Secreted ORF8 induces monocytic pro-inflammatory cytokines through NLRP3 pathways in patients with severe COVID-19

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1. Version published to 10.1016/j.isci.2023.106929

   Jun 1, 2023
2. 

   ScreenIT

   Dec 8, 2021

   SciScore for 10.1101/2021.12.02.470978: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Samples for these studies were requested and approved by the Task Force Review Committee and the Mayo Clinic Institutional Review
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The amount of sample equivalent to 1ml of culture supernatant was loaded into a 4-20% gradient gel followed by western blotting per routine probing with anti-Strep II antibody at 1:1000 dilution (#71590, Millipore) and developed on a Li-Cor scanner.	anti-Strep II suggested: (Millipore Cat# 71591-3, RRID:AB_11214448)
   Detection of ORF8 protein in sera of patients newly …

   SciScore for 10.1101/2021.12.02.470978: (What is this?)

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

   Table 1: Rigor

   Ethics	IRB: Samples for these studies were requested and approved by the Task Force Review Committee and the Mayo Clinic Institutional Review
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The amount of sample equivalent to 1ml of culture supernatant was loaded into a 4-20% gradient gel followed by western blotting per routine probing with anti-Strep II antibody at 1:1000 dilution (#71590, Millipore) and developed on a Li-Cor scanner.	anti-Strep II suggested: (Millipore Cat# 71591-3, RRID:AB_11214448)
   Detection of ORF8 protein in sera of patients newly infected with SARS-CoV-2: For detection ORF8 protein in serum samples from patients newly infected with SARS-CoV-2, typically four microliters of serum directly mixed with 37 microliters of 4x Laemmli buffer with 5% of BME, boiled for 3 min prior to load to 10-20% SDS-PAGE gels, the blots were probed with 1:5000 diluted rabbit anti-ORF8 antibody (MyBioSource, Cat# MBS3014575) at 4°C overnight.	anti-ORF8 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Construct DNAs were used to make stable expression cell lines in HEK293 cells by lentiviral transduction, transient expression in HeLa cells using lipofectamine method or transient expression in HEK293F cells with HyCell TransFx media (Cytiva) method, respectively.	HeLa suggested: None
   Detection of SARS-CoV-2 protein secretion: To detect protein secretion, HEK293 cells expressing the protein of interest were seeded at 70% confluency in a T25 flask in 4ml of DMEM/10% FCS.	HEK293 suggested: None
   supernatant: Transfected HEK293F cells were grown for 3-4 days at a cell density below 6×106 cells per ml.	HEK293F suggested: None
   Recombinant DNA
   Sentences	Resources
   Expression and purification of ORF8 from E. Coli: DNA encoding SARS CoV-2 ORF8 codon-optimized for expression in E. coli was cloned into pMCSG7 30 and transformed into Rosetta2 cells (EMD), and protein expression was induced with 100 µM IPTG overnight at 15°C. E. coli culture was pelleted, lysed, and protein purified as described previously 31, except that cells were lysed by sonication with a Branson sonicator in place of Dounce homogenization.	pMCSG7 suggested: RRID:Addgene_83505)
   Software and Algorithms
   Sentences	Resources
   Single cell RNA sequencing Data Analysis: We used the Seurat package (v4.0.1) to perform integrated analyses of single cells.	Seurat suggested: (SEURAT, RRID:SCR_007322)
   KEGG pathway enrichment analysis was performed on the genes that were differentially expressed significantly using the ClusterProfiler package.	KEGG suggested: (KEGG, RRID:SCR_012773) ClusterProfiler suggested: (clusterProfiler, RRID:SCR_016884)
   Analysis of ORF8 expression and survival in patients with Covid-19: Overall survival was calculated from the time of Covid-19 diagnosis, using the Kaplan-Meir method on JMP 14.0 software (SAS Institute, Cary, NC).	SAS Institute suggested: (Statistical Analysis System, RRID:SCR_008567)

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

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   Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04540120	Recruiting	Safety and Efficacy of Dapansutrile for Treatment of Moderat…

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

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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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3. 

   Version published to 10.1101/2021.12.02.470978 on bioRxiv

   Dec 3, 2021