dataesr/scientific-paragraphs-categorization huggingface dataset:
Oraganoids were cultured in 48-well plates, 100 crypts per well. RNA was isolated using the RNeasy Mini kit and the RNase free DNase kit (Qiagen). Gene expression was analyzed using primers purchased from Sigma. Data were normalized to the B2M housekeeping gene expression. qRT-PCR. RNA was isolated using the RNeasy Mini kit and the RNase free DNase kit (Qiagen). RT-PCR reactions were performed overnight using the SuperScript II reverse transcriptase (ThermoFisher) and the oligo(dT)18 primers (ThermoFisher), as recommended by the supplier. Gene-specific primers were designed and purchased from Sigma (HBD1, CAGGTGGTAACTTTCTCACAGG/AATAGAGACATTGCCCTCCACT; HBD2, GCCATGAGGGTCTTGTATCTC/TTAAGGCAGGTAACAGGATCG; HBD3, TTTGGTGCCTGTTCC AGGTCAT/GCCGCCTCTGACTCTGCAATAATA; IL1B, TACGATCACTGAACTGCACGCT/TCTTTC AACACGCAGGACAGGT; IL8, AAGAAACCACCGGAAGGAACCA/ATTTCTGTGTTGGCGCAGTGTG; TNF, AAACAACCCTCAGACGCCACAT/AGTGCTCATGGTGTCCTTTCCA; MYC, CAAACTTGAACAGCTA CGGAAC/TTCATAGGTGATTGCTCAGGAC).
The qRT-PCR reactions were carried out in a 20 μL final volume containing 8 μL of cDNA (diluted at 1/50), 2 μL of primers (0. 2 μM each) and 10 μL of Power SYBR Green mix (ThermoFisher). Reactions were run on a QuantStudio 7 (ThermoFisher) with recommended universal thermal cycling parameters. Each sample reaction was run in duplicate on the same plate. Relative gene expression quantification was performed using the comparative Ct method. Data were normalized to the B2M housekeeping gene expression. ELISA. We used the ELISA kits for HBD1 (900-K202, PeproTech), and IL-8 (900-K18, PeproTech), as recommended by the supplier. Absorbance was measured on a M200PRO fluorimeter (Tecan). Immunoblotting. Total cell lysates were harvested by removing growth medium and adding Nonidet P-40 lysis buffer [25 mM Tris HCl (pH 7.
Statistics. Statistical analysis was performed on GraphPad Prism 5 (GraphPad software). Results are presented as a mean of at least 4 independent experiments. Error bars represent the SD. Statistical comparisons were performed using the two-tailed Mann-Whitney u test or the Welch t test. A p-value < 0. 05 was considered significant. Pilot studies were performed and used for estimation of the sample size to ensure an adequate power. In most of our experiments, 4 to 6 samples were sufficient to see differences between experimental conditions. Samples were randomly assigned to experimental conditions, to processing order and to positions on plates where applicable. No blinding was done.
The region of Santander is located in the northeast of Colombia and comprises the states of Santander and Norte de Santander. Previous studies reported changes in the predominance of serotypes over time in this region  [11, [13] [14] [15]. The argument over whether there is a genetic basis or there are random fluctuations that explain the temporal distribution of serotypes has not been settled yet. Previous studies  [16] [17] [18] [19]  that focused on the molecular epidemiology of dengue in Colombia included sequence data collected up to 2008 and did not include DENV-4. Consequently, many aspects of the local epidemiology remain unclear. In the present study, we obtained 143 new envelope gene sequences from serum samples collected in the region of Santander and six sequences from serum samples collected in other regions between 1998 and 2015.
Full-length envelope gene (E-gene) sequencing Viral RNA was extracted directly from supernatants of DENV-infected C6/36 cells using the QIAamp Viral RNA mini kit (Qiagen). The RNA was transcribed to cDNA using RevertAid reverse transcriptase (Thermo Scientific, USA) and random hexamer primers (Thermo Scientific). The full E-gene (~1485 pb) was amplified by PCR using DENV serotype specific primers [S1  instructions. Each reaction of DNA amplification produced overlapping amplicons of 863-1520 nucleotides in length. Amplicons were sequenced using a Sanger sequencing commercial service (Macrogen DNA Sequencing Service, Seoul Korea). Sequence assembly was performed with CLC Genomics Workbench 4. 5 (CLC Bio, Denmark) and sequences were deposited in GenBank [S2 Table ].
Data selection Available full-length E-gene accessions with known location and sampling date were retrieved from GenBank (last accessed in March 2017). The total dataset (n = 9. 669) was aggregated by serotype (3676 of DENV-1, 2925 of DENV-2, 1746 of DENV-3 and 1322 of DENV-4) and analyzed separately. Identical sequences from the same country and year were removed using the UCLUST algorithm in the USEARCH v. 10. 0. 240 software  [26]. The filtered datasets (3452 of DENV-1, 2789 of DENV-2, 1645 of DENV-3 and 1100 of DENV 4 Sequences) were combined with novel Colombian isolates, aligned using MUSCLE v. 3. 8  [27]  and visualized using Bioedit v. 7. 2. 5 software  [28]. Preliminary phylogenetic analyses were done using Maximum Likelihood (ML) methods with a non-parametric bootstrap using PhyML v.
3. 1 software  [29]  in order to identify genotypes and to focus the analysis in the genotypes that were observed in Colombia [S2 Fig]. Sequences from the same year were downsampled when they fell within a monophyletic group by country and were overrepresented. The resulting datasets (DENV-1 = 118, DENV-2 = 118, DENV-3 = 66 and DENV-4 = 77) had only sequences from the region of the Americas because the phylogenetic trees showed a single introduction of each genotype into this region from non-American countries. A Likelihood Mapping Analysis in TREE-PUZZLE v. 5. 3  [30]  software and a substitution saturation analysis in DAMBE v. 6  [31]  showed that there was enough phylogenetic information in each dataset [S1 Fig]. All datasets were free of recombination signal following a pairwise homoplastic index test using SplitsTree v.
Data analysis Haplotype diversity and nucleotide diversity were calculated using the software DNAsp v. 5  [33]. For each dataset, the best-fit model of nucleotide substitution was selected based on Akaike Information Criterion (AIC) using the APE package v. 3. 4. 2  [34]  and the R v. 3. 3. 0 software  [35] ; The Tamura and Nei plus invariant and discrete gamma models were the best-fit nucleotide substitution models for DENV-1 and -2, and the generalized time reversible plus invariant and discrete gamma models were the best-fit nucleotide substitution models for DENV-3 and -4. Regression of root-to-tip genetic distance against sampling time [S3 Table ].
using TempEst v 1. 5  [36] , showed that there was sufficient temporal signal in each dataset to proceed with phylogenetic molecular clock analyses. The best-fit clock model (strict vs. relaxed) and the best-fit demographic model (among constant size, exponential growth, skyride and skygrid models), were selected via path sampling (PS) and stepping-stone (SS) methods (100 steps with 5 million iterations each)  [37]. In all cases, the uncorrelated lognormal (UCLN) relaxed-clock model was preferred. For the model-based demographic inference, the non-parametric skygrid model was preferred for DENV-1 and DENV-3, and the parametric Exponential growth model for DENV-2 and DENV-4 [S4 Table ]. Viral demographic curves were reconstructed for each serotype using either the isolates from Colombia (n = 235) sampled between 1982 and 2015 or the isolates from the region of Santander (n = 160) collected between 1998 and 2015 in order to have a measure of dengue genetic diversity and its fluctuations over time.
A Bayes factor test (BF > 9) was used to recognize well-supported diffusion rates using the SpreaD3 v0. 9. 6 software  [39] ; well-supported diffusion routes concerning Colombia were compared among schemes. Bayesian approach was implemented in BEAST v1. 8. 3 software package  [40]. For every analysis, five independent Markov Chain Monte Carlo (MCMC) were used with at least 100 million generations each. Samples were combined and diagnosed using Tracer v1. 6 (http:// tree. bio. ed. ac. uk/software/tracer) until the reached effective sample sizes over 200 for all parameters  [41]. Maximum clade credibility trees (MCC) were summarized using TreeAnotator v1. 8. 1.
DENV serotypes demographic history The Bayesian demographic reconstructions for Colombia are shown in DENV-4. There were oscillations in the case of DENV-1 and more pronounced ones in the case of DENV-3, but the general trend is of increased diversity over time. In the case of DENV-3, there is an increase in diversity peaking during the period 2001-2005, a sharp drop after that and a steady increase from 2009 onwards. The demographic reconstruction for the region of Santander, which accounted for 82% of the Colombian sequence data, followed the same dynamics as that recovered with the dataset from the Country. There was an agreement between the increase of Ne of DENV-3 and the re-introduction of this serotype into the country in 2001 that led to high DENV-3 prevalence in the following years.
Discussion This study is the first large-scale analysis of the spatial and temporal dynamics of DENV in Colombia during the period 1998-2015 using newly sequenced and curated genetic data retrieved from GenBank. The novel sequences were sampled in distinct locations of Colombia, but most of them came from the region of Santander. We documented the frequent introduction of dengue lineages into Colombia, the ongoing co-circulation of multiple serotypes and lineages within the country and the local viral population dynamics. Our phylogenetic analysis confirmed the circulation of one genotype per serotype (DENV-1: Genotype V, DENV-2: Asian/American Genotype, DENV-3: Genotype III and DENV-4: Genotype II) and it is consistent with the results of other studies  [16] [17] [18] [19]. The presence of DENV-4 genotype I was documented in Brazil in 2013  [47] , but we did not find evidence of its circulation in Colombia.
Despite multiple strain introductions of DENV-1, DENV-2 and DENV-4, our data indicated that only one lineage predominates up to date, suggesting the turnover of viral lineages over time. With the data at hand we could not address if such turnovers are due to stochastic die-off or other factors; however, the re-emergence of DENV-2 in an dengue-endemic setting from Indonesia was due to the loss or decrease of herd immunity during a 5-year period where DENV-1 predominated  [48]. Similarly, changing serotype prevalence has been associated with lineage extinction and lineage replacements in Thailand and with dengue epidemics in Singapore  [49, 50, 51, 52]. Here, we recorded two distinct lineages of DENV-3 that apparently were introduced at the same time, that had the same level of circulation and that overlapped in time and space.
Acknowledgments This project was possible thanks to support from coordinators in healthcare institutions and diagnostic laboratories in Santander and Ocaña. We thank  Luis Angel Villar  from  Unidad de Epidemiologı ´a Clı ´nica, Escuela de Medicina, Universidad Industrial de Santander , for his supporting virological surveillance activities in 2014-2016. Financial support: This study was supported by the  Colombian Government: Science, Technology and Innovation Fund  of  Sistema General de Regalias, Santander, Casanare, Valle del Cauca  ( BPIN 2013000100011 ).
For intracellular staining, cells were first stained for viability and extracellular markers, permeabilized and fixed with Perm/Fix reagent (BD Biosciences) before labeling with the appropriate antibodies. Samples were acquired with a LSRII device (BD Bioscience) using FACSDiva™ software (version 6. 1. 3, BD Biosciences) and analyzed with FlowJo Software (V7. 6. 5, TreeStar). Application settings and rainbow beads (BD Bioscience) were used to monitor and adjust the laser intensities.
96 well-selected TaqMan assays (Applied Biosystems, Thermo Fisher Scientific), selected according to their scientific relevance to multiple sclerosis, and were added at a concentration of 18 mM each to ensure gene specific reverse transcription and pre-amplification (a list of the genes is provided in Table  S2 ). The expression levels of each of the 96 genes of interest were quantified using microfluidic qPCR analysis on the Fluidigm ® Bio-mark™ HD system according to the manufacturer's instructions and expressed as a function of the Ct level. Raw expression data were processed with the Fluidigm ® Real-Time PCR Analysis software and analyzed with the Fluidigm ® Singular Analysis Toolset in R.
Acknowledgments The authors would like to thank all of the patients who agreed to participate in this study and who provided their time and access to their biological samples. We thank  P. O. Couraud  ( Cochin Institute ) for providing us with the hCMEC/D3 cell line and the protocol. Tissue samples and associated clinical and neuropathological data were supplied by the  Multiple Sclerosis Society Tissue Bank , funded by the  Multiple Sclerosis Society of Great Britain and Northern Ireland , registered charity 207495. This work has been supported by a grant from the  ARSEP Foundation  and by the  ANTARES Association.
Tumor Control by a Combination of Monalizumab and Cetuximab in Patients with SCCHN We found that combinations of NKG2A-blocking mAbs with other IO treatments, such as anti-PD-1 mAbs, anti-PD-L1 mAbs or cetuximab, had additive effects on anti-tumor immunity in preclinical experimental settings in vitro and in vivo. These results provide a scientific rationale for evaluations of the efficacy and safety of monalizumab in cancer patients. SCCHN tumors were strongly positive for HLA-E and were infiltrated with CD8 + T cells and NK cells, which may express NKG2A (Figures  4A,  4B,  and 4D ). Cetuximab is used in the standard care regimen for SCCHN  (Vermorken et al. , 2007). We therefore assessed the safety and efficacy of the combination of monalizumab and cetuximab in patients with previously treated recurrent or metastatic (R/M) SCCHN in a phase II clinical trial (NCT02643550).
Importantly, such a clinical trial is ongoing (NCT02671435) and very recently preliminary safety and efficacy data were reported  (Segal et al. , 2018). Briefly, the dose escalation part of the study demonstrated the feasibility of combining the two agents with no new safety signals noted beyond the known safety profile for each individual agent. The initial clinical activity data from a cohort expansion in pretreated (median of three previous lines of systemic therapy) microsatellite stable colorectal cancer (MSS CRC; n = 39) demonstrated an ORR of 8% (median duration of response of 16. 1 weeks) and a disease control rate (DCR) at 16 weeks of 31%  (Segal et al. , 2018). Although these results are very preliminary, they are an example of potential therapeutic opportunities for immunotherapy in MSS CRC, a setting in which immune checkpoint-based therapy has, so far, failed to demonstrate any consistent and meaningful clinical benefit.
Other antibodies are also used to stimulate the immune system via ADCC. One such antibody is cetuximab, which is used in metastatic CRC and SCCHN. We showed in vitro that NKG2A blockade with monalizumab boosts NK cell-mediated ADCC against cetuximab-coated SCCHN tumor cells. Consistent with these data, treatment with a combination of monalizumab and cetuximab was found to be effective in the NCT02643550 phase II clinical trial for SCCHN. Importantly, little, if any, toxicity was associated with blocking NKG2A and especially no signs of auto-immunity. The encouraging results obtained for SCCHN in NCT02643550 and for MSS CRC in NCT02671435 require consolidation in further clinical trials, but they constitute a key step toward the use of monalizumab in combination treatments against cancer. Once the samples from all the patients enrolled in the monalizumab clinical trials will be accessible, translational studies will be performed.
Preparation of human TILs Tissues were mechanically dissociated before enzymatic digestion using collagenase I (2 mg/mL, Sigma C0130), DNase I (25mg/mL, Roche 10104159001) and hyaluronidase (2mg/mL, SIGMA H3506). Red cells from dissociated tissues and whole blood were lysed (eBioscience 00-4300-54) and dead cells stained with Live/Dead Aqua. Cells were stained with the antibodies detailed in the Key Resources Table. Samples were analyzed by flow cytometry (BD LSR FORTESSA) using the BD FACS DIVA software 8. 0. Data were analyzed on FlowJo V10. 02. Functional assay with CD8 + T cells (cytotoxic assay) Flu-specific CD8 + T cell lytic activity was assessed against K562 HLA-E HLA-A2 PD-L1 cells using a standard chromium release assay. K562 cells co-expressing HLA-E HLA-A2 and PD-L1 were first pulsed with 1 mg/mL flu peptide for 1 h at 37 C and then washed three times before loading with 51 Cr for 1 h.
After washing, cells were plated with CD8 + T cells at 2:1 effector-to-target (E:T) cell ratio in presence of anti-human CD64 antibody (10 mg/mL) and in presence or absence of monalizumab (20 mg/mL), durvalumab (20 mg/mL) or isotype control antibodies. After 4 h at 37 C, 50 mL of supernatant was harvested and counted in a Topcount microplate scintillation gamma counter. Data were acquired in counts per minute. Specific lysis was calculated according to the formula [(test release À spontaneous release)/(maximum release À spontaneous release)] 3 100. Spontaneous release represents 51 Cr release from target cells in medium alone, and maximum release is the 51 Cr release from target cells lysed in medium 2% Triton X-100, each measured in at least six replicate wells.
Trial design Phase II study of monalizumab, a first-in-class NKG2A monoclonal antibody, in combination with cetuximab in previously treated recurrent or metastatic squamous cell carcinoma of the head and neck (NCT02643550) is a multicenter, international (US and France), open label, single arm study to evaluate the antitumor activity of monalizumab in combination with cetuximab. Five dose levels of monalizumab (0. 4, 1, 2, 4, 10 mg/kg every 2 weeks) in combination with the approved dosage of cetuximab (400 mg/m 2 load then 250 mg/m 2 weekly) were explored. The highest dose tested (10 mg/kg) was used for the phase II cohort expansion; the overall phase II study will include 40 patients. The trial consisted of 2 parts: a phase Ib dose escalation part of monalizumab combined with cetuximab followed by a cohort expansion phase II part of cetuximab + monalizumab.
QUANTIFICATION AND STATISTICAL ANALYSIS Details on statistics used can be found in figure legends. All statistical analyses were performed using GraphPad Prism software version 7 (GraphPad). Kaplan Meier was used for survival representations. When sample size was large enough, normality of populations was tested using d'Agostino-Pearson omnibus normality test. When normality was not reached, statistical significance between two paired samples populations was determined using Wilcoxon matched-pair signed rank test, statistical analyses for three or more groups. N represents the number of samples used in the experiments. Samples are shown with means or medians with or without error bars showing the SD. Significance was assumed with *p < 0. 05; **p % 0. 01; ***p < 0. 001, ****p < 0. 0001.
ACKNOWLEDGMENTS We thank  Mohammed Dar  ( Mediumme ) for critically reviewing the manuscript;  Francois Romagne(MI-mAbs ),  Fabien Chanuc ,  Nicolas Fuseri , and  Flavien Caraguel  ( Innate Pharma ) for help and advice during these studies. The E. V. lab was supported by funding from the  European Research Council (ERC)  under the European Union's  Horizon 2020 research and innovation program  ( TILC , grant agreement no. 694502 ), the  Agence Nationale de la Recherche ,  Equipe Labellisee ''La Ligue,'' Ligue Nationale contre le Cancer, MSDAvenir, Innate Pharma , and institutional grants to the  CIML (INSERM, CNRS, and Aix-Marseille University ) and  Marseille Immunopole.
Material and methods Dat'AIDS cohort is a French multicentric prospective cohort involving 19 HIV reference centres in France (Clinicaltrials. gov ref NCT02898987). These reference centres maintain prospective databases of all HIV infected patients seeking care in the centres and providing written consent. The data collection has been approved by the French national commission on informatics and liberty (CNIL). The database is implemented via an electronic medical  [14]. Dat'AIDS cohort collects sociodemographic and clinical data, medical and ARV history, immunological and virological status at regular 3-to 6-month intervals during routine clinical assessment. For the purpose of the study we selected all HIV patients aged 50 year-old and over at the last visit, with at least one visit since 2004 and still on follow-up at the censoring date (Dec 31 st 2015).
Patients were classified as "elderly" if they were aged between 50 and 74 and "geriatric" if they were aged 75 or older at the latest visit. Medical history collected data on past or current diabetes (insulin-dependent or non insulin-dependent diabetes), hypertension (confirmed blood pressure above 140/90 mm Hg or hypertension treatment), dyslipidaemia (hypercholesterolemia and/or hypertriglyceridemia or treatment with lipid lowering agent), history of cardiovascular disease (ischemic cardiovascular disease or stroke), depression, and cancer. Renal insufficiency was defined by a confirmed estimated glomerular filtration rate below 60 ml/ min/1. 73 m 2 , as calculated using the Modification of Diet in Renal Disease (MDRD) formula. Continuous variables were described by their medians and interquartile ranges (IQR) and compared between groups using a Mann-Whitney test.
Results Among the 37,511 HIV infected adult patients included in the Dat'AIDS cohort with at least one visit since 2004 and still on follow-up at the 31 December 2015, 17 008 (45. 3%) were older than 50 years (S1 Dataset). Among them 16,436 subjects were aged between 50 and 75 years and classified in the elderly group and 572 subjects were aged 75 years or older and classified in the geriatric group. The geriatric group represented 1. 5% of the whole population. Sociodemographics and clinical characteristics of the elderly and geriatric groups are presented in Table  1. At the last visit, there was no difference in the proportion of HIV-1 infected subjects on ART, 97. 3% and 97. 2% in the geriatric and the elderly group, respectively, with a duration of known HIV infection slightly shorter in the geriatric group than in the elderly group (18.
Acknowledgments Members of the Dat'AIDS study group:  C. Drobacheff-Thie  ´baut,  A. Foltzer ,  K. Bouiller ,  L. Hustache-Mathieu ,  C. Chirouze ,  Q. Lepiller ,  F. Bozon ,  O Babre ,  P. Muret  ( Besancon )  H. Laurichesse ,  O. Lesens ,  M. Vidal ,  N. Mrozek ,  C. Aumeran ,  O. Baud ,  V. Corbin ,  P. Letertre ,  S. Casanova ,  C. Jacomet  ( Clermont-Ferrand )  B. Hoen ,  I. Lamaury ,  I. Fabre ,  E. Curlier ,  R. Ouissa ,  K. Schepers ,  C. Herrmann-Storck ,  N. Dournon (Guadeloupe) D. Merrien ,  P. Perre ,  T. Guimard ,  O. Bollangier ,  S. Leautez ,  M. Morrier  ( La Roche sur Yon )  F. Ader ,  F. Biron ,  A. Boibieux ,  L. Cotte ,  T. Ferry ,  P Miailhes , T.
To sort out the reservoir, we captured 72 rodents from different locations in Waziristan, such as around houses, in cornfields, and in wild plantations surrounding the houses of CL patients, and analyzed liver and spleen samples by PCR. In addition, we collected sand flies from within 1. 5 km of CL patients' houses for molecular analysis  (6). We determined the prevalence rate of CL by a formula described previously  (7)  and performed statistical analyses using statistical software SAS Enterprise Guide (version 4. 2; SAS Institute, Cary, NC, USA) by univariate analysis of variance with statistical significance at p<0. 05. Prevalence according to sex was consistent with previous findings, indicating that CL infections were more prone to develop in males (Table ) because of more social activity and interaction with IDPs, whereas females always remain covered because of Islamic rules and thus are less prone to sand fly bites.