1. SciScore for 10.1101/2021.11.16.21266115: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIRB: Mary’s Hospital Research Ethics Committee (09/H0712/59).
    Consent: All participants gave written, informed consent.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Mouse IgG2a monoclonal anti-human ICAM-1 antibody (antibody R6.5) was produced from the hybridoma cells (ATCC HB-9580, mouse hybridoma).
    Mouse IgG2a monoclonal anti-human ICAM-1 antibody
    suggested: None
    anti-human ICAM-1
    suggested: None
    To block ICAM-1, a receptor responsible for RV-A16 infection of HBECs, anti-ICAM-1 antibodies were added to the apical and basolateral compartment, 3h prior RV-A16 infection at the dose of 10ug/mL (Fig. S1A).
    suggested: None
    Protein samples precipitated from the apical supernatants were analyzed with the use of goat anti-IL-1β antibodies (1:1000, R&D Systems, Minneapolis, USA), rabbit anti-caspase-1 (1:1000, Cell Signaling Technology, Danvers, USA), and HRP conjugated anti-goat (1:10,000, Santa Cruz Biotechnology, Santa Cruz, USA), and anti-rabbit (1:10,000, Jackson ImmunoResearch, West Grove, USA) antibodies.
    suggested: None
    suggested: None
    , magnetized, and incubated with 10 μg of anti-ASC antibodies (Santa Cruz Biotechnology, Santa Cruz, USA) overnight at 4°C, followed by ASC immunoprecipitation with 100 μl of Protein A beads (Bio-Rad, Hercules, USA) for 2h in RT.
    suggested: None
    Co-IP samples and input (protein not bound to the beads) were collected and together with the cell lysates were further analyzed with the Western Blot protocol with the use of mouse anti-human RIG-I antibodies (1:200, Santa Cruz Biotechnology, Santa Cruz, USA) or rabbit anti-human MDA5 antibodies (1:1000, Abcam, Cambridge, UK) and HRP conjugated anti-mouse or anti-rabbit antibodies (1:10,000, Jackson ImmunoResearch, West Grove, USA).
    anti-human RIG-I
    suggested: (LSBio (LifeSpan Cat# LS-C19644-1000, RRID:AB_839891)
    anti-human MDA5
    suggested: None
    Subsequently, samples were incubated with the goat anti-mouse Alexa Fluor 488 (for ASC), and the goat anti-mouse Alexa Fluor 546 (for IL-1β and RIG-I) secondary antibodies at the concentrations of 1:2000 (Invitrogen, Waltham, USA) for 60 minutes at RT.
    suggested: None
    suggested: None
    Primary antibodies for NLRP3 (5 µg/mL, mouse anti-NLRP3, Adipogen, San Diego, USA)
    suggested: None
    Primary antibodies for IL-1β (10 μg/mL, mouse anti-IL1β, Abcam, Cambridge, UK),
    suggested: None
    Subsequently, samples were incubated with the goat anti-rabbit Alexa Fluor 488 (for caspase-1), and the goat anti-mouse Alexa Fluor 546 (for IL-1β and RIG-I) secondary antibodies for 60 minutes in RT in the concentration of 1:1000 (Invitrogen, Waltham, USA).
    suggested: None
    Experimental Models: Cell Lines
    Briefly, HeLa cells were infected with the virus serial dilutions from 10-2 to 10-8 in duplicates.
    suggested: None
    Co-culture of electroporated BHK-21 cells with susceptible Vero E6 cells produced passage 0 of SARS-CoV-2 virus.
    suggested: ATCC Cat# CRL-6281, RRID:CVCL_1914)
    Passage 0 was used to infect Vero E6 cells to generate passage 1 working stocks, which were used for all experiments.
    Vero E6
    suggested: RRID:CVCL_XD71)
    THP-1 cell culture: THP-1-XBlue cells (Invivogen, San Diego, USA) were defrosted in 32 mL of RPMI-1640 medium (Sigma-Aldrich, St. Louis, USA) supplemented with the Penicillin/Streptomycin/Kanamycin, MEM vitamins, Na-Pyruvate/MEM Non-essential Amino Acid Solution and heat-inactivated FCS (cRPMI medium) in the 75cm2 T-flask, and cultured for 1 day in the humidified incubator at 37°C with 5% CO2.
    suggested: None
    suggested: RRID:CVCL_X582)
    Gene expression (5ng of cDNA/well) was assessed by RT-PCR using i) SYBR Green PCR Master Mix (ThermoFisher Scientific, Waltham, USA) for DDX58, IFNB, IFNL1, IL1B, MDA5, and ii) TaqMan assays for RV-A16 and SARS- CoV-2 Protein N, Protein S, ORF1AB detection (ThermoFisher Scientific, Waltham, USA) and was performed on the QuantStudio 7 Flex Real-Time PCR System (ThermoFisher Scientific, Waltham, USA).
    suggested: None
    Software and Algorithms
    Quantification of the protein expression was performed in Fiji Software. 128 Briefly, an area of the peak of the protein of interest was measured in triplicates, and average was used to calculate the ratio between expression of the protein of interest and β-actin (protein/β-actin).
    suggested: (Fiji, RRID:SCR_002285)
    The mRNA expression data are publicly available at the Gene Expression Omnibus platform (https://www.ncbi.nlm.nih.gov/) under the accession number: GSE6114166.
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    suggested: (GENSAT at NCBI - Gene Expression Nervous System Atlas, RRID:SCR_003923)
    05 calculated for the entire gene lists in each project using the edgeR R package130.
    suggested: (edgeR, RRID:SCR_012802)
    Differentially expressed probe was identified by the limma R package with empirical Bayes estimation.
    suggested: (LIMMA, RRID:SCR_010943)
    Additionally, top 100 genes upregulated after RV-A16 infection in the HBECs from control individuals and patients with asthma from GSE6114166 were analyzed for the enriched pathways using Metacore software version 20.3.70200
    suggested: (MetaCore, RRID:SCR_008125)
    The statistical comparison of protein expression between groups was performed with the Bioconductor limma package131.
    suggested: (Bioconductor, RRID:SCR_006442)
    Additionally, for Target 96 Inflammation panel data are presented as: i) heatmaps of curated signatures of inflammasome-mediated immune responses and antiviral responses (Supplementary Table S10) and ii) protein interactions and pathways analysis prepared using the STRING (version 11.0)132, and further processed with the Cytoscape software (version 3.8.2)133 (Supplementary Table S3).
    suggested: (STRING, RRID:SCR_005223)
    suggested: (Cytoscape, RRID:SCR_003032)

    Results from OddPub: Thank you for sharing your code and data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    An appropriate balance between activation of RIG-I epithelial inflammasome and subsequent IL-1β/IL-1 receptor (IL1R) signaling with RIG-I-dependent type I/III IFN responses should lead to the limitation of viral replication, efficient virus clearance and timely resolution of airway inflammation52. Indeed, we observed here that in the bronchial epithelium of healthy subjects at early time points during RV infection there was an activation of RIG-I inflammasome and inflammasome-mediated immune responses, together with efficient type I/III IFN and ISG-responses. Importantly all of these responses were actively inhibited or went back to the pre-infection state, already 4 days after in vivo infection. In contrast, in epithelium of patients with asthma, there was enhanced RIG-I inflammasome activation accompanied by augmented inflammasome/IL1R- mediated proinflammatory responses starting early after infection and still non-resolved in vivo 4 days after infection. Overactivation of epithelial RIG-I inflammasome and subsequent increases in mature IL-1β release might be at least partially responsible for the delayed and sustained type I/III IFN/ISG responses. We demonstrated this here by blocking caspase-1 with YVAD which led to an increase in IFN-β (IFNB) and RIG-I (DDX58) mRNA together with IFN-responsive chemokines such as CXCL10, CXCL11, CCL3, and CCL4. Our findings are in line with early observations showing that IL-1β is able to attenuate transcription and translation of type I ...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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