Bromodomain and Extraterminal Inhibition Blocks Inflammation-Induced Cardiac Dysfunction and SARS-CoV-2 Infection (Pre-Clinical)

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   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.08.23.258574: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	Mice were randomized and received either vehicle 30% (m/v) Kolliphor 15 HS (Sigma) in PBS or 2 mg per 30 g mouse body weight of INCB054329 at 20 mg/ml in the Kolliphor solution (66.7 mg/kg).
   Blinding	For some experiments apabetalone and RVX-2157 were sent blinded by Resverogix.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Single cells were separated using a 100 μm strainer and labelled with CD31 antibody (1:200, M082329-2, DAKO) at 4°C for 45 min followed by 30 min staining with a goat anti-mouse secondary antibody conjugated to AlexaFluor 488 or 555 (1:400, A-11001 and …

   SciScore for 10.1101/2020.08.23.258574: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	Mice were randomized and received either vehicle 30% (m/v) Kolliphor 15 HS (Sigma) in PBS or 2 mg per 30 g mouse body weight of INCB054329 at 20 mg/ml in the Kolliphor solution (66.7 mg/kg).
   Blinding	For some experiments apabetalone and RVX-2157 were sent blinded by Resverogix.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Single cells were separated using a 100 μm strainer and labelled with CD31 antibody (1:200, M082329-2, DAKO) at 4°C for 45 min followed by 30 min staining with a goat anti-mouse secondary antibody conjugated to AlexaFluor 488 or 555 (1:400, A-11001 and A-21422, ThermoFisher Scientific).	CD31 suggested: None anti-mouse suggested: (Thermo Fisher Scientific Cat# A-11001, RRID:AB_2534069)
   Cells were stained with primary antibodies CD31 (1:200, M082329-2, DAKO), NG2 (1:200, 14-6504-82, ThermoFisher Scientific) and cardiac troponin T (1:400, ab45932, Abcam) in 5% FBS and 0.25% TritonX-100 Blocking Buffer at 4°C overnight on a rocker.	NG2 suggested: (Thermo Fisher Scientific Cat# 14-6504-82, RRID:AB_10870987) cardiac troponin T suggested: (Abcam Cat# ab45932, RRID:AB_956386)
   Cells were washed twice for 1 h with Blocking Buffer and labelled with secondary antibodies goat anti-mouse IgG1 AlexaFluor 488 (1:400, A-21121),	anti-mouse IgG1 suggested: (Molecular Probes Cat# A-21121, RRID:AB_2535764)
   After 1 h blocking using a 1:1 mix of LI-COR Odyssey Blocking Buffer (LI-COR Biotechnology) and PBS, membranes were incubated overnight on a platform shaker with primary antibodies for ACE2 (1:200, R&D Systems, AF933) and GAPDH (1:1000, Cell Signaling Technologies, 97166S).	ACE2 suggested: (LSBio (LifeSpan Cat# LS-C347-1000, RRID:AB_1271963) GAPDH suggested: (Cell Signaling Technology Cat# 97166, RRID:AB_2756824)
   Membranes were washed 5 times 3 minutes in PBS with 0.5% Tween, prior to incubation with IRDye® secondary antibodies (1:10000 for IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody, 926-32210, and 680RD Donkey anti-Goat IgG Secondary Antibody, LI-COR Biotechnology, 925-68074) for 1 h at room temperature.	anti-Goat IgG suggested: (LI-COR Biosciences Cat# 925-68074, RRID:AB_2650427)
   For ACE2 assays the following was used for control, 1:200 Goat IgG Alexa Fluor 647-conjugated antibody, and assay 1:200 anti-human ACE2 AlexFluor 647 conjugated antibody and 1:200 anti-human CD90 (all RnD Systems) and were incubated for 60 min at 4°C in Binding Buffer.	anti-human ACE2 suggested: None anti-human CD90 suggested: None
   Both conditions were then incubated with 1:400 goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 555 (ThermoFisher Scientific) in Binding Buffer for 45 min at 4°C.	anti-mouse IgG suggested: None
   Both conditions were then incubated with 1:400 F(ab’)2-goat anti-human IgG Fc secondary antibody conjugated to Alexa Fluor 488 and 1:400 goat anti-mouse IgG secondary antibody conjugated to Alexa Fluor 555 (both ThermoFisher Scientific) in Binding Buffer for 45 min at 4°C.	anti-human IgG suggested: (GenWay Biotech Inc. Cat# GWB-73F555, RRID:AB_10273468)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   SARS-CoV-2 K18-hACE2 mouse infection model: Female K18-hACE2 mice were lightly anesthetized using isoflurane and 50 μl of SARS-CoV-2 at 5 × 104 TCID50 per mouse was administered via intranasal inoculation (i.n.).	K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
   Software and Algorithms
   Sentences	Resources
   Videos were then analysed with a custom written Matlab program (Mills et al., 2017).	Matlab suggested: (MATLAB, RRID:SCR_001622)
   MS data processing: RAW MS data was processed in the MaxQuant software environment (Cox and Mann, 2008)	MaxQuant suggested: (MaxQuant, RRID:SCR_014485)
   Data analysis was performed using the Perseus software package (Tyanova and Cox, 2018).	Perseus suggested: (Perseus, RRID:SCR_015753)
   Default options were used with CellRanger and a custom made pre mRNA reference using GRCh38 v3.0.0 was used to map the reads and for count quantification with the CellRanger counts tool.	CellRanger suggested: None
   All pre-processing and filtering steps of the datasets were subsequently carried out via the Python package Scanpy (https://scanpy.readthedocs.io/en/stable/) (Wolf et al., 2018).	Python suggested: (IPython, RRID:SCR_001658)
   hCO comparison to bulk nuclei RNA sequencing data for PCA: Nuclear RNA-seq dataset generated from sorted cardiomyocyte nuclei at two stages (fetal and adult) was obtained from BioProject ID: PRJNA353755 (Gilsbach et al., 2018).	BioProject suggested: (NCBI BioProject, RRID:SCR_004801)
   Annotations and genome files (hg38) were obtained from Ensembl (release 102).	Ensembl suggested: (Ensembl, RRID:SCR_002344)
   Subsequent analyses of the count data were performed in the R statistical programming language with the Bioconductor packages edgeR (Robinson et al., 2010) and the annotation package org.	Bioconductor suggested: (Bioconductor, RRID:SCR_006442)
   Additionally, ribosomal and mitochondrial genes as well as pseudogenes, and genes with no annotation (Entrez Gene identification) were removed before normalization and statistical analysis.	Entrez Gene suggested: (Entrez Gene, RRID:SCR_002473)
   Sequence reads were trimmed for adapter sequences using Cutadapt version1.9 (Martin, 2011) and aligned using STAR version 2.5.2a (Dobin et al., 2013) to the Mus Musculus GRCm38 assembly with the gene, transcript, and exon features of Ensembl (release 102) gene model, and the SARS-CoV-2 Ensembl genome assembly ASM985889v3.	Cutadapt suggested: (cutadapt, RRID:SCR_011841) STAR suggested: (STAR, RRID:SCR_015899)
   Quality control metrics were computed using RNA-SeQC version 1.1.8 (DeLuca et al., 2012) and expression was estimated using RSEM version 1.2.30 (Li and Dewey, 2011).	RNA-SeQC suggested: (RNA-SeQC, RRID:SCR_005120) RSEM suggested: (RSEM, RRID:SCR_013027)
   Trimmed mean of M-values (TMM) normalization and differential expression analysis were performed using the R package edgeR (Robinson et al., 2010).	edgeR suggested: (edgeR, RRID:SCR_012802)
   To estimate SARS-CoV-2 replication levels, sequence reads were aligned to SARS-CoV-2 only, and samtools (Li et al., 2009) version 1.9 was used to estimate the mapping rate of the reads to the viral genes.	samtools suggested: (SAMTOOLS, RRID:SCR_002105)
   Comparison of different RNA-seq data: KEGG and Encode TF analyses was performed using Enrichr (Kuleshov et al., 2016).	KEGG suggested: (KEGG, RRID:SCR_012773)
   Networks were generated through the use of Ingenuity Pathway Analysis (IPA) on DEGs (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis).	Ingenuity Pathway Analysis suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)
   Statistics were performed using GraphPad Prism v8 unless noted.	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: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT01067820	Completed	ApoA-I Synthesis Stimulation and Intravascular Ultrasound fo…

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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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   Read the original source
2. 

   Version published to 10.1101/2020.08.23.258574v3 on bioRxiv

   Feb 15, 2021
3. 

   preLights

   Nov 27, 2020

   Excerpt

   ‘In the eye of the (Cytokine) Storm’: COVID-19 inflammation and heart function in 3D-engineered cardiac tissues

   Read the original source
4. 

   Version published to 10.1101/2020.08.23.258574v2 on bioRxiv

   Oct 16, 2020
5. 

   Version published to 10.1101/2020.08.23.258574v1 on bioRxiv

   Aug 24, 2020