dataesr/scientific-paragraphs-categorization huggingface dataset:
None of the H9N2 isolates tested showed cytopathic effect (CPE) in infected CEF monolayer in the absence of trypsin whereas CPE could be observed by 72 h post infection in presence of trypsin, in 29 out of 40 samples. This clearly indicated that the Tunisian H9N2 isolates are of low pathogenicity, which is further supported by sequencing data results. Our findings also demonstrated that Ag EIA has higher sensitivity than virus detection through egg and cell culture inoculations and underlined its potential and value in IA routine diagnostic. Positive cases were then subtyped by both HI test using specific H9 polyclonal antisera and rRT-PCR assay followed by conventional RT-PCR using subtype-specific primers for the genes H9 and N2, respectively. The rRT-PCR assay for M and H9 genes revealed significantly higher numbers of positive samples, and confirmed all the positive results revealed by AgELISA.
Acknowledgments This work has been developed under the  MEV09  project and the research platform of  Veterinary of Epidemiology and Microbiology Laboratory. We would like to thank  FAO  for providing reference sera and technical support. We are also grateful to all field veterinarians for their help in filling out the questionnaire and collecting the animal samples, also  Dr Ondrej Libiger  ( PhD, Research Associate, Scripps Translational Science Institute California, USA ) for copyediting the manuscript.
Reporter Visualization and Immunohistochemistry Whole intestines were mounted and opened along the mesenteric line and stained for LacZ activity  [17]. Expression patterns were determined from at least three animals per experimental group using a standard dissecting microscope. The total numbers of stained crypts were counted within the 2-5 cm region distally from the stomach using imageJ software (http://rsbweb. nih. gov/ij/). For immunohistochemistry (IHC), tissue was fixed in ice cold 10% formalin and processed into wax blocks by conventional means. Section were cut at 5 lm, dewaxed, and rehydrated into phosphate buffered saline. Staining was performed using the Envisionþ mouse or rabbit kit (Cat No# K4007 & K4011, Dako, U. K. , http://www. dako. com) according to manufacturer's instructions.
ACKNOWLEDGMENTS We thank  Mark Bishop ,  Lucie Stocking ,  Matthew Zverev , and  Derek Scarborough  for technical assistance;  Aliaksei Holik  for scientific discussion;  Sylvie Robine  and  D. Louvard  for reviewing the manuscript. This work was supported by  Cancer Research UK. L. P. would like to thank  Drs. Jo Cable  and  Jo Lello  for their kind financial contribution to help complete this work.
Furthermore, the IGF-1R pathway has been implicated in resistance to DNA damaging agents, suggesting its role in DNA damage repair  (5) (6) (7) (8). Previous studies on in vitro models of SCLC suggest that inhibition of the IGF-IR pathway might enhance the sensitivity of SCLC to chemotherapy through the inhibition of PI3K-Akt activation  (9, 10). However, these studies did not include in vivo validation and comprehensive molecular analyses on the interaction between chemotherapy and IGF-1R inhibitors. As a result, there is little rationale to support the four phase I/II studies that are currently investigating agents targeting the IGF-1R/IGF axis in patients with SCLC: OSI-906 vs. Topotecan (NCT01387386 & NCT01533181, phase II trials), Cisplatin Etoposide and Cixutumumab (NCT00887159, phase II trial), Pasireotide and Topotecan (NCT01417806, phase II trial).
Gene Expression analysis All samples were obtained from H526 xenografts in nude mice. Total RNA was isolated from tissues using the RNAlater protocol. Affymetrix human genome U133 Plus 2. 0 arrays were employed for expression profiling and the information concerning each probe on the array was extracted from the image data according to the manufacturer's instructions. The raw intensity values from the gene expression files were imported into the R statistical Software which is used for all data input, diagnostic plots, normalization (RMA), and quality checking steps of the analysis process. Generalized linear models were computed (Limma, Bioconductor) to determine the differentially expressed probe sets (> 2 fold change, adjusted p values were computed for multiple testing (Benjamini and Hochberg's method).
Fisher's exact tests were computed to test the overlap with gene sets from selected molecular signature databases (Gene Ontology gene sets and Canonical Pathways (KEGG, Biocarta and Reactome)). Only overlaps with a p value of <0. 05 significance were retained for further analysis. The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www. ncbi. nlm. nih. gov/geo (accession no. GSE45626). The RMA normalized gene expression data were uploaded to the Synapse public portal (https://synapse. sagebase. org) for convenience under the following entity: "IGF1R targeting in SCLC (R1507)" (synapse ID syn1588571). Extensive information on Synapse can be found at: https://sagebionetworks. jira. com/wiki/display/SYND/. Further information regarding the use of the Synapse R Client is available on the synapse wiki: https://sagebionetworks.
Phospho-protein arrays Human Phospho-RTK arrays were processed according to the manufacturer's instructions (2010 Cell Signaling Technology, Inc. , Pathscan® RTK signaling antibody array kit #7982). Briefly, 50mg of protein lysates were incubated with blocked membranes that were subsequently washed and exposed to a chemiluminescent reagent and to an X-ray film. Dot images were captured by an ImageQuant 350 CCD camera controlled by ImageQuant 350 capture software (GE Healthcare), at 3, 5 and 6 minutes exposure time. The antibody directed against pIGF-1R in this kit is selective and does not cross-react with pINSR (the phosphorylated form of the insulin receptor).
Statistical analysis The description of the results are based on classic statistical methods: median, range and 95% confidence intervals. For clonogenic assays and WST-1 proliferation assays, data are presented as the mean ± SEM from a minimum of three independent experiments and were compared using the Student's t test. The synergy effect between different conditions was assessed using the two-way ANOVA interaction test. Tumor sizes were compared using Student's t tests. Tumor growth delay (days) was analyzed using the Kaplan Meier method, considering any event as the moment when the tumor volume had attained 5-fold the baseline volume (RTV5). For all the tests performed, a p value of less than 0. 05 was considered statistically significant, and all tests were two-tailed. The statistical analyses were conducted using the R statistical software and Bioconductor packages (affy, limma).
R1507 downregulated IGF-1R protein expression and disrupted its downstream signaling in selected SCLC cell lines Three SCLC cell lines were selected according to their high basal IGF1R protein expression and their specific mutation profiles: H146 cells are mutated for MAP2K4 (c. 814_891del78), whereas H69 cells are mutated for both PIK3CA (c. 317_325del9) and c-Met (c. 2962C>T) (Cosmic database). WB analyses showed a down-regulation of IGF-1R protein expression in the presence of R1507 (200nM) in all the selected cell lines (Fig. 1C ). R1507 decreased IGF-1-induced downstream effector pAKT in H526 and H146 cells but not in H69 cells (Fig. 1D ). R1507 also inhibited ligand-induced phosphorylation of MEK in H526 cells. However, the MAPK pathway (pMAPK, pMEK) appeared to be stimulated by R1507 in a dose-dependent manner in H146 cells.
The overall regulation of gene expression induced by R1507 in H526 xenografts at the following time points: baseline (vehicle), R1507 day 1 and R1507 day 7. A total of 2396 probe sets were differentially expressed at R1507 day 1, whereas only 254 probe sets were differentially expressed at R1507 day 7. Interestingly, only 6. 7% (n= 156) of the initially deregulated probe sets (R1507 day 1) remained differentially expressed at R1507 day 7 (Fig. 5B , Supplementary Fig. 3 ). To investigate the biological basis of the gene expression alterations, we computed the overlap between the differentially expressed probes and gene set databases. The following pathways were enriched in the R1507-Day 1 condition: Cell-to-Cell Adhesion Signaling, Map Kinase Pathway, Beta-Arrestin and Vitamin D Receptor (Supplementary table.
Integrated genomic and functional analysis revealed the involvement of NER and mitochondrial apoptotic pathways in the response to CDDP-and IR-R1507 combinations To identify the molecular mechanisms involved in the response to CDDP-and IR-R1507 combinations, we performed global gene expression profiling on mice bearing H526 xenografts treated with the following treatment conditions: vehicle, R1507 CDDP, IR, CDDP-R1507 and IR-R1507 (Fig. 6A, B , Supplementary Fig. 5&6 , Supplementary table. 2). To investigate the biological basis of the gene expression variation, we computed the overlap between the differentially expressed probes and gene set databases. The probes differentially expressed exclusively in the CDDP-R1507 combination were associated with downregulation of the NER pathway (Nucleotide Excision Repair) (P=0.
Acknowledgments None to be reported. Grant support: This work was supported by a grant  U54CA149237  from the  Integrative Cancer Biology Program of the National Cancer Institute  ( C. Ferté ) and by a fellowship from the  Ministère de l'Enseignement Superieur et de la Recherche (France)  ( C. Ferté ). A significant financial grant was also provided by  Roche Diagnostics (Penzberg, Germany)  to support this work ( INSERM U1030 ).
Background: Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). Methods: Treatment: gefitinib 250 mg day À 1 until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples. Results: Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70. 8%; adenocarcinoma 97.
MATERIALS AND METHODS Study design and patients. The gefitinib follow-up measure study (NCT01203917) was a prospective, open-label, multicentre, singlearm study to characterise the efficacy, safety and tolerability of gefitinib (250 mg day À 1 ) as a first-line treatment of Caucasian patients with EGFR mutation-positive, locally advanced or metastatic NSCLC. The primary end point was ORR (investigator assessment). Secondary end points included PFS, disease control rate (DCR), overall survival (OS), safety and tolerability, and correlation between clinical characteristics and baseline tumour EGFR mutation status. Pre-planned exploratory objectives included the comparison of baseline tumour and plasma EGFR mutation status in all screened patients with evaluable results (Exploratory Biomarker Objective I).
Data for Exploratory Biomarker Objective I is reported here and in the Supplementary Appendix. Statistical analysis. Data were analysed using a data cutoff at 6 months after the last patient had started study treatment  (15 February 2012; data cutoff: 15 August 2012). It was estimated that 1250 Caucasian patients with advanced NSCLC would have to be screened to obtain 100 patients with eligible EGFR mutationpositive tumours for gefitinib treatment. A total of 100 patients would ensure precise ORR estimation, with the ORR 95% CI no more than 10% above or 10% below the observed ORR (e. g. if the observed ORR was 50%, the CI would be within 40% to 60%). The ORR (primary end point) was calculated from investigator data and summarised in the full analysis set (FAS; all screened patients with an eligible, positive EGFR mutation status who received X1 dose of gefitinib), with 95% CIs (Wilson score intervals).
The trial is registered with ClinicalTrials. gov (NCT01203917). Role of the funding source. This study was funded by AstraZeneca. AstraZeneca employees participated in the conception and design of the study, collection and assembly of data, data analysis and interpretation. The corresponding author had full access to all the study data and final responsibility for the decision to submit for publication. The DCR was 90. 6% (95% CI: 83. 5-94. 8%). Median PFS was 9. 7 months (95% CI: 8. 5-11. 0%), with 38. 5% of patients progression-free at 12 months (Figure  2A ). Median OS was 19. 2 months (95% CI: 17. 0-not calculable; 27% maturity) (Figure  2B ). Safety and tolerability. Safety and tolerability data (EFS population) are summarised in Table  3. Median duration of exposure was 245 days (8. 0 months).
Although investigator and central-review variability is not uncommon and has been observed previously in oncology studies  (Tang et al, 2010) , in our study the difference between investigator (70%) and central-review (50%) ORRs was investigated further. When the ORR data from our study were analysed post-hoc, 17 patients were assessed as having no measurable disease at baseline by central review. If these 17 patients are excluded from the ORR analysis, the central-review ORR is 60%. This retrospective analysis may, therefore, help to explain differences between investigator and central-review results. Since the presence of measurable disease was an inclusion criterion for this study, this result may be considered to represent the likely outcome had central review been the principal designation of ORR.
Sample and data collection Paired urine and blood samples were collected at enrollment and up to 18 hours later by standard methods and centrifuged. Plasma (EDTA), serum and urine supernatants were frozen, shipped on dry ice, stored at ≤-70°C and thawed immediately prior to analysis. Clinical data including patient demographics, prior health history, serum creatinine, and hourly urine output as available in the hospital record were collected. Samples were analyzed at Astute Medical by technicians blinded to clinical data. Password-protected, anonymized clinical data collected with electronic case-report forms resided on servers at independent sites (Acumen Healthcare Solutions, Plymouth, MN, USA and Medidata Solutions, New York, NY, USA for Discovery and Sapphire studies, respectively).
Subject characteristics and event rates We enrolled 744 subjects in the validation cohort, 460 (62%) from North American sites and 284 (38%) from Europe (Figure  1 ). Sixteen patients (2%) were excluded from the analysis cohort because of withdrawal of consent, loss to follow-up, or invalid or missing test results leaving 728 subjects for the analysis. Demographic information for the analysis cohort is depicted in Table   2  and Table  S1  in Additional file 1). 4 ). Relative risk for AKI (KDIGO 2 to 3 within 12 hours) was also examined by tertile (data not shown). Compared to the lowest tertile, subjects with a result in the middle tertile had a 3-fold relative risk (P <0. 001) and those in the highest tertile had a nearly 10-fold relative risk (P <0. 001).
Acknowledgements The authors would like to acknowledge the many staff, coordinators, and investigators whose work was essential to completion of this study. A complete list of Sapphire investigators and support personnel is provided in the online appendix and is available online [31]. This study was funded by  Astute Medical. For the Discovery study , the Mayo cohort was paid based on patients enrolled while the Vienna and Duke cohorts were paid a research grant per annum. Sapphire study sites were paid based on patient enrollment and data entry. The study sponsor (Astute Medical) was primarily responsible for trial design, data collection, data analysis, and data interpretation, with crucial assistance from the trial lead investigators (JK, LC, KK) and the independent biostatisticians (MW, JS, JH).
Molecular cloning of S. mansoni sirtuins Sirtuins encoded in the S. mansoni genome were identified by screening using Hidden Markov Model profiles derived from the Pfam database  [35]. Predicted protein sequences were manually annotated by integrating data from InterProScan  [36]  and reverse PSI-BLAST analysis. In order to verify and complete the predicted sequences, we carried out 59 and 39 RACE (GeneRacer Kit, Invitrogen) using oligonucleotides (Table  S1 ) based on these sequences and generated full length cDNA sequences. The integrity of the sequences was verified by performing PCR using the Advantage 2 Polymerase mix according to the manufacturer's procedure (Clontech) and oligonucleotides encompassing the coding region. The PCR product was purified from agarose gels using the extraction kit Wizard SVGel and PCR clean up system (Promega) and inserted into pCR2.
1-TOPO before transformation of chemically competent Escherichia coli cells (One Shot T0P10, Invitrogen). Selected clones were sequenced by GATC Biotech. Sequence analyses and alignments were performed using the LASERGENE package (DNAStar) and the BioEdit v7. 1. 11 package (http://www. mbio. ncsu. edu/BioEdit/bioedit. html). In silico analyses of S. mansoni sirtuins Sirtuin conserved catalytic domain (Pfam: PF02146) sequences, delimited based on alignments in  [15] , were aligned using BioEdit v7. 1. 11 and the Clustal W program implemented therein. In order to determine the relationship of S. mansoni sirtuins to defined classes and families, we included sirtuin sequences from vertebrates, ecdysozoan invertebrates and one other lophotrochozoan (Clonorchis sinensis) in the analysis. An unrooted phylogram was generated using the MEGA4 neighbour-joining method  [37]  based on a Poisson correction substitution model and the figure was generated using FigTree (http://tree.
Quantitative RT-PCR Complementary DNAs were obtained by reverse transcription of total RNA using the Thermoscript RT-PCR System (Invitrogen) and used as templates in triplicate assays for PCR amplification using the SYBR Green PCR Master Mix and ABI PRISM 7000 sequence detection system (Applied Biosystems). Primers specific for S. mansoni sirtuins (Table  S1 ) were designed by the Primer Express Program (Applied Biosystems) and used or amplication in triplicate assays. Measurements of real time PCR efficiency for each primer pair allowed the ratios of expression to be calculated using the 2 2DDCt ratio  [39]  with S. mansoni a-tubulin as the reference transcript  [40].
Molecular cloning and characterization of S. mansoni sirtuins Five protein sequences corresponding to sirtuins were identified in the S. mansoni predicted proteome  [14]  and initial sequence similarity searches using Blastp  [47]  provisionally identified them as potential homologs of mammalian sirtuins. The S. mansoni proteins include Sirt1 (Smp_138640), Sirt2 (Smp_084140), Sirt5 (Smp_055090), Sirt6 (Smp_134630) and Sirt7 (Smp_024670). Each of the corresponding coding sequences was present on a separate genome scaffold and each corresponded to a single copy gene. In order to verify the predicted sequences and to detect eventual splicing isoforms we carried out 59 and 39 RACE PCR using oligonucleotides based on the predicted coding sequences. This allowed us to confirm the existence of each of the predicted proteins as transcripts and to correct a number of assembly errors in the predicted sequences.
The corrected sequences (including splice variants) have been submitted to the NCBI with accession numbers ABG78545 and KC993850 to KC993857. The alignment of the catalytic domain (Pfam: PF02146) of S. mansoni sirtuins (SmSirt1, 2, 5, 6 and 7) with homologues from other species is shown in Fig. 1A, B  and C. This alignment shows that although the schistosome sirtuin sequences diverge from those of vertebrates or ecdysozoan invertebrates, crucial residues involved in NAD+ binding, acetyl-lysine peptide binding or zinc binding are generally conserved, suggesting functional conservation of these sirtuins in the parasite. The SmSirt1 transcript (1832 nt) encodes a protein of 568 aa. The alignment of the catalytic domain of SmSirt1 (aa183-466) with orthologues from C. sinensis, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens, is shown in Fig.
Accession numbers The corrected sequences (including splice variants) of the S. mansoni sirtuins have been submitted to the NCBI with accession numbers ABG78545 (SmSirt1) KC993850 (SmSirt2), KC993851 (SmSirt5 isoform 1), KC993852 (SmSirt5 isoform 2), KC993853 (SmSirt5 isoform 3), KC993854 (SmSirt5 isoform 4), KC993855 (SmSirt5 isoform 5), KC993856 (SmSirt6) and KC993857 (SmSirt7).
This work was financed by the  European Community  (http://ec. europa. eu/)grant  SEtTReND  ( FP7-Health contract no. 241865 ). The work was also supported by institutional funds from the  Centre National de la Recherche Scientifique  (CNRS, http://www. cnrs. fr/), the  Institut National de la Santeet de la Recherche Medicale  (INSERM, http://www. inserm. fr/)and the  Universitede Lille 2  (http://www. univ-lille2. fr/). In-vitro studies on hSirts in the Jung lab are funded by the  Deutsche Forschungsgemeinschaft  ( Ju295/8-1 , http://www. dfg. de/). GO was also supported by contracts  CNPq  ( 306879/2009-3 ,  480576/2010-6  and  573839/2008-5 , http://www. cnpq. br/)and  FAPEMIG  ( PPM-00439-10 , http://www. fapemig. br/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
In Oxford, half of the cohort consisted of 'health conscious' subjects from England, Wales, Scotland, and Northern Ireland. The cohorts of France, Norway, Utrecht, and Naples include women only  [18]. Participants were recruited between 1992 and 2000 depending on the study center. At recruitment, men were 40 to 70 and women 35 to 70 years old  [18]. All participants gave written informed consent to use their questionnaire data and the Internal Review Boards of the International Agency for Research on Cancer and all EPIC recruitment centers approved the analyses based on EPIC participants. Of 511,781 apparently healthy participants at baseline, we excluded individuals with a ratio for energy intake versus energy expenditure in the top or bottom 1% (n = 10,197) and those with self-reported cancer, stroke or myocardial infarction at baseline (n = 29,300).
Exposure assessment Following the results of several methodological studies conducted in the early 1990s, habitual diet over the previous twelve months was measured at recruitment by country-specific instruments designed to capture local dietary habits and to provide high compliance  [18]. Seven countries adopted an extensive self-administered dietary questionnaire, which can provide data on up to 300 to 350 food items per country. In Greece, Spain and Ragusa, the dietary questionnaire was very similar in content to the above, but was administered by direct interview. A food frequency questionnaire (FFQ) and a seven-day food record were adopted in the UK. In Malmö, Sweden, a quantitative questionnaire combined with a seven-day menu book and an interview was used. Baseline food consumption, as well as ethanol and energy intake, was calculated from the dietary instruments applied in each center.
We also explored meat intake in models without adjusting for total energy intake. Additionally adjusting for fruit and vegetable consumption did not appreciably change the observed associations and was not included in the main models. In order to improve the comparability of dietary data across the participating centers, dietary intakes from the questionnaires were calibrated using a standardized 24-hour dietary recall  [24, 25] , thus, partly correcting for over-and underestimation of dietary intakes  [26]. A 24-hour dietary recall was collected from an 8% random sample of each center's participants. Dietary intakes were calibrated using a fixed effects linear model in which gender-and center-specific 24-hour dietary recall data were regressed on the questionnaire data controlling for weight, height, age, day of the week, and season of the year.