SARS-CoV-2 infects human adipose tissue and elicits an inflammatory response consistent with severe COVID-19

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1. 

   ScreenIT

   Nov 19, 2021

   SciScore for 10.1101/2021.10.24.465626: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: The protocol was approved by the Stanford Institutional Review Board and all subjects gave written, informed consent. Consent: The protocol was approved by the Stanford Institutional Review Board and all subjects gave written, informed consent. Field Sample Permit: Sample preparation for Luminex: Supernatants from mock or infected SVC were collected after 24 hpi into low-binding protein collection tubes.
   Sex as a biological variable	not detected.
   Randomization	This function generates an average module score by calculating the mean expression of each gene in the module corrected for expression of a random set of similarly expressing genes.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell …

   SciScore for 10.1101/2021.10.24.465626: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: The protocol was approved by the Stanford Institutional Review Board and all subjects gave written, informed consent. Consent: The protocol was approved by the Stanford Institutional Review Board and all subjects gave written, informed consent. Field Sample Permit: Sample preparation for Luminex: Supernatants from mock or infected SVC were collected after 24 hpi into low-binding protein collection tubes.
   Sex as a biological variable	not detected.
   Randomization	This function generates an average module score by calculating the mean expression of each gene in the module corrected for expression of a random set of similarly expressing genes.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: VeroE6 cells were obtained from ATCC and were mycoplasma free.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Virus and cell lines: The USA WA1/2020 strain of SARS-CoV-2 was obtained from BEI Resources, passaged in VeroE6 cells, and tittered by Avicel (FMC Biopolymer) overlay plaque assay on VeroE6 cells.	VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Validation of Remdesivir treatments in A549-ACE2 cells.	A549-ACE2 suggested: None
   Recombinant DNA
   Sentences	Resources
   A standard curve for Ct values and genome copy numbers was obtained using pET21b+ plasmid with the N-gene inserts.	pET21b+ suggested: RRID:Addgene_92208)
   Software and Algorithms
   Sentences	Resources
   Alignment and preprocessing of scRNA-seq data: The quality of the raw FASTQ data was examined with FASTQC and then aligned (cell ranger count) to a custom genome including human genome (hg38) and the complete genome sequence of SARS-CoV-2 (2019-nCOV/USA-WA1/2020) (GenBank: MN985325.1) using the “Cell Ranger” software package v6.0.0 (10x Genomics).	FASTQC suggested: (FastQC, RRID:SCR_014583) Ranger” suggested: None
   Briefly, count matrices were merged and loaded into Seurat with SARS-CoV-2 counts removed and appended to the metadata.	SARS-CoV-2 suggested: (BioLegend Cat# 946101, RRID:AB_2892515)
   Heatmaps were generated using ComplexHeatmap, Seurat and pheatmap packages, Violin plots were generated using ggplot2, and dotplots were generated using the Seurat packages in R.	ComplexHeatmap suggested: (ComplexHeatmap, RRID:SCR_017270) pheatmap suggested: (pheatmap, RRID:SCR_016418) ggplot2 suggested: (ggplot2, RRID:SCR_014601)
   Statistical analysis: GraphPad Prism version 9.1.0 (216) and R (4.0.4) were used for statistical analysis.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   GO and KEGG term enrichment for markers of C12 macrophages.	KEGG suggested: (KEGG, RRID:SCR_012773)
   Data from scRNA-seq will be deposited with the Gene Expression Omnibus.	Gene Expression Omnibus suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

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   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).

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Our study has several limitations. Our numbers of replicates were limited for some assays, such as the evaluation of cytokine secretion following infection of mature adipocytes. Nonetheless, we observed significant indications of inflammatory responses. It is possible that we were unable to fully wash input virus following infection of mature adipocytes due to the high lipid content, size, and fragility of the cells, falsely increasing the viral signal. However, our detection of sgRNA, the time-dependent increase in viral RNA accumulation that was inhibited by remdesivir, and the detection of SARS-CoV-2 RNA in autopsy samples all provide orthogonal support for true infection of adipocytes. Our autopsy studies were limited in number, and we were only able to perform confirmatory ISH on epicardium, not in the subcutaneous, omental, or pericardial fat due to limited autopsy tissue availability. All experiments were performed with the WA-01 strain of SARS-CoV-2 and no experiments with its variants were performed, and plaque assays to confirm viral production were not performed. As tissue donors were obese, an important area of future investigation will be to study the effects of SARS-CoV-2 infection on lean adipose tissue, as well as to study the adipose tissue of those with ‘long COVID’. Overall, here we provide evidence that two cell types within human adipose tissue are permissive to SARS-CoV-2 infection. This adds to data showing susceptibility of other tissues including hear...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   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).

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

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   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.

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

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2. 

   Version published to 10.1101/2021.10.24.465626 on bioRxiv

   Oct 25, 2021