SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

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1. 

   ScreenIT

   Dec 16, 2021

   SciScore for 10.1101/2021.12.10.472112: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: All experiments and procedures were pre-approved by the UC Berkeley Animal Care and Use Committee, Protocol AUP-2014-08-6638-2.
   Sex as a biological variable	EXPERIMENTAL MODELS AND SUBJECT DETAILS: Mice: Six- to eight-week-old wild-type C57BL/6J female mice were purchased from the Jackson Laboratory and housed at the University of California, Berkeley Animal Facility under specific pathogen-free conditions.
   Randomization	For immunofluorescence microscopy experiments, images of random fields were captured.
   Blinding	Researchers were not blinded during experiments.
   Power Analysis	not detected.
   Cell Line Authentication	Authentication: Identified DEGs were analyzed by STRING to identify predicted …

   SciScore for 10.1101/2021.12.10.472112: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: All experiments and procedures were pre-approved by the UC Berkeley Animal Care and Use Committee, Protocol AUP-2014-08-6638-2.
   Sex as a biological variable	EXPERIMENTAL MODELS AND SUBJECT DETAILS: Mice: Six- to eight-week-old wild-type C57BL/6J female mice were purchased from the Jackson Laboratory and housed at the University of California, Berkeley Animal Facility under specific pathogen-free conditions.
   Randomization	For immunofluorescence microscopy experiments, images of random fields were captured.
   Blinding	Researchers were not blinded during experiments.
   Power Analysis	not detected.
   Cell Line Authentication	Authentication: Identified DEGs were analyzed by STRING to identify predicted protein-protein interaction networks as well as enriched pathway analysis (https://string-db.org/).

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Afterwards, plates were incubated with biotinylated chicken anti-human TGF-β1 detection antibody and then with streptavidin-horseradish peroxidase (HRP) for signal detection with tetramethylbenzidine (TMB) substrate.	anti-human TGF-β1 suggested: None
   , heparin (Sigma, H3393), anti-Spike (Genetex, 1A9, GTX632604), anti-Spike (Absolute Antibody, CR3022), rabbit anti-TGFBR1 (Thermo Fisher Scientific, PA5-32631).	anti-Spike suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418) GTX632604 suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418) CR3022 suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080) anti-TGFBR1 suggested: (Thermo Fisher Scientific Cat# PA5-32631, RRID:AB_2550094) PA5-32631 suggested: (Thermo Fisher Scientific Cat# PA5-32631, RRID:AB_2550094)
   The following antibodies were used in this study: goat anti-ACE2 (R&D Systems, AF933)	anti-ACE2 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Vero-E6 cells were used for SARS-CoV-2 titration and maintained in D10 media at 37°C with 5% CO2.	Vero-E6 suggested: None
   In brief, 15-cm2 dishes of HEK293T cells were transfected with a plasmid encoding SARS-CoV-2 S (Wuhan-Hu-1, Accession #QHD43416.1) using 45 μg total DNA.	HEK293T suggested: None
   In brief, lentivirus vectors were transfected into 293T cells using a lipofectamine 3000 transfection protocol according to the manufacturer’s instructions, along with a packaging vector (psPAX2) and a pseudotyping vector (pMD2.G).	293T suggested: None
   In brief, 6×104 HPMEC or 2×105 Calu-3 were seeded in 300 μL into the apical chambers of 24-well transwell polycarbonate membrane inserts (Transwell permeable support, 0.4 μM, 6.5 mm insert; Corning) and 1.5 mL of medium was added to the basolateral chamber.	Calu-3 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   EXPERIMENTAL MODELS AND SUBJECT DETAILS: Mice: Six- to eight-week-old wild-type C57BL/6J female mice were purchased from the Jackson Laboratory and housed at the University of California, Berkeley Animal Facility under specific pathogen-free conditions.	C57BL/6J suggested: RRID:IMSR_JAX:000664)
   The hACE2 encoding plasmid was a gift from Hyeryun Choe (Addgene plasmid #1786; http://n2t.net/addgene:1786; RRID:Addgene_1786) (66)	RRID:Addgene_1786 suggested: None
   Recombinant DNA
   Sentences	Resources
   The hACE2 encoding plasmid was a gift from Hyeryun Choe (Addgene plasmid #1786; http://n2t.net/addgene:1786; RRID:Addgene_1786) (66)	detected: RRID:Addgene_1786)
   At 24 hours post-transfection, VSV-G expressing VSV-ΔG-rLuc pseudotyped virions were used to infect the transfected HEK293T cells.	VSV-G suggested: RRID:Addgene_138479)
   In brief, lentivirus vectors were transfected into 293T cells using a lipofectamine 3000 transfection protocol according to the manufacturer’s instructions, along with a packaging vector (psPAX2) and a pseudotyping vector (pMD2.G).	psPAX2 suggested: RRID:Addgene_12260) pMD2 . G suggested: None
   CRISPR-Cas9 Knockout: To produce gene-specific knockout cell lines, we utilized a CRISPR-Cas9 pipeline based on the lentiCRISPR v2 lentivirus construct obtained from Feng Zhang (Addgene plasmid # 52961; http://n2t.net/addgene:52961;RRID:Addgene_52961), as previously described (71).	detected: RRID:Addgene_52961)
   Software and Algorithms
   Sentences	Resources
   SARS-CoV-2 S stabilized trimers from diverse viral variants were purchased from the Native Antigen Company including B.1.1.7	B.1.1.7 suggested: None
   Leakage was quantified around the injection sites using Image Studio software (LI-COR Biosciences).	Image Studio suggested: (Image Studio Lite, RRID:SCR_013715)
   Following sequencing of sample libraries, quality control was performed on the fastq files to ensure that sequencing reads met preestablished cutoffs for number of reads and quality using FastQC (version 0.11.8) (73) and MultiQC (version 1.8) (74).	FastQC suggested: (FastQC, RRID:SCR_014583) MultiQC suggested: (MultiQC, RRID:SCR_014982)
   Quality filtering and adapter trimming were performed using BBduk tools (version 38.76, https://sourceforge.net/projects/bbmap).	https://sourceforge.net/projects/bbmap suggested: (BBmap, RRID:SCR_016965)
   Remaining reads were aligned to the ENSEMBL GRCh38 human reference genome assembly (release 33) using STAR (version 2.7.0f) (75), and gene frequencies were counted using featureCounts (version 2.0.0) within the Subread package (76).	ENSEMBL suggested: (Ensembl, RRID:SCR_002344) STAR suggested: (STAR, RRID:SCR_004463) featureCounts suggested: (featureCounts, RRID:SCR_012919)
   Comparative analysis of DEGs was performed using a negative binomial distribution model used by DESeq2 (version 1.28.1) (77) as implemented in R (version 4.0.3).	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   Hierarchical clustering of DEGs and visualization were performed using the ComplexHeatmap (version 2.4.2) and pheatmap package (version 1.0.12).	ComplexHeatmap suggested: (ComplexHeatmap, RRID:SCR_017270) pheatmap suggested: (pheatmap, RRID:SCR_016418)
   Identified DEGs were analyzed by STRING to identify predicted protein-protein interaction networks as well as enriched pathway analysis (https://string-db.org/).	STRING suggested: (STRING, RRID:SCR_005223)
   Statistics: All data were plotted and quantitative analyses performed using GraphPad Prism 8 software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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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: No clinical trial numbers were referenced.

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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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   Read the original source
2. 

   Version published to 10.1101/2021.12.10.472112v1 on bioRxiv

   Dec 13, 2021