dataesr/scientific-paragraphs-categorization huggingface dataset:
By extension, the patient will be more or less prone not only to CDI but also to severe forms of CDI because of his or her underlying clinical condition  [26]. We did not capture or stratify data to be able to apply an analysis of the propensity of this. Despite these limitations and to progress on this debate, it would be interesting to continue with prospective studies to provide clear answers using systematic visits and feces screening as better evaluations of the use of other drugs which could increase the risks of CDI episodes. Unfortunately, since 5 December 2020, CT availability has been internationally suspended due to Ralstonia pickettii contamination in several batches  [27]. A global voluntary recall was performed, but there is still no planned date for availability.
Genetic determinants of gastrointestinal adverse events with colchicine There were 767 participants randomized to colchicine who were included in the genetic analyses for gastrointestinal adverse events, of those, 187 had a gastrointestinal event. The ABCB1 rs1045642 variant and the CYP3A4 metaboliser phenotype were not associated with gastrointestinal adverse events (P=0. 97 and P=0. 31 respectively). We found 22 genetic variants significantly associated with gastrointestinal events at 2 loci located on chromosomes 6 and 10 (Figure  1 ). ). When conditioning on rs6916345, no additional genetic variants remained significant at P<5×10 -8 in the region and rs6916345 had the highest probability of being causal by CAVIAR analysis (Data Supplement). The minor allele (A) was associated with gastrointestinal events in the colchicine group (hazard ratio (HR)=1.
requests. The analytic methods and study materials may be made available to other researchers for purposes of reproducing the results or replicating the procedure. Summary statistics are available publicly for download and visualisation via PheWEB 4 at statgen. org/pheweb/colcot. The COLCOT clinical trial was registered at www. clinicaltrials. gov under the unique identifier NCT02551094. The study protocol was approved by the Montreal Heart Institute research ethics committee and complies with the Declaration of Helsinki. Written informed consent was obtained from all participating subjects. Full Methods are available in the Data Supplement of the article. Downloaded from http://ahajournals. org by on February 10, 2021.
Acknowledgments: We acknowledge the technical support of  Diane Valois  and  Isabelle Fillion  for the genotyping work and of  Yannik Couture  and  Sylvain Versailles  for blood and DNA sample preparation. We thank the patients and staff who supported this study. Sources of Funding: This work was supported by the  Health Collaboration Acceleration Fund from the Government of Quebec  [to J. -C. T. ]. M. -A. L. is supported by a  Frederick Banting and Charles Best Canada Graduate Scholarship Doctoral Award  from the  Canadian Institutes  of.
SMPD3 expression, mutations, and DNA methylation in human melanoma SMPD3 expression in patients with primary and metastatic melanoma was evaluated using the Oncomine  (20)  and The Cancer Genome Atlas (TCGA) melanoma  (21)  databases; each dataset was evaluated separately (Fig. 1 ). TCGA genomic and clinical data were downloaded from the UCSC cancer genome browser project (http:// www. genome. ucsc. edu/). Among the 422 patients with melanoma from the TCGA dataset, the analysis population consisted in 342 patients with distant metastasis for whom there were overlapping RNA sequencing and clinical data (Supplementary Data File S1). No other inclusion/exclusion criteria were applied for this analysis. Differentially expressed genes (DEG) in human metastatic melanoma samples exhibiting high (n ¼ 68) versus low (n ¼ 68) SMPD3 expression were identified according to a fold-change (FC) threshold of 2 and a P value < 0.
05 adjusted with the Benjamini-Hochberg procedure. Voom transformation and DEG analysis were performed with R packages egdeR and limma, respectively. The top 200 genes upregulated (FC > 7. 75) in human metastatic melanoma samples exhibiting high SMPD3 expression level group were selected for pathway analysis. This was performed with Autocompare ZE based on the Zelen exact test for P value estimation (ref. 22 ; available at https://sites. google. com/site/ fredsoftwares/products/autocompare-ze) using C5 database from the    (26, 27). To generate the B16K1 cell line, B16F10 cells were engineered to stably express the MHC class I molecule H-2Kb  (27). B16K1 cells were cultured in DMEM medium (Gibco, #61965-026) containing 10% heatinactivated FCS and were authenticated in February 2012 by the Leibniz Institute DSMZ GmbH.
In vitro PD-L1 expression B16K1 cells (0. 3 Â 10 5 ) expressing high or low levels of nSMase2 were plated in 6-well plates and treated with vehicle [PBS (Eurobio Scientific, #CSP1PBS01-01)], 0. 1% BSA (Euromedex, #1035-70-C), 50 ng/mL recombinant murine TNF (Peprotech, #315-01A), and 100 U of recombinant murine IFNg (Peprotech, #315-05) alone or combined in DMEM 10% FCS for 72 hours. Cells were detached using Versene solution (Gibco, #15040-033), PD-L1 expression (BD Biosciences, MIH5) was assessed by flow cytometry on an LSRFortessa X-20 (BD Biosciences), and data were analyzed with the FlowJow10 software.
For the staining of intracellular targets, cells were first stained for membrane markers, then fixed and permeabilized using the "Foxp3 fixation/permeabilization staining buffer set" (eBioscience, #00-5523-00) before staining for Foxp3 or TNF and IFNg. Samples were acquired using an LSRFortessa X-20 (BD Biosciences), and data were analyzed with the Diva or FloJow 10 softwares. Gating strategies are depicted in Supplementary Data File S2.
Statistical analysis Each experiment was designed to use the minimum number of mice or samples required to obtain informative results and sufficient material for subsequent studies. No specific statistical tests were used to predetermine the sample size. For animal experimentation, we used at least 5 mice per group, and experiments were typically performed twice, unless otherwise stated in the figure legends. Statistical significance of difference between groups was evaluated using the GraphPad Prism 7 software. Briefly, we tested whether the values come from a Gaussian distribution using a D'Agostino-Pearson omnibus normality test. When passing the normality test, a Student t test was used. Otherwise, a Mann-Whitney U test was used. For statistical significance of animal survival, the log-rank test was used.
SMPD3 expression in human melanoma biopsies correlates with a CD8 þ T-cell gene signature Analysis from the Oncomine database indicated that low levels of SMPD3 transcripts are found in human metastatic melanoma as compared with primary tumors and normal skin (Fig. 1A ), suggesting that nSMase2 downregulation is likely associated with melanoma progression. ISH experiments on skin sections from patients with advanced melanoma using RNAscope technology revealed robust and low SMPD3 expression in the tumor-adjacent stratum granulosum and cutaneous melanoma, respectively (Fig. 1B ; Supplementary Fig. S1A-S1D ). Moreover, a majority of tumor biopsies from the TCGA cohort of patients with advanced melanoma expressed SMPD3 at low levels (Fig. 1C ). Because gene hypermethylation has been shown to downregulate SMPD3 expression in solid tumors (9), we analyzed the methylation status of SMPD3 in the TCGA melanoma cohort.
2E ). Because nSMase2 overexpression did not decrease in vitro proliferation of B16K1 cells but impaired tumor growth in vivo, we hypothesized that high nSMase2 expression in melanoma cells could trigger a remodeling of the tumor microenvironment. Building upon the data obtained using human samples from the TCGA dataset, we analyzed the immune response in mice engrafted with B16K1 melanoma cells expressing nSMase2 at low or high levels. Leukocytes (CD45 þ ) and T cells (Thy1 þ ) frequencies were significantly increased in melanomas that expressed nSMase2 at high levels (Fig. 2F ). Among T cells, the proportion of CD8 þ TILs was 3-fold higher in tumors expressing nSMase2 at high levels than in tumors expressing low levels of the enzyme (Fig. 2F ). To determine whether this increase in CD8 þ TILs may be important in mediating the deleterious impact of nSMase2 on melanoma growth, nSMase2 high and nSMase2 low B16K1 cells were engrafted in CD8adeficient mice.
Discussion The present study provides the evidence that (i) SMPD3 is expressed at low levels in most human metastatic melanoma samples and (ii) low SMPD3 expression is associated with shortened overall survival in patients. High SMPD3 expression was associated with "Immune system process" and "Lymphocyte activation" gene signatures, and melanoma samples expressing SMPD3 at high levels exhibited a gene signature of TILs. In agreement with the data obtained from human melanoma databases, high nSMase2 expression increased CD8 þ TIL frequency and the CD8 þ T-cell-dependent immune response to decrease melanoma growth in WT mice but not in mice lacking CD8 þ T cells. This demonstrates that nSMase2 antitumorigenic properties are fully dependent on its ability to stimulate adaptive immunity. Collectively, our data reveal that SMPD3 downregulation or mutation facilitates melanoma progression by contributing to melanoma immune escape.
In addition, SMPD3 expression in melanoma samples may serve as a novel biomarker to predict the clinical response to immunotherapy. This is currently being evaluated in a prospective clinical trial (IMMUSPHINX: NCT03627026) conducted in our institute and others, and the primary objective of this clinical trial is to evaluate whether SL metabolites and/or SL-metabolizing enzymes are putative biomarkers to predict response/resistance in patients with advanced melanoma treated with anti-PD-1 therapy alone or in combination with anti-CTLA-4 therapy.
Acknowledgments The authors are grateful to the flow cytometry, microscopy, and animal facilities of the  I2MC  ( INSERM U1048 , Toulouse, France) and CRCT (INSERM  U1037, Toulouse, France ) for their technical assistance. They thank  Drs. F. Sabourdy  and  S. Carpentier  ( INSERM U1037, Toulouse, France ) performing mutation analysis and technical assistance, respectively. They also thank  Prof. J. van Meerwijk  ( INSERM U1043, Toulouse, France ) for the kind gift of CD8-deficient mice. They also thank  Prof. L. Nieto  ( IPBS, Toulouse, France ) for sEVs analysis. They thank  Drs. V. Douin-Echinard  ( INSERM U1048 ,  Toulouse, France) ,  J. C. Gu  ery ( INSERM U1043, Toulouse, France ),  J. Torrisani ,  J. J. Fourni  e,  L. Martinet , and  A. F. Tilkin-Mariam  e ( INSERM U1037, Toulouse, France ) for fruitful discussion.
This work was supported by  INSERM, Paul Sabatier University (Toulouse III) ,  Ligue Nationale contre le Cancer  (LNCC, Equipe Labellis ee 2013),  Ligue R egionale contre le Cancer (Midi-Pyr en ees) ,  INSERM Transfert, Canc eropôle Grand Sud-Ouest ,  Rotary Toulouse Clubs, Fondation Toulouse Cancer Sant e, and Fondation ARC  (Equipe Labellis ee 2019). The research leading to these results has received funding from the  Transcan-2 Research Program , which is a transnational R&D program jointly funded by national funding organizations within the framework of the  ERA-NET Transcan-2  ( IMMUSPHINX  project). A. Montfort was a recipient of the " Prestige" fellowship program  from the  Marie Curie Actions  ( PCOFUND-GA-2013-609102 ) and is a recipient of a fellowship from  Fondation de France. The costs of publication of this article were defrayed in part by the payment of page charges.
A, SMPD3 expression analysis in normal human skin (n ¼ 4), primary (P. Mel. , n ¼ 14), and metastatic (M. Mel. , n ¼ 40) melanoma samples from the Oncomine database. Ã , P < 0. 05; ÃÃ , P < 0. 01. B, SMPD3 expression analyzed by ISH on a skin sample from a patient with advanced melanoma. Pictures are representative of staining carried out on samples from five patients. C, SMPD3 expression analysis in tumor biopsies from patients with metastatic melanoma (TCGA melanoma cohort; n ¼ 342). RSEM, RNA-Seq by Expectation Maximization. D, Neutral Sphingomyelinase 2 in Melanoma Immunotherapy AACRJournals. org Cancer Immunol Res; 9(5) May 2021.
The dashed horizontal line represents the assay's limit of detection (30 copies/mL) with undetectable events plotted as 15 copies/mL. d The frequency of immune activation (HLA-DR + CD38 + ) was quantified in memory (CD95 + ) CD4 + T-cells in PBMCs and e representative flow cytometry plots are given at critical time points (as indicated above) for a cytokine-treated (RQm14) and control RMs (RSp14; 16 of 260 single-replicate stains). f The expression of IFN-stimulated genes (ISGs) was measured by RNA-seq in PBMCs at 2 h post-rIFNα and was calculated as a log 2 fold-change between rIFNα-treated (n = 6) and control RMs (n = 5), which is represented as a double-gradient heatmap. The size of each data point corresponds inversely to the log 10 -transformed nominal p value with significant (p < 0.
05) adjusted p values indicated by a black border. Using DESeq2, data were analyzed with a two-sided (95% CI) Wald test using the Benjamini-Hochberg method for multiple comparisons. By flow cytometry, the frequency of g HLA-E + CD4 + T-cells and h NKG2a/c + CD8 + T-cells was quantified of CD45 + lymphocytes in PBMCs. i NK cells isolated from PBMCs were cultured alone or the presence of K562-HLA-E * 0101 cells either unloaded or loaded with SIVmac 239/251 Env peptides. The SIV-Env-specific, HLA-E-restricted activity was calculated as the log 10  cell responses by SIV-Gag-stimulated IFN-γ ELISpot (Supplementary Fig. 10e ). At d13 post ATI, the distribution of NK cell maturation subsets in PBMCs was similar to that observed during ART with higher levels of terminally differentiated (NKG2a/ c low CD16 + , stage 3) NK cells with strong, innate degranulation activity (i.
i. or d374 p. i. ), as was cellassociated SIV-DNA (log 10 copies per 10 6 cells) in d PBMCs, e LN, and f RB. a-f Data from individual RMs are overlaid against the mean ± SEM (in red): control (ART-only, black square; n = 5 RMs) and cytokine-treated (ART + IL-21 + IFNα, blue circle; n = 8 RMs). g From cryo-preserved, magnetically isolated LN CD4 + cells, the number of infectious units per million (IUPM) cells were measured with a limiting dilution quantitative viral outgrowth assay (QVOA; 3 serial dilutions plated in triplicate) in control (n = 4) and cytokine-treated RMs (n = 6). a-g Treatment phases are indicated with the following background shading: IL-21 (orange), rIFNα (red), and ART (gray). Data were analyzed with two-sided (95%), two-way ANOVA with Bonferroni's correction with comparisons relative to controls and time.
ARTICLE Flow cytometric analysis. Fourteen-parameter flow cytometric analysis was performed on fresh PBMCs and mononuclear cells derived from LN biopsies and RB punches. Samples were stained utilizing standard procedures employing clones of anti-human mAbs that we have shown to be cross-reactive in RMs  20, 44, 46, 47  and are validated in databases maintained by NHP Reagent Resource (MassBiologics: https:// www. nhpreagents. org/ReactivityDatabase). The following antibodies were utilized for the longitudinal staining panel per T-cell and B-cell characterization: anti-CD21-PE (clone B-ly4, 10 μL, cat. 555422), anti-CD28-PE-CF594 (clone CD28. 2, 5 μL, cat. 562296), anti-CD95-PE-Cy5 (clone DX2, 10 μL, cat. 559773), anti-CCR7-PE-Cy7 (clone 3D12, 7. 5 μL, cat. 557648), anti-CD45RA-APC (clone 5H9, 10 μL, cat.
12-5825-82 from eBioscience; Supplementary Fig. 1d ). Stain volumes are given per 10 6 live mononuclear cells or per 4-5 processed RB punches. Cells were fixed and permeabilized with a Cytofix/Cytoperm kit (BD Biosciences). Flow cytometry acquisition was performed on a minimum of 120,000 live CD3 + T cells for PBMC and LN, and a minimum of 50,000 live CD3 + T cells in RB. Data acquisition was performed on an LSR II (BD Biosciences) using FACS DiVa software (v8. 0. 1) and analyzed using FlowJo software (version 9. 9. 6; TreeStar). Gates for activation (HLA-DR + CD38 + ) and proliferation in memory CD4 + T cells and memory CD8 + T cells were set based on their expression within the corresponding naive subset. Cryo-preserved PBMCs were stained as previously described  37  to characterize NK cell subsets using a panel of the following mAbs: anti-CD3-V500 (clone SP34-2, 5 μL, cat.
L34957) from Thermo Fisher Scientific (Supplementary Fig. 3a ). Stain volumes are given per test (approximately 5-10 RB punches). Cells were fixed and permeabilized with a Cytofix/Cytoperm kit (BD Biosciences). The intracellular stain was incubated for 1 h at room temperature in the dark. Data acquisition was performed on a minimum of 50,000 live CD3 + T cells on an LSR II (BD Biosciences) using FACS DiVa software (v8. 0. 1) and analyzed using FlowJo software (version 9. 9. 6; TreeStar). Determination of plasma viremia. Six-replicate reaction hybrid qRT-PCR assay was performed to determine SIV-RNA copies per mL of EDTA plasma (i. e. , viral loads) as previously described  48  with a limit of detection of 30 copies/mL. While primers target a highly conserved viral sequence in SIVmac 239 gag (Supplementary Table  2 ), unconventional neutral bases are incorporated to avoid biasing in measuring off-target viral sequences.
5. 2b) with default alignment parameters  62  (https://www. unmc. edu/ rhesusgenechip/index. htm). Rates of increase in log 10 SIV-RNA copies per mL of plasma post ATI were obtained using a mixed-effects linear model specifying that data follow a linear regression over time, with a random intercept for each animal. The mean slope and mean linear increase were estimated and compared between treatment conditions within the framework of the mixed-effects linear model  68. DESeq2 version 1. 22. 1R package  69  was used to produce normalized read counts and compute the differential expression estimation using the Wald test. Multiple-test correction was performed with the Benjamini-Hochberg method and a FDR <0. 05 was used to indicate statistical significance. Statistics.
Acknowledgements We thank  Stephanie Ehnert  and  Christopher Souder  from  Research Resources , and  Sherrie Jean  and  Jennifer Wood  from  Veterinary Medicine at Yerkes National Primate Research Center (YNPRC ) for providing animal and veterinary care, respectively. We gratefully acknowledge  Katie Kitrinos  at  ViiV Healthcare  for supplying DTG, and  Romas Geleziunas  at  Gilead Sciences  for supplying FTC and TDF. Both the rhesus rIL-21-IgFc and rhesus IFNα-IgFc were kindly supplied by  Kenneth Rogers  at the  Resource for Nonhuman Primate Immune Reagents. We would also like to thank  Mathias Lichterfeld ,  Pilar Garcia Broncano , and  Xiaodong Lian  of the  Ragon Institute , and  William Bosche  at  Leidos Biomedical Research  for technical assistance. The SIVmac 239 strain used to infect the RMs was kindly provided by  Koen Van Rompay  of  UC-Davis , and the 174xCEM cell line was supplied by  Dr.
Data availability Materials provided via MTA were supplied without restrictions on use. Source data are provided for the performed correlations, as are the statistical readouts for the correlations and RNA-seq analyses. RNA-seq data related to Figs. 1f  and 5a , and Supplementary Fig. 4  are publicly available in GenBank (https://www. ncbi. nlm. nih. gov/geo/query/acc. cgi) under GEO accession GSE163443 with subseries GSE163440, GSE163441, and GSE163442. The MacaM version 7 assembly of the Indian rhesus macaque genomic reference is publicly available at https://www. unmc. edu/rhesusgenechip/index. htm. Source Data are provided with this paper.
f The expression of IFN-stimulated genes (ISGs) was measured by RNA-seq in PBMCs at 2 h post-rIFNα and was calculated as a log 2 fold-change between rIFNα-treated (n = 6) and control RMs (n = 5), which is represented as a double-gradient heatmap. The size of each data point corresponds inversely to the log 10 -transformed nominal p value with significant (p < 0. 05) adjusted p values indicated by a black border. Using DESeq2, data were analyzed with a two-sided (95% CI) Wald test using the Benjamini-Hochberg method for multiple comparisons. By flow cytometry, the frequency of g HLA-E + CD4 + T-cells and h NKG2a/c + CD8 + T-cells was quantified of CD45 + lymphocytes in PBMCs. i NK cells isolated from PBMCs were cultured alone or the presence of K562-HLA-E * 0101 cells either unloaded or loaded with SIVmac 239/251 Env peptides.
IM-1001/MAN0003188). Alternative products might be used, but methodology might need to be adjusted. Representative data provided in this protocol was obtained using a mouse monoclonal antibody to CAS9 (dilution 1:2,000, incubation overnight (>12 h) at 4 C) and an HRP mouse monoclonal antibody to b-actin (dilution 1:20,000, incubation for 2 h at room temperature). Troubleshooting 1 4. Validation of gene editing efficacy of selected CAS9-expressing iniDC clones a. Based on CAS9-expression detected by WB, label selected clones that express high level of CAS9 in the seed plates. Trypsinize those clones as described in step 2c and stop trypsin with 150 mL CG medium, transfer all content to one well of a 6-well plate, supply 2 mL of CG medium (containing 10 mg/mL blasticidin), and maintain in an incubator until cells reach 50%-70% confluency.
Collectively, these data demonstrate that the ox-DNA was of mitochondrial origin. We then used an ELISA to quantitate ox-mtDNA in patient plasma and compared levels in paired patient BM and PB plasma. Log 10 -transformed, glucose-adjusted (to account for possible changes arising from hyperglycemia-induced inflammasome activation  30, 31  ) ox-mtDNA was detected in both PB and BM plasma with significantly higher levels found in BM plasma (P 5 1. 0 3 10 23 ), with a positive correlation among paired samples (r 5 0. 339; P 5 4. 0 3 10 23 ; n 5 71) (Figure  3 B-C ). We next quantified ox-mtDNA in PB plasma from MDS patients compared with age-matched healthy donors (Table  1 ). 32 We found no robust differences in log 10 -transformed, glucose-adjusted ox-mtDNA levels based on sex, race, co-existing autoimmune diseases, alcohol consumption, or tobacco usage (supplemental Figure  8 ).
Acknowledgments The authors thank laboratory interns  Alexandra R. Calescibetta ,  Nina S. Hong ,  Tommy Pham , and  Robert P. Dalton  for their assistance, and they acknowledge  Eric Lau  and Kenneth Wright who were instrumental in preparing this manuscript. The visual abstract was created with BioRender. This study was partially funded by the  Eastern Cooperative Oncology Group/American College of Radiology Imaging Network Cancer Research Group Study  ( E2905   Intergroup Study ), by grants from the  National Institutes of Health, National Cancer Institute (NCI)  ( CA180820 ) and the  NCI-designated Comprehensive Cancer Center  ( P30-CA076292 ), from the  Leukemia & Lymphoma Society  ( 7018-19 ), and from the  Flow Cytometry, Microscopy , and  Tissue Core Facilities  at the H. Lee  Moffitt Cancer Center & Research Institute.
Cell Culture and Treatments Human aortic endothelial cells (HAECs) from 4 different male donors were obtained from Promocell. HAECs were grown in endothelial cell growth media following the manufacturer's instructions (Promocell). All assays were done at 37 • C, 95% sterile air, and 5% CO 2 in a saturation-humidified incubator. Cells were used between passages 3-5. HAECs were treated with recombinant SARS-CoV2 Spike protein (1 µg/mL, R&D Systems) for 24 to 72 h for protein studies. The duration and the dose of the treatment was chosen based on previous studies (data not shown). Cells were treated with Spironolactone (10 -6 M, Sigma Aldrich), Eplerenone (10 -5 M, Sigma Aldrich), or the Galectin-3 inhibitor G3P-01 (1 mg/mL, G3 Pharmaceuticals) 1 h before Spike stimulation to assess the preventive effect of the aforementioned MRAs and the Gal-3 inhibitor.