Coronavirus infection and PARP expression dysregulate the NAD metabolome: An actionable component of innate immunity

  1. Collin D. Heer
  2. Daniel J. Sanderson
  3. Lynden S. Voth
  4. Yousef M.O. Alhammad
  5. Mark S. Schmidt
  6. Samuel A.J. Trammell
  7. Stanley Perlman
  8. Michael S. Cohen
  9. Anthony R. Fehr
  10. Charles Brenner

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  1. ScreenIT

    SciScore for 10.1101/2020.04.17.047480: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Mice: Animal studies were approved by the University of Kansas Institutional Animal Care and Use Committee (IACUC) as directed by the Guide for the Care and Use of Laboratory Animals (Protocol #252-01).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableBriefly, samples were obtained from nasopharyngeal swabs of from 430 male and female individuals with SARS-CoV-2 infection and 54 controls (32).
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Blots were blocked with 5% Milk-PBST for 30 min, incubated O/N in primary antibody (Rabbit Pan-ADPr 1:1000, Cell Signaling E6F6A; Rabbit GFP 1:1000, Chromotek PABG1-100; Mouse Tubulin 1:1000; Cell Signaling DM1A).
    GFP
    suggested: (ChromoTek Cat# PABG1-20, RRID:AB_2749857)
    Tubulin
    suggested: (LSBio (LifeSpan Cat# LS-C89856-1000, RRID:AB_1940111)
    Primary incubation was followed with HRP-conjugated secondary antibodies (Rabbit-HRP 1:10000, Jackson Laboratories 111-035-144; Mouse-HRP 1:5000, Invitrogen 62-6520).
    Rabbit-HRP
    suggested: None
    Mouse-HRP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    NHBE (Fig. 1B) and A549 (low MOI) (Fig.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    Cell culture: DBT, 17Cl-1, HEK293T, and HeLa cells expressing the MHV receptor carcinoembryonic antigen-related cell adhesion molecule 1 (a gift from Dr. Thomas Gallagher, Loyola University, Chicago, IL) were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), HEPES, sodium pyruvate, non-essential amino acids, L-glutamine, penicillin and streptomycin.
    HeLa
    suggested: None
    For analysis of the NAD metabolome HEK293T cells were transfected with 1µg of pEGFP-C1 Empty Vector or pEGFP-C1-CMV-PARP10 using CalPhos Mammalian Transfection Kit (Takara Bio).
    HEK293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Pathogen-free C57BL/6 mice were purchased from Jackson Laboratories and maintained in the animal care facility at the University of Kansas.
    C57BL/6
    suggested: None
    Software and Algorithms
    SentencesResources
    RNA was collected, and differential gene expression analysis was performed using the DESeq2 package.
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Graphs were generated using GraphPad Prism v8.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    However, there are a few caveats. First, that at pharmacological doses, NAM has the potential to function as a PARP inhibitor (41). Second, NAMPT is considered a driver of pulmonary vascular remodeling and potentially a target to be inhibited to maintain lung health of some people at risk for COVID-19 (42). Thus, in order to maximize the likelihood of success in human CoV prevention and treatment trials, care should be taken to carefully compare efficacy and dose-dependence of NR, SBI and NAM with respect to control of cytokine storm and antiviral activities in vivo. The cellular results presented herein warrant the testing of NAD boosting agents in the context of in vivo CoV infections. In addition to animal trials, the safety of various forms of vitamin B3 should allow rapid clinical assessments of NAD boosters to be evaluated in two placebo-controlled contexts. First, we suggest that improved NAD status could help blunt the severity of infection by sustaining PARP-dependent IFN signaling in the face of the self-limiting nature of cellular NAD during infection and by limiting the storm of inflammatory cytokines that is typically associated with serious disease (43). In a small placebo-controlled clinical trial designed to address the oral safety and activity of Niagen NR in older men, it was discovered that 1 gram of NR per day depresses levels of IL-6, IL-5, and IL-2 (22). Based on these findings, we suggest that NAD boosters be tested on hospitalized and nonhospitalized C...

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

    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.

  2. Version published to 10.1074/jbc.ra120.015138
  3. Version published to 10.1101/2020.04.17.047480v6 on bioRxiv
  4. Version published to 10.1101/2020.04.17.047480v5 on bioRxiv
  5. ScreenIT

    SciScore for 10.1101/2020.04.17.047480: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Blots were blocked with 5% Milk-PBST for 30 min, incubated O/N in primary antibody (Rabbit Pan-ADPr 1:1000, Cell Signaling E6F6A; Rabbit GFP 1:1000, Chromotek PABG1-100; Mouse Tubulin 1:1000; Cell Signaling DM1A).
    GFP
    suggested: (ChromoTek Cat# PABG1-20, AB_2749857)
          <div style="margin-bottom:8px">
        <div><b>Tubulin</b></div>
        <div>suggested: (LifeSpan Cat# LS-C89856-1000, <a href="https://scicrunch.org/resources/Any/search?q=AB_1940111">AB_1940111</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary incubation was followed with HRP-conjugated secondary antibodies (Rabbit-HRP 1:10000, Jackson Laboratories 111-035-144; Mouse-HRP 1:5000, Invitrogen 62-6520).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Rabbit-HRP</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Mouse-HRP</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK 293T cells were grown with the indicated expression plasmids for GFP or PARP10 and treated with small activate NAMPT (10 M SBI-797812) or inhibit PARP1 and PARP2 (300 nM Veliparib).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK 293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PARP10 and GFP-expressing HEK293 cells were used to show that veliparib and SBI-797812 differ in their ability to promote the autoMARylated form of PARP10.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Genes with “Status = Low” or “Status = Outlier” were not considered for analysis, leaving 56 genes from the A549 dataset and 52 genes from the NHBE dataset for analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>A549</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">DBT, 17Cl-1, HEK293T, and HeLa cells expressing the MHV receptor carcinoembryonic antigen-related cell adhesion molecule 1 (a gift from Dr. Thomas Gallagher, Loyola University, Chicago, IL) were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), HEPES, sodium pyruvate, non-essential amino acids, L-glutamine, penicillin and streptomycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HeLa</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed using DESeq2 to calculate fold change and p-values (Supplementary Material 4 and 5).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>DESeq2</b></div>
        <div>suggested: (DESeq, <a href="https://scicrunch.org/resources/Any/search?q=SCR_000154">SCR_000154</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Ten genes did not have ferret orthologs in Ensembl and one gene exhibited undetectable expression (TDO2) leaving 60 ferret genes for analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Ensembl</b></div>
        <div>suggested: (Ensembl, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002344">SCR_002344</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Graphs were generated using GraphPad Prism v8.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GraphPad Prism</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    </td></tr></table>

    Results from Barzooka: We also found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  6. Version published to 10.1101/2020.04.17.047480v4 on bioRxiv
  7. Version published to 10.1101/2020.04.17.047480v3 on bioRxiv
  8. Version published to 10.1101/2020.04.17.047480v1 on bioRxiv
  9. Version published to 10.1101/2020.04.17.047480v2 on bioRxiv