dataesr/scientific-paragraphs-categorization huggingface dataset:
described an antibody against a nonfunctional P2RX7 variant that is promising to treat basal cell carcinoma  31. It would be interesting to combine HEI3090 with the therapeutic P2RX7 antibody and assay the efficacy of this combo treatment. Antineoplastic action of eATP was previously explored using ATP administration in cancer patients and abandoned for lack of convincing results  32, 33. Extracellular ATP is naturally degraded to adenosine by ectoenzymes, and adenosine is an immunosuppressive molecule  34. To inhibit the production of adenosine, blocking antibodies against CD39 and CD73 ectoenzymes were produced and tested in mouse cancer models but also in ongoing clinical trials (NCT03454451). This strategy seems to be promising, at least, in mice tumor models. In a first study, Perrot et al.
For all panels, the optimal cutoff is determined on KMplot. The p value (log-rank, Mantel Cox test), the hazard ratio, and number of patients are indicated. Source data are provided as a Source Data file. ADC adenocarcinoma, HR hazard ratio. predictive markers. Using TCGA dataset, we uncovered that P2RX7 expression is correlated to CD274 (PD-L1) expression and "hot" immunophenotype signatures in NSCLC patients. In addition, patients with high P2RX7 and low CD274 or high CD274 and low P2RX7 have a better overall survival than patients with high CD274 and high P2RX7. This result suggests that immunotherapies may be efficient in double positive patients and questions the ability of P2RX7 to represent a valuable biomarker for αPD-1/PD-L1 therapies. In this context, we showed in another study  26  that the expression of P2RX7B splice variant in tumor immune cells is associated with less infiltrated tumors in lung adenocarcinoma.
For intracellular cytokines, staining was performed after stimulation of single-cell suspensions with Phorbol 12-myristate 13acetate (PMA at 50 ng mL -1 , Sigma), ionomycin (0. 5 μg mL -1 , Sigma) and 1 μL mL -1 Golgi Plug™ (BD Biosciences) for 4 h at 37 °C 5% CO 2. Cells were incubated with Live/Dead Near-IR stain (Invitrogen), according to the manufacturer's protocol prior to Ab surface staining. Then, intracellular staining was performed using Cytofix/Cytoperm™ kit (BD biosciences) following the manufacturer's instructions. The production of IFN-γ and IL-10 was simultaneously analyzed in CD45 + , NK, CD4 + T, or CD8 + T cells. Data files were acquired and analyzed on Aria III using Diva software (BD Biosciences) or on the CytoFlex LX (Beckman Coulter) and analyzed using FlowJo software (LLC).
Gating strategies are shown in Supplementary Fig. 12. Immunohistological analysis of tumors. Collected tumors or lungs were fixed in 3% formamide for 16 h prior inclusion in paraffin. We used the following antibodies: anti-CD3, anti-CD8, anti-IL-18, αPD-L1, and anti-Ki67 (see Supplementary Table  1 ). After staining, slides were captured and analyzed using NDP view2software. For the analyses, five zones per tumor were randomly selected and cells were counted using ImageJ software. Results are expressed as number of positive cells per total cell number. Characterization of lung lesions in the LSL-KRas G12D mouse model. At the end of the treatment mice were sacrificed, exsanguinated and lungs processed for histologic and immunological analyses. After deparaffinization, HE stains were performed and slides were captured and analyzed using NDP view2 software.
Tumor burden was calculated by determining the mean of total tumor area per lung using the NDP view2 software. To count the cells and determine the percentage of Ki67-positive cells within lesions, ten lesions per lung, from grade 2 to 5 according to the Sutherland scoring 50 , were randomly selected, their perimeter was determined, and positive and negative nuclei were counted using ImageJ software. Results are expressed as number of cells per mm 2 and the percentage of Ki67positive cells. Ex vivo macrophages and BMDC stimulation. Peritoneal lavage was done with RPMI 1640 medium on WT or p2rx7 -/-mice. 4. 10 5 macrophages were seeded in a 96-well plate overnight in RPMI 1640 containing 10% FBS, 2% sodium pyruvate, 1% penicillin/streptomycin, and 50-µM β-mercaptoethanol. After two washes with the complete medium, cells were primed for 4 h with 100 ng/ml LPS (Sigma-Aldrich) at 37 °C and then stimulated for 30 min at 37 °C with ATP (Sigma-Aldrich) with or without 50 µM of HEI3090 or with 10 µM nigericin.
The bands intensity values were normalized to that of β-actin using ImageJ software. Full scan blots are presented in the Source Data file. Cell lines. The LLC cell line (ATCC CRL-1642), the melanoma B16-F10 (ATCC CRL-6475) cell line, and HEK293T cells (ATCC CRL-3216) were used in this study. Cells were cultured in DMEM medium supplemented with 10% FBS and 100 U/mL penicillin and 100 mg/mL streptomycin at 37 °C in a humid atmosphere containing 5% CO 2 and routinely checked for mycoplasma contamination. Cells were used between passages 5 and 15. Calcium uptake assay. 20. 10 3 HEK293T-mP2RX7 C57BL/6J or HEK293T-pcDNA6 cells were seeded per well on a poly-L-Lysine (Sigma-Aldrich) 96 well-coated plate in complete medium. Twenty-four hours later, cells were washed in sucrose buffer (300 mM sucrose, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 10 mM Glucose, 20 mM HEPES, pH 7-7.
Data availability In silico data used in this study are available in the Tumor Comprehensive Genome Atlas (TCGA) project database, at https://software. broadinstitute. org/morpheus/. We used the TCGA Lung Adenocarcinoma (LUAD) cohort. The Gene Set Enrichment Analysis (GSEA) signatures sets are available at https://www. gsea-msigdb. org/gsea/msigdb/index. jsp and into the folder named « GSEA_SIGNATURE_SET » as source data files named:« GO_POSITIVE_REGULATION_OF_ADAPTIVE_IMMUNE_RESPONSE »« GO_POSITIVE_REGULATION_OF_CYTOKINE_PRODUCTION »« GO_POSITIVE_REGULATION_OF_T_CELL_MEDIATED_IMMUNITY »« GO_RESPONSE_TO_TUMOR_CELL »The GSEA analyses results are available into the folder named « GSEA_ANALYSES » as Excel Data Sheets named:« ANALYSIS_GO_POSITIVE_REGULATION_OF_ADAPTIVE_IMMUNE_RESPONSE »« ANALYSIS_GO_POSITIVE_REGULATION_OF_CYTOKINE_PRODUCTION »« ANALYSIS_GO_POSITIVE_REGULATION_OF_T_CELL_MEDIATED_IMMUNITY »« ANALYSIS_GO_RESPONSE_TO_TUMOR_CELL »Source data are available as a Source Data file.
Acknowledgements The authors wish to thank  Prof L. Counillon  for valuable discussions,  Anne Laure Rossi  for technical expertise and graphical art, and  Alexandre Gallerand  for BMDC preparation. The authors greatly acknowledge the  IRCAN's Animal core facility  and IRCAN's Flow Cytometry Facility that is supported by  FEDER, Ministère de l'Enseignement Supérieur, Région Provence Alpes-Côte d'Azur ,  Conseil Départemental 06 ,  ITMO Cancer Aviesan  (plan cancer),  Canceropole PACA, CNRS, and Inserm. The funding sources for this work were  Institut National du Cancer (INCa, plan Cancer) ,  Canceropole PACA, Bristol-Myers Squibb Foundation for Research in Immuno-Oncology , the  Ligue Nationale Contre le Cancer , the  French Government (National Research Agency, ANR)  through the  "Investments for the Future" programs LABEX SIGNALIFE   ANR-11-LABX-0028  and  IDEX  UCAJedi  ANR-15-IDEX-01  and the  Centre National de la Recherche Scientifique (CNRS) , the  Institut National de la Santé et Recherche Médicale (INSERM)  and the  University of Orleans , The  Region Centre Val de Loire  ( 2003-00085470 ), the  Conseil General du Loiret and European Regional Development Fund (FEDER  No.
1 in the presence of 10 peptides (P1 to P8, P1scr, and Ppen as controls) at 10 µM for 2 h. Then, the virus was removed, and cultures were washed, incubated for 48 h, before supernatant was collected to measure virus replication by ELISA. Data are combined from 3 to 6 independent experiments and expressed as compared to untreated SARS CoV-2infected Vero-E6 cells. b SARS-CoV-2 titer reduction in Vero-E6. Cells were infected with SARS-CoV-2/PSL2020 P#2 stock in triplicate at a multiplicity of infection (MOI) of 0. 1 in the presence of different concentrations (from 0. 01 to 10 µM) of peptides P8, P9, and P10 for 2 h. Then the virus was removed, and cultures were washed and incubated for 72 h to measure virus production by plaque assay. c Cell cytotoxicity in Vero-E6 cells. Cell viability was measured by MTT assays after treatment with vehicle 0, 0.
3 in the presence of six different concentrations (from 0. 01 to 10 µM) of peptides P1, P1 scr, P7, P8, P9, P10, for 2 h. Then the virus was removed, and cultures were washed in, incubated for 48 h, before supernatant was collected to measure virus replication by ELISA. Data are combined from 3 to 6 independent experiments and expressed as percent of inhibition compared to untreated SARS CoV-2-infected Vero-E6 cells. Data fitted in the sigmoidal dose-response curve represent the means (±SD) of at least three independent experiments and are expressed as percent of inhibition compared to untreated SARS CoV-2-infected Vero-E6 cells. f Cell cytotoxicity in Calu-3 cells. Cell viability was measured by MTT assays after treatment with vehicle 0, 0. 1, 1, or 10 µM of P8, P9, or P10 for 24, 48, or 72 h.
d. , 5 μm, 200 Å) at a flow rate of 14 mL min -1 and 20 mL min -1 , respectively. Small-scale crudes (<30 mg) were purified using semipreparative Ace column (Ace 5, C18, 250 mm × 10 mm i. d. , 5 μm, 300 Å) at a flow rate of 5 mL min -1. Purification gradients were chosen to get a ramp of ~1% solution B per minute in the interest area, and UV detection was done at 220 and 280 nm. Peptide fractions from purification were analyzed by LC-MS (method A or B depending on retention time) or by analytical HPLC on a Dionex system consisting of an automated LC system (Ultimate 3000) equipped with an autosampler, a pump block composed of two ternary gradient pumps, and a dual wavelength detector, piloted by Chromeleon software. All LC-MS or HPLC analyses were performed on C18 columns. The pure fractions were gathered according to their purity and then freeze-dried using an Alpha 2/4 freeze-dryer from Bioblock Scientific to get the expected peptide as a white powder.
6 mm i. d. , 2. 5 μm), eluting with 0. 1% formic acid in water (solvent A) and 0. 1% formic acid in acetonitrile (solvent B), using the following elution gradient: 0-3. 2 min, 5-100% B; 3. 2-4 min, 100% B. Flow rate was 1. 8 mL min -1 at 40 °C. The mass spectra were recorded on a Waters ZQ mass spectrometer using electrospray positive ionization [ES+ to give (MH)+ molecular ions] or electrospray negative ionization [ES-to give (MH)-molecular ions] modes. The cone voltage was 20 V. CD spectroscopy. CD spectra were recorded on a Jasco J-815 CD spectropolarimeter equipped with a Peltier temperature controller. Data were obtained at 25 °C over a wavelength range between 185 nm and 270 nm, using a wavelength interval of 0. 2 nm and a scan rate of 20 nm min -1. Peptide samples were prepared at a concentration of 60 µM in 50 mM sodium phosphate buffer, pH 7.
Data were reference subtracted and fitted to a 1:1 binding model using Octet Data Analysis Software v10. 0 (ForteBio). All figures are representative of at least two independent experiments. K D values were calculated from Supplementary Table  6. Inhibition of infection was calculated comparing viral concentration in each case with that of untreated SARS-CoV-2-infected cells. Toxicity study on Calu-3 and Vero-E6 cells. Cell cytotoxicity was measured using MTT assays and fluorescent activated cell sorting (FACS). Calu-3 and Vero-E6 were seeded in 96-well (for MTT assays) or 24-well (for FACS) plates and let adhere. When ~40% confluence was reached, the wells were washed, and new medium containing vehicle (water, 5% final volume) or different concentrations of the indicated peptide was added.
MTT (2 mM) was added to each well after 24 h, 48 h, or 72 h of treatment and incubated 4 h at 37 °C. Supernatants were then discarded and formazan salts were dissolved in DMSO to read plate absorbance at 570 nM. Absorbance in each well was normalized with those treated with vehicle. For FACS analyses, cells were harvested from the wells with the help of accutase, pelleted, and stained with Annexin-V-APC (0. 1 μg mL -1 ) and propidium iodide (0. 5 µg mL -1 ) in annexin binding buffer (10 mM Hepes pH 7. 4, 140 mM NaCl, and 2. 5 mM CaCl 2 ). Cells were sorted in a FACScalibur flow cytometer and data was analyzed using FlowJo 10. 0 software, considering cell death as the sum of Ann-V + /PI -and Ann-V + /PI + events. Biolayer Interferometry experiments. Fc-tagged 2019-nCoV RBD-SD1 (Sanyou Biopharmaceuticals Co.
0. 1. For BLI experiments, data were reference subtracted and fit to a 1:1 binding model using Octet Data Analysis Software v10. 0 (ForteBio). K D values were extracted from 3 independent determinations (P9 and P10) validated by a statistical coefficient determination (R 2 ) > 0. 95. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Acknowledgements This work was supported by private funds (P. K. ),  SATT-Lutech, Kaybiotix  (LGM PhD grant), and  French Research Ministry  (EO PhD grant). P. K. is grateful to  SATT-Lutech  team supporting this project, to  Fabrice Viviani  and  Akanksha Gangar from Oncodesign  for their unwavering support. The authors thank  David Boutolleau  from the  Virology Department, Pitié-Salpêtrière, AP-HP , where was diagnosed the BAL SARS-COV-2 infection. Philippe Karoyan dedicates this work to  Gérard Chassaing  on the occasion of his 75th birthday. "There are no borders in science. The only limit is our imagination. ".
Data availability The complex between hACE2 and the surface spike protein of SARS-CoV-2 data used for peptide design is available in RCSB PDB with the identifier doi: 10. 2210/pdb6M0J/pdb  14. The randomly selected RBD amino-acid sequences from China (QOH25833, QIG55857, QHR63290, QHR63250, QJG65957, QJG65956, and QJG65951), USA (QIV65044, QJD23847, QJU11481, QJD24531, QJD25193, QJD25529, and QJA17180), and France (QJT73034, QJT73010, QJT72902, QJT72806, QJT72794, QJT72722, QJT72710, QJT72626, QJT72614, and QJT72554), used for sequence alignment are available in the Genebank database at ncbi. nlm. nih. gov. The amino acid sequence of the Fc-tagged 2019-nCoV RBD-SD1 was provided by Sanyou bio with purchase (sanyoubio. com/EN/2019-nCoV. php) and is depicted in Supplementary Fig. 1a. The datasets generated and/or analyzed during the current study are in the manuscript and the raw data are available from the corresponding author on reasonable and justified request.
Code availability The predictive data supporting our work was generated using publicly available software. For sequence alignment, the Weblogo software (available at https://weblogo. berkeley. edu) and Clustal Omega (available at https://ebi. ac. uk/Tools/msa/clustalo/) were used. The Agadir helical content predictor is available at https://agadir. crg. es. Peptide antigenicity can be determined at https://imed. med. ucm. es/Tools/antigenic. pl. Online analysis for peptide CD spectra and using DichroWeb server and the CDSSTR algorithm is available at https://dichroweb. cryst. bbk. ac. uk. The HELIQUEST web server used to calculate peptide hydrophobicity and hydrophobic moment is available at https:// heliquest. ipmc. cnrs. fr.
The combined data from the cell-based cross-resistance analysis and enzymatic assays with purified wt SFV or CHIKV nsP1 suggested that the various compounds can be divided into distinct classes based on their modes of action. Therefore, we set out to perform molecular docking studies with the individual nsP1 inhibitors and the CHIKV nsP1 cryo-EM structure, in order to understand the structural basis of nsP1 inhibition and to obtain more insights into the potential mechanisms of action of these compounds at the molecular level. Predicted CHIKV nsP1 binding pockets and poses of endogenous ligands. Using the ICM Pocket Finder method  (30, 31)  and the available CHIKV nsP1 cryo-EM structure representing a dodecameric ring (Fig. 4A  and B ), we identified an elongated ligand binding site, referred to as pocket 1 or the main binding pocket, which is depicted in Fig.
Mutations in this region of nsP1 could arise due to a compensatory effect increasing SAM binding or SAM access to the SAM-binding site. Consequently, compensatory mutations would counteract the inhibition by CHVB compounds. The SAM analogue sinefungin was also docked at the predicted main binding pocket, but its pose is not considered here, since it was expected to bind in a manner similar to that of SAM. Based on the docking results presented in  FIG 6  Key amino acid residues linked to resistance to CHIKV nsP1 inhibitors, mapped to the CHIKV nsP1 cryo-EM structure (PDB code 6Z0V), represented as three consecutive nsP1 protomers: n, n11, and n-1. Residues are color-coded as follows: green, catalytic residues H37 and D63; blue, residues P34 and T246, involved in resistance to the MADTP series; orange, residues G230 and K299, conferring resistance to the FHNA series.
Residues F450 to S458 (purple) flank the area where residues S454 and W456, involved in resistance to the CHVB series, would be located. Orange and green dotted volumes represent the proposed binding pockets of GTP and SAM, respectively, for reference. Table  3 , it could be that sinefungin preferentially drifts to the GTP-binding site because it is more solvent exposed than the SAM-binding site in the current CHIKV nsP1 conformation. The docking results for the FHNA series support the hypothesis that they target a different region and function of nsP1, since they preferentially bind to pocket 2  FIG 7  Predicted binding mode of the CHVB and MADTP series in the CHIKV nsP1 SAM-binding site of the main binding pocket (PDB code 6Z0V). (A) CHVB-066 (purple) and CHVB-032 (pink) in complex with CHIKV nsP1, occupying the SAM-binding site (green dots).
Lastly, sinefungin does not seem to bind in a manner similar to those of the inhibitors of the MADTP, CHVB, and FHNA series. Even though sinefungin structurally resembles SAM, it preferentially docked at the GTP-binding site (Table  3 ). However, the docking results for sinefungin need to be interpreted with caution because the SAM's binding pose in itself is not very reliable in the current CHIKV nsP1 conformation. As mentioned below, in Materials and Methods, SAM and sinefungin were docked differently (i. e. , using different docking grids or reference residues). In addition, more experimental data would need to be obtained to explain why sinefungin stimulates GTase activity and does not serve as a competitive SAM inhibitor. In summary, we identified several classes of CHIKV nsP1 inhibitors with unique modes of action by our cell-based and biochemical assays as well as molecular-docking studies.
describing the process of CHIKV nsP1 oligomerization into structurally novel capping rings challenge the biochemical data from these assays  (28). Confirmatory experiments with oligomeric wt and mutant CHIKV nsP1 would need to be performed to validate the results obtained with other forms of alphavirus nsP1. It still remains very puzzling why compounds docking at the active site of CHIKV nsP1, including the MADTP and CHVB series and sinefungin, are not active against the related alphavirus SFV (Table  1 ). Previously, it was shown that FHA and FHNA, which were active against both CHIKV and SFV, were inactive against SINV  (22) , and the MADTP and CHVB series were also found to be inactive against other alphaviruses  (21, 24). The current opinion in the field holds that the ring-shaped, membrane-associated nsP1 complex is conserved among alphaviruses.
Besides its enzymatic functions, important for RNA capping, the nsP1 complex plays a key role in anchoring the replication complex (nsP1 to -4) to membranes (4). The nsP1 capping ring appears to interact with nsP4 on both the inner and outer sides of the pore  (28). Taking our findings together, this study predicts the mode of action of several CHIKV nsP1 inhibitors. We emphasize that our conclusions are based on a combined interpretation of docking poses and mutagenesis data. Nevertheless, other docking possibilities may exist, especially if different conformations of CHIKV nsP1 (i. e. , other atomic structures in complex with either endogenous or targeted ligands, or monomeric nsP1) are considered. Our combined data suggest that compounds belonging to the MADTP and CHVB series likely interfere with CHIKV nsP1 functions directly via competitive or noncompetitive inhibition of SAM binding.
MATERIALS AND METHODS Cells and virus strains. Vero E6 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 8% fetal calf serum (FCS) and penicillin-streptomycin. Infection assays were performed in Eagle's minimum essential medium (EMEM) supplemented with 2% FCS, 2 mM L-glutamine, and penicillin-streptomycin. CHIKV LS3 (GenBank accession no. KC149888) is an infectious clone-derived virus  (35). CHIKV nsP1-P34S is a reverse-engineered LS3-derived mutant that is resistant to MADTP compounds  (21). CHIKV nsP1-G230R1K299E is a reverse-engineered LS3-derived mutant resistant to FHA and FHNA  (22). CHIKV nsP1-S454G1W456R is a reverse-engineered LS3-derived mutant resistant to CHVB-032 and CHVB-066  (24). Semliki Forest virus (SFV) strain SFV4 was used in cytopathic effect (CPE) reduction assays to assess the antiviral spectrum of nsP1 inhibitors.
After overnight exposure, the 32 P-labeled covalent m 7 GMP-nsP1 intermediate products were visualized with a Typhoon imager (Amersham). System preparation and CHIKV nsP1 molecular docking. Docking was performed using ICM Pro software, v3. 9-1b (Molsoft LLC, San Diego, CA)  (37, 38). The apo CHIKV nsP1 cryo-EM structure representing a dodecameric ring (PDB code 6Z0V) was prepared by adding and optimizing the positions of hydrogen atoms, as well as the orientation and protonation states of histidine and cysteine residues and the orientation of glutamine and asparagine residues. "Chain A" in the cryo-EM structure was used as the main nsP1 monomeric structure (referred to as n), and chains n11 and n-1, flanking chain n, were considered in order to acknowledge the complexed nature of active nsP1.
Statistical analysis. Statistical analysis was performed, and figures were generated, using Graph-Pad Prism, v8. Oncode Institute, the Netherlands. R. J. and J. R. were supported by the ATIP-Avenir program and the Bettencourt Shueller Foundation. M. -J. P. -P. acknowledges the financial support of AEI (grant PID2019-105117RR-C22).
1994 ) containing 10% foetal calf serum, and the cells were transferred from the plate to FACS-tubes. Cells were centrifuged at 216 g at 4 °C for 5 min and the supernatant was removed. After this procedure, cells were resuspended in 500 µL HPMI, and the intracellular fluorescence intensity of HL-60 cells was monitored (on channel FITC LP505; emission at λ = 505 nm; LP 505, BP 530/30) by flow cytometry (BD LSR II, BD Bioscience, San Jose, CA, equipped with 488 nm; Coherent Sapphire, 22 mW laser. ) which is proportional to the cellular uptake. Data were analysed with FACSDiVa 5. 0 software. HeLa cells (8. 0 × 10 4 /well) in α-MEM( +) were cultured in 24-well microplates overnight at 37 °C. Cells were treated with fluorescently-labelled peptides (2. 5, 5 and 10 μM) in α-MEM( +) and α-MEM without 10% BS [α-MEM(-)], respectively, for 30 min at 37 °C.
The precipitated purple crystals were dissolved in 100 μL DMSO and the absorbance was determined at λ = 540 nm and λ = 620 nm using ELISA plate reader (iEM-SReader, Labsystems, Finland). The percent of cytostasis was calculated using the following equation: Cytostatic effect (%) = [1 -(OD treated /OD control )] × 100; where OD treated and OD control correspond to the optical densities of the treated and the untreated control cells, respectively. In each case three independent experiments were carried out with four parallel measurements. The 50% inhibitory concentration (IC 50 ) values were determined from the dose-response curves. The curves were defined using MicrocalTM Origin (version 9. 2) software: cytostasis was plotted as a function of concentration, fitted to a sigmoidal curve, and based on this curve, the IC 50 value was determined.