The impact of hypoxia on B cells in COVID-19

  1. Prasanti Kotagiri
  2. Federica Mescia
  3. Aimee L. Hanson
  4. Lorinda Turner
  5. Laura Bergamaschi
  6. Ana Peñalver
  7. Nathan Richoz
  8. Stephen D. Moore
  9. Brian M. Ortmann
  10. Benjamin J. Dunmore
  11. Michael D. Morgan
  12. Zewen Kelvin Tuong
  13. Berthold Göttgens
  14. Mark Toshner
  15. Christoph Hess
  16. Patrick. H. Maxwell
  17. Menna. R. Clatworthy
  18. James A. Nathan
  19. John R. Bradley
  20. Paul A. Lyons
  21. Natalie Burrows
  22. Kenneth G.C. Smith

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Abstract

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  1. Version published to 10.1016/j.ebiom.2022.103878
  2. ScreenIT

    SciScore for 10.1101/2021.07.12.21260360: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethical approval was obtained from the East of England – Cambridge Central Research Ethics Committee (
    Consent: All participants provided informed consent.
    IACUC: All procedures were ethically approved by the University of Cambridge Animal Welfare and Ethical Review Body and complied with the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012, under the authority of a UK Home Office Licence.
    Sex as a biological variablenot detected.
    RandomizationRandomization was genetic and, where possible, investigators were blinded to the genetic status.
    BlindingRandomization was genetic and, where possible, investigators were blinded to the genetic status.
    Power Analysisnot detected.

    Table 2: Resources

    Experimental Models: Organisms/Strains
    SentencesResources
    Animal Models: Mice: Vhl−/− mice(Haase et al., 2001) were crossed with Cd79a-cre (Mb1-cre)(Hobeika et al., 2006) or Cd19-cre (JAX, stock no. 004126) to delete Vhl in the B cell lineage.
    Vhl−/−
    suggested: None
    Cd19-cre
    suggested: None
    The deletion efficiency for mature B cells was 52% (95% CI 30-75%) in Vhl-/-Cd19-cre mice and 98% (95% CI, 97–99%) in Vhl-/-Mb1-cre mice(Burrows et al., 2020).
    Vhl-/-Cd19-cre
    suggested: None
    Vhl-/-Mb1-cre
    suggested: None
    These mice, along with C57BL/6J mice (JAX, stock no. 000664) were housed in specific pathogen-free animal facilities (at 20–23 °C, with 40–60% humidity, 12-h light:12-h dark cycle).
    C57BL/6J
    suggested: None
    Software and Algorithms
    SentencesResources
    Samples were gated in FlowJo v10.2 and number of cells falling within each gate was recorded.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Read quality was assessed using FastQC v.
    FastQC
    suggested: (FastQC, RRID:SCR_014583)
    Babraham Bioinformatics, UK) and BBSplit (BBMap v.38.67(BBMap -
    BBMap
    suggested: (BBmap, RRID:SCR_016965)
    Alignment was performed using HISAT2 v.
    HISAT2
    suggested: (HISAT2, RRID:SCR_015530)
    Count matrices were generated using featureCounts (Rsubreads package)(Liao, Smyth and Shi, 2019) and stored as a DGEList object (EdgeR package)(Robinson, McCarthy and Smyth, 2009) for further analysis.
    featureCounts
    suggested: (featureCounts, RRID:SCR_012919)
    Counts were filtered using filterByExpr (EdgeR) with a gene count threshold of 10 CPM and the minimum number of samples set at the size of the smallest disease group.
    EdgeR
    suggested: (edgeR, RRID:SCR_012802)
    Gene set enrichment analysis (GSEA)(Subramanian et al., 2005) was used to identify biological pathways enriched in COVID-19 severity groups relative to healthy controls.
    Gene set enrichment analysis
    suggested: (Gene Set Enrichment Analysis, RRID:SCR_003199)
    Sequence analysis: Raw reads were filtered for base quality using a median Phred score of ≥32 (http://sourceforge.net/projects/quasr/).
    http://sourceforge.net/projects/quasr/
    suggested: (QUASR, RRID:SCR_006820)
    Sequences without complete reading frames and non-immunoglobulin sequences were removed and only reads with significant similarity to reference IGHV and J genes from the IMGT database using BLAST were retained.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    Murine BCR amplification and sequencing: BCR amplification and sequencing was performed as described in Burrows et al.(Burrows et al., 2020) Data are available at the Sequence Research Archive (SRA) database (BioProject accession nos.
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)
    Raw reads were filtered as Burrows et al.(Burrows et al., 2020) Ig gene sequence annotations were performed in IMGT V-QUEST, where somatic hypermutation repertoire and isotype usage differences were performed by custom scripts in python, and statistics were performed in R using Wilcoxon tests for significance (non-parametric test of differences between distributions).
    python
    suggested: (IPython, RRID:SCR_001658)
    Raw imaging data were processed using Imaris.
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    The image analysis was performed in ImageJ.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    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

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.07.12.21260360v1 on medRxiv