type table: by typing in an MLVA allelic profile, it will report both the MLVA type and MLVA complex. The tool can be set to report the exact and closest matching profiles. MLVA of MRSA is by far the most intensely used MLVA scheme in RIVM. By May 2012, the MRSA MLVA database contained MLVA profiles of nearly 29,000 isolates and 3,351 different profiles and 28 MLVA complexes were recognised among these isolates. For MRSA, virtually all isolates are sent to RIVM for molecular typing as part of the national MRSA surveillance.
The S. pneumoniae database is the second largest MLVA database at RIVM. Although smaller, it still contains profiles of approximately 4,000 isolates. In all MLVA schemes used in RIVM, assessment of the number of repeats in each locus is performed by sizing of the fluorescently labelled PCR products on an automated DNA sequencer. Each unique MLVA profile is given a MLVA type designation, e. g. MT21, and profiles are used for clustering and assignment of MLVA complexes. The use of fluorescent labels also allows for the simultaneous MLVA and detection of particular genes. This was used in the MRSA MLVA protocol, in which primer sets were included to detect the mecA and lukF genes. Although separation of the PCR products is performed on a DNA sequencer, standardisation may pose a problem for MLVA.
Norway In Norway, the Norwegian Institute of Public Health (NIPH) is the primary facility for nationwide surveillance of food-borne infections. MLVA is used extensively as the primary routine genotyping tool for a number of enteropathogenic bacteria with the exception of Campylobacter spp. (for which other methods are applied), giving the NIPH an up-to-date overview of the spread and introduction of these pathogens in Norway. NIPH genotypes and maintains databases for E. coli, S. Typhimurium, Shigella spp. Yersinia enterocolitica and Listeria monocytogenes. For typing E. coli, three different protocols are in use: two designed for E. coli O157:H7 and sorbitol-fermenting O157:H-strains (unpublished), as well as a generic MLVA assay able to genotype all serotypes of E. coli using 10 loci  [44].
As of 17 December 2012, a total of 2,411 whole bacterial genomes were listed by the National Center for Biotechnology Information (NCBI)  [54] , where all sequences may be downloaded and examined for VNTR content. Thus, MLVA assay development can be performed regardless of access to in-house sequencing (although this is an advantage). The nature of MLVA makes it a practical system for rapid sharing and digital storing of results, as can be seen by the online databases that are already operational in Europe. This has been achieved in a relatively short time frame: a S. Typhimurium MLVA protocol was first published in 2004  [1]  and by September 2011, standardised protocols were available in Europe  [53]. In comparison, PFGE was first described in the early 80s and it was not until 2004 that PulseNet Europe was established, using protocols standardised in the United States  [55].
g. cycloheximide, CHX), to determine which stimuli induced cell death in our model. TNF-a and TRAIL robustly triggered cell death at low concentrations, whereas FasL did not (Figure  1c ). By contrast, none of the three cytokines triggered cell death in control cells. Experimentations also showed that cell death occured within 6 h of TNF-a stimulation (Figures  1d  and e ). In addition, we assessed the extent of cell death achieved in the various culture conditions by performing an annexin V-FITC/propidium iodide (PI) assay and evaluated the percentage of apoptotic dead cells (annexin V-positive, PIpositive) by flow cytometry. The viability of cells was determined following transfection with EVER2 plasmid construct or empty vector as control. Data from a representative experiment are shown in Supplementary Figure  S1.
Indeed, EVER2 disrupts the TRAF2/cIAPs interaction in a dose-dependent manner (Figure  5c ). Importantly, this interaction is known to be indispensable for RIPK1 ubiquitination, an essential biochemical event for maintenance of RIPK1 in complex I. 35  These observations are in accordance with our data showing a decreased recruitment of RIPK1 in complex I in the EVER2expressing cells, whereas no RIPK1 degradation was observed, as shown by western blots of whole-cell lysates (Figures  4c  and 5a ). As EVER2 impaired complex I formation, we assessed the impact of EVER2 expression on TNF-a-induced NF-kB activation. EVER2 expression resulted in much lower levels of NF-kB activity upon TNF-a stimulation than that observed in control conditions (Figure  5d ). These data were substantiated by differential analysis of TNF-a-induced NF-kB activation following transfection with various EVER2 fragments (Figure  5e ).
Cells were plated in 24-well plates, grown to 50-80% confluence, and transfected for 5 h by the PEI (polyethylenimine) method, as previously reported. 7 For NF-kB luciferase reporter assays, we used 0. 3 mg per well NF-kB-firefly luciferase reporter plasmid (Stratagene, San Diego, CA, USA) and 0. 3 mg EVER2-Flag plasmid. The Renilla luciferase pRLTK vector (0. 03 mg) was used as an internal control for transfection efficiency. Luciferase activity was measured with the Dual-Luciferase Reporter Assay kit (Promega, Madison, CA, USA) and a Centro XS  3  LB 960 luminometer (Berthold Technologies, Bad Wildbad, Germany). Data were normalized with respect to Renilla luciferase activity and protein concentration, as determined in the Bio-Rad protein assay (Bio-Rad). Semi-quantitative PCR. We isolated mRNA with the mmacs mRNA isolation kit (Miltenyi Biotec, Paris, France) and generated cDNA by reverse transcription with the Superscript II First-Strand system (Invitrogen).
Apoptotic cell death was also determined by flow cytometry with an Annexin-V/PI viability assay. Floating and adherent cells were collected and stained with Annexin V-FITC kit (Miltenyi Biotec) according to the manufacturer's instructions. Cell death was detected using a CyAn flow cytometer (Beckman Coulter, Villepinte, France), and the data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). High-throughput Gaussia protein complementation assay (HT-GPCA). ORF sequences corresponding to proteins of interest were inserted into the SPICA-N1 and SPICA-N2 vectors for expression in fusion with fragments 1-93 (Gluc1) and 94-169 (Gluc2) of Gaussia princeps luciferase, as previously described. 25 HEK-293T cells were used to seed 96-well plates at a concentration of 3. 5 Â 10 4 cells per well.
After 24 h, cells were transfected with 120 ng of SPICA-N1 and SPICA-N2 constructs. The firefly luciferase pFLTK vector (0. 03 mg) was used as an internal control for transfection efficiency. Cells were harvested 24 h after infection by incubation with 30 ml of Renilla Lysis Buffer (Promega, E2820) for 30 min, and Renilla luciferase activity was measured with a Berthold Centro XS LB960 luminometer. Results are expressed as a fold-change normalized with respect to the sum for controls, as previously described. 25 Data were normalized with respect to firefly luciferase activity and the final result is the mean normalized luminescence ratio (NLR) for triplicate experiments. Statistics. The statistical significance of differences was determined with Student's t-test. For all analyses, Po0. 05 was considered significant.
Acknowledgements. This work was supported by grants from the  ANR  (contract no. 026 01 ),  ARC  (contract no. A09/1/5031 ) and the  Ligue Nationale contre le Cancer  (contract no. RS07/75-75 ) and by a donation from  ODYSSE RE Holdings Corp. G GAUD  was supported by a fellowship from the  ANR  (contract no. 026 01 ). We also thank  Alex Edelman  and Associates for correcting the English version of the manuscript.
Peroxidase was revealed by incubation with 0. 05% 3,39-diaminobenzidine tetrahydrochloride containing 0. 008% hydrogen peroxide, and sections were Nissl counter-stained. Tyrosine hydroxylase TH1 neurons revealed with the chromogen diaminobenzidine were analysed by bright field microscopy using a Nikon Optiphot-2 microscope (Nikon France, Champigny/Marne, France) equipped with the Explora Nova VisioScan T4. 18 software (La Rochelle, France) as previously described  15. TH1 cell bodies were quantified stereologically on regularly spaced sections covering the whole mesencephalon from the rostral pole of the substantia nigra to the locus coeruleus using the VisioScan stereology tool  19. The investigator performing the quantification was blinded to the treatment groups during analysis. The total number of SN TH 1 neurons varied from 5860 to 8030 in saline treated WT and 7205 to 8070 in saline treated knock-outs, respectively.
For microglia quantification, similarly adjacent (6-8) serial sections were used. An observer blind to the samples identity quantified the number of Iba1 or MHC II-positive cells in the SN as described above. Statistical analysis. Data are expressed as mean 6 S. E. M. Statistical analysis was performed using two-way ANOVA with genotype as one and saline/MPTP treatment as second independent variable, followed by post-hoc Student-Newman-Keuls test for all pairwise comparisons.
Acknowledgements C. N. was supported by a postdoctoral grant from the  Deutsche Forschungsgemeinschaft, (DFG) ,  Germany. D. A. F. was supported by a postdoctoral grant from the  Michael J. Fox Foundation (MJFF)  and a research grant of the  University Medical Center Giessen and Marburg (UKGM). A. O. was supported by the  Mildred-Scheel-Stiftung. T. L. was supported by a Master grant from the  Fondation pour la Recherche Medicale (FRM). C. H. was supported by an M. D. thesis grant by the  Boehringer Ingelheim Fond (BIF). E. C. H. and S. H. are investigators at the  Centre National pour la Recherche Scientifique (CNRS). A. H. was supported by a '' Poste Vert'' (Accueil de Chercheurs Etrangers ) from the  Institut National de la Santeet de la Recherche Medicale (INSERM).
Thirty-four patients participated in a pharmacokinetic substudy with more frequent sampling at week 20; 26 patients had sufficient data for analysis (tocilizumab-SC, n=13; tocilizumab-IV, n=13) (see supplementary figure S5, available online only). At steady state from weeks 20 to 24, the mean AUC 20-24wk was 30 168 μg h/mL for tocilizumab-SC and 41 304 μg h/mL for tocilizumab-IV. The mean C max values (±SD) at week 20 were 52. 7 (±27. 3) μg/mL for tocilizumab-SC and 233 (±117) μg/mL for tocilizumab-IV. CRP levels decreased in both groups after the first dose of tocilizumab (see supplementary figure S6, available online only). Thereafter, in both groups, CRP remained below the upper limit of normal (ULN; 0. 99 mg/dL) to week 24. The time course of CRP for tocilizumab-SC was comparable to that for tocilizumab-IV, although there was a trend towards slightly lower CRP levels in the tocilizumab-SC group.
Acknowledgements The authors wish to thank all investigators and patients who participated in the study and all members of the WA22762 study team. Support for third-party writing assistance for this manuscript, furnished by  Denise Kenski , PhD of Health Interactions, was provided by  F. Hoffmann-La Roche, Ltd. Contributors LR and MB designed the study, analysed and interpreted the data. PL analysed and interpreted the data. GRB, AR-R, AC, SH, PL, DF, MJR, GR, CL and EFM were involved in generating the data at their clinical research sites. All authors were involved in writing the manuscript and approved it. Funding This study was funded by  Roche. F. Hoffmann-La Roche, Ltd (Roche)  sponsored the study, participated in the design of the study as well as in the collection, analysis and interpretation of the data.
After 24 h, cells and parasites were washed once in PBS 1Â (pH 7. 4) to eliminate extracellular parasites. Then the intracellular multiplication of tachyzoites was observed (8-32 tachyzoites per rosace) until the fourth day post-infection when tachyzoites were harvested from culture and the Toxoplasma stage preparation was verified by qRT-PCR by using tachyzoites markers and bradyzoites markers (Supplemental data 1). Parasites used in this study were cultivated from passages 3-9 on Vero cells. For 2D-DIGE analysis, four samples of parasites came from the same cell culture passage. For qRT-PCR analysis, we used six samples: one sample came from the same passage as in 2D-DIGE and the five other samples came from additional passages. Then, parasites were separated from host cells by filtration through 3 lm polycarbonate filters (Whatman International).
Image analysis and statistics Proteins were visualized using Ettan DIGE Imager 1. 0 (GE Healthcare) with CyDye filters. For the Cy2, Cy3 and Cy5 fluorochromes, the excitation wavelengths were respectively 480/30, 540/25 and 633/30 nm and emission wavelengths were 530/40, 595/25 and 680/30 nm. All gels were scanned at 100 lm resolution. Image analysis was carried out with DeCyder 2D software ver-sion 6. 5 (GE Healthcare). The DIA (differential in-gel analysis module) was used to co-detect, normalize with the internal standard from each 2D-gels and differentially quantitate the protein spots in the image. The matching of the different maps from each gel and the statistical analysis of protein variation between samples (resistant/sensitive) was performed by the BVA (Biological Variation Analysis Module)  (Alban et al.
1% (v/v) FA for 5 min. The column was then equilibrated in 97. 9% water/2% acetonitrile/0. 1% (v/v) FA for 5 min (total run time per sample was 50 min). Ionized peptides were analyzed in the mass spectrometer (0-106 m/z, global and Msx) using the ''triple play'' mode, consisting initially of a survey (MS) spectrum from which the three most abundant ions were determined (threshold = 200-500 TIC). Collision energy was set at 35% for 30 min. The charge state of each ion was then assigned from the C13 isotope envelope ''zoom scan'' and finally subjected to a third MS/MS scan. The LTQ was tuned using a 500 fmol/lL solution of glufibrinopeptide (m/z 785. 5, [M = 2H] 2+ ) and calibrated according to the manufacturer's instructions. The resulting MS/MS spectra (data files) were merged into an mgf file, which was submitted to Mascot searching.