ACE2 Is Expressed in Immune Cells That Infiltrate the Placenta in Infection-Associated Preterm Birth

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1. Version published to 10.3390/cells10071724

   Jul 8, 2021
2. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.09.27.20201590: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   Consent: All tissues and peripheral blood were provided by the Research Centre for Women’s and Infants’ Health Bio Bank program at Sinai Health System and are collected following informed written consent (process n# 20-0101-E) and in adherence with the policies of the Sinai Health System and the University of Toronto Research Ethics Board and in accordance with the Helsinki declaration on the use of human tissues. IRB: All tissues and peripheral blood were provided by the Research Centre for Women’s and Infants’ Health Bio Bank program at Sinai Health System and are collected following informed written consent (process n# 20-0101-E) and in adherence with the …

   SciScore for 10.1101/2020.09.27.20201590: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: All tissues and peripheral blood were provided by the Research Centre for Women’s and Infants’ Health Bio Bank program at Sinai Health System and are collected following informed written consent (process n# 20-0101-E) and in adherence with the policies of the Sinai Health System and the University of Toronto Research Ethics Board and in accordance with the Helsinki declaration on the use of human tissues. IRB: All tissues and peripheral blood were provided by the Research Centre for Women’s and Infants’ Health Bio Bank program at Sinai Health System and are collected following informed written consent (process n# 20-0101-E) and in adherence with the policies of the Sinai Health System and the University of Toronto Research Ethics Board and in accordance with the Helsinki declaration on the use of human tissues.
   Randomization	Explants were cultured for 24 hours and then randomly divided into treatment groups.
   Blinding	Statistical Analysis: All analyses were conducted blind to the experimental conditions.
   Power Analysis	not detected.
   Sex as a biological variable	Ethical Approval: This is a cross-sectional study involving placental tissue collection from pregnancies (25.3-36.0 weeks’ gestation) complicated by preterm birth (PTB; N=12) or preterm birth with chorioamnionitis (ChA; N=12) or from term pregnancies (>37 weeks’ gestation) from healthy women in labour (vaginal delivery, SVD; n=12) or not in labour (elective caesarean section, ELCS; N=12).

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Briefly, slides were deparaffinized, rehydrated, and subjected to heat mediated antigen retrieval with 10mM sodium citrate Ph6.0 for CD206 and neutrophil primary antibodies, or 1mM EDTA Ph9.0 for MHC-II, CD68 and ACE2.	MHC-II, CD68 suggested: None ACE2 suggested: None
   After blocking with Dako protein block (Dako, Mississauga, ON, Canada), the slides were incubated overnight at 4°C with primary antibodies: anti-rabbit ACE2 (1:200, ab15348, Abcam, Toronto, ON, Canada	anti-rabbit ACE2 suggested: (Abcam Cat# ab15348, RRID:AB_301861)
   , anti-rabbit neutrophil (NE)(1:100, ab21595, abcam) was added instead of the primary antibody.	anti-rabbit neutrophil ( NE) suggested: None
   Negative controls were performed using either isotype Mouse IgG1 or rabbit IgG/IgG1 antibodies.	IgG1 suggested: None rabbit IgG/IgG1 suggested: None
   Subsequently, ACE2 + CD68 slides were washed three times and incubated with fluorescent secondary antibodies, using either the anti-mouse Alexa 488 (1:1000) or the anti-rabbit Alexa 594 (1:1000) secondary antibodies (Thermo Fisher Scientific and counterstained with 1 μg/mL of DAPI, for 1 hour).	anti-mouse suggested: None anti-rabbit suggested: None
   For the localization of ACE2+MHC-II (M1), ACE2+CD206 (M2) and ACE2+Neutrophil elastase (NE) (primary antibodies the same species), immunofluorescence experiments were performed as described previously (Salio et al., 2005; Dunk et al., 2018; Choudhury et al., 2019).	M1 suggested: (Fitzgerald Industries International Cat# 10C-CR2005M1, RRID:AB_1282560) ACE2+Neutrophil elastase suggested: None
   Slides were incubated with primary antibodies anti rabbit-MHC II (M1) (1:100, Abcam), anti-rabbit mannose receptor (CD206, M2,1:100, Abcam) anti-rabbit NE (1:100, Abcam) and anti-rabbit IgG1 (Abcam) added as an isotype control overnight at 4°C.	anti rabbit-MHC II ( M1 ) suggested: None anti-rabbit mannose receptor suggested: None CD206 suggested: None anti-rabbit NE suggested: None anti-rabbit IgG1 suggested: None
   The primary antibodies used were anti-rabbit ACE2 (dilution 1:1000; Abcam, ab108209, Toronto, ON, Canada) and anti-goat β-actin (dilution 1:2000; Santa Cruz Biotechnology, Dallas, TX, USA).	anti-goat β-actin suggested: None
   The PVDF membranes were subsequently incubated for 1 h with HRP-linked anti-goat secondary antibody (GE Healthcare Bio-Science	anti-goat suggested: None
   To detect the expression of ACE2 protein, surface staining was conducted by Alexa Fluor® 700-conjugated mouse anti-human ACE-2 (R&D Systems) and APC-H7 mouse anti-human CD45 (BD Biosciences) antibodies.	anti-human ACE-2 suggested: None anti-human CD45 suggested: None
   Software and Algorithms
   Sentences	Resources
   The protein band intensity was quantified using Image Lab™ software.	Image Lab™ suggested: (Image Lab Software, RRID:SCR_014210)
   Data analyses were performed by FlowJo V10 (TreeStar) or Kaluza 2 (Beckman Coulter) software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Data analyses were performed with Prism version 8 (GraphPad Software Inc.	Prism suggested: (PRISM, RRID:SCR_005375)
   , San Diego, CA, USA) qPCR data were assessed for normal distribution using D’Agostino and Pearson or the Shapiro-Wilk test; outliers were identified using “QuickCalcs” Outlier calculator program (version 7.0; GraphPad Software, Inc.	GraphPad 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 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: Please consider improving the rainbow (“jet”) colormap(s) used on page 14. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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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.
   • Thank you for including a protocol registration statement.

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3. 

   Version published to 10.1101/2020.09.27.20201590v2 on medRxiv

   Sep 30, 2020
4. 

   Version published to 10.1101/2020.09.27.20201590v1 on medRxiv

   Sep 29, 2020