SARS-CoV-2 Receptor Angiotensin I-Converting Enzyme Type 2 (ACE2) Is Expressed in Human Pancreatic β-Cells and in the Human Pancreas Microvasculature

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1. Version published to 10.3389/fendo.2020.596898

   Nov 13, 2020
2. 

   ScreenIT

   Jul 25, 2020

   SciScore for 10.1101/2020.07.23.208041: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   Consent: In INNODIA EUnPOD network, pancreata not suitable for organ transplantation were obtained with informed written consent by organ donors’ next-of -kin and processed with the approval of the local ethics committee of the Pisa University. IRB: 2.6 RNA extraction from cells and tissues: For gene expression evaluation, total RNA was extracted from approximately 3.0×105 EndoC-βH1 or from fresh lung tissue (0.5×0.5×0.5cm), obtained from a lung tumor surgery donor by dissecting a not affected portion of the tissue (obtained with informed written consent of the patient and approved by the local Ethics Committee at the University of Siena).

   Randomization

   not detected.

   SciScore for 10.1101/2020.07.23.208041: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	Consent: In INNODIA EUnPOD network, pancreata not suitable for organ transplantation were obtained with informed written consent by organ donors’ next-of -kin and processed with the approval of the local ethics committee of the Pisa University. IRB: 2.6 RNA extraction from cells and tissues: For gene expression evaluation, total RNA was extracted from approximately 3.0×105 EndoC-βH1 or from fresh lung tissue (0.5×0.5×0.5cm), obtained from a lung tumor surgery donor by dissecting a not affected portion of the tissue (obtained with informed written consent of the patient and approved by the local Ethics Committee at the University of Siena).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The next day, sections were incubated with secondary antibody polyclonal rabbit anti-mouse HRP-conjugate (cat.P0260, Dako, Agilent Technologies, Santa Clara, CA, USA) diluted 1:100 in PBS 1× for 1h at room temperature (RT).	anti-mouse HRP-conjugate ( cat.P0260 suggested: None
   In order to further evaluate ACE2 expression in pancreas sections, the same ACE2 IHC protocol was applied to other 2 primary antibodies anti-Human ACE2: monoclonal rabbit anti-Human ACE2 (cat.	anti-Human ACE2: monoclonal rabbit anti-Human ACE2 suggested: None anti-Human suggested: None
   The secondary antibody in both cases was polyclonal goat antirabbit HRP-conjugate (cat. 111-036-003, Jackson ImmunoResearch, Philadelphia, PA, USA), diluted 1:1000 in PBS 1× for 1h at room temperature (RT).	antirabbit suggested: None
   All the 3 primary antibodies anti-Human ACE2, with respective secondary antibodies, were also used to perform a positive control staining in FFPE human lung sections (7-μm thickness), in order to double check the specificity of the primary antibodies.	anti-Human ACE2 suggested: None
   Subsequently, sections were incubated with goat anti-guinea pig Alexa-Fluor 555 conjugate (cat. A21435, Molecular Probe, ThermoFisher Scientific, Waltham, MA, USA) diluted 1:500 in PBS 1X, goat anti-rabbit Alexa-Fluor 647 conjugate (cat. A21245, Molecular Probe, ThermoFisher Scientific, Waltham, MA, USA) diluted 1:500 in PBS 1X and goat anti-mouse 488 conjugate (cat.A11029 - Molecular Probe, ThermoFisher Scientific, Waltham, MA, USA) diluted 1:500 in PBS 1X, as secondary antibodies for 1h.	anti-guinea pig suggested: (Innovative Research Cat# A21435, RRID:AB_1500610) anti-rabbit suggested: (Molecular Probes Cat# A-21245, RRID:AB_141775) anti-mouse suggested: (Molecular Probes Cat# A-11029, RRID:AB_138404)
   A negative control with only secondary antibody incubation (no MAB933 primary antibody control sample) was also included in order to exclude any background artifacts or fluorochrome overlaps (Figure S1B).	MAB933 suggested: (R and D Systems Cat# MAB933, RRID:AB_2223153)
   EndoC-βH1 cells were incubated with antibody polyclonal Guinea Pig anti-Human Insulin (cat.	anti-Human Insulin suggested: None
   To identify ACE2, three different antibodies were used: #Ab108252, #Ab15348 (Abcam) and #MAB933 (R&D system) were respectively diluted 1:1000, 1:500 and 1:250 in 5% non-fat dry milk in TBST1X and incubated o/n at +4°C and then with Goat anti-rabbit (#111-036-003, Jackson Laboratories) or Goat anti-Mouse (#115-036-003, Jackson Laboratories) diluted 1:5000 in 2% non-fat dry milk in TBST1X 1 h RT.	ACE2 suggested: (LSBio (LifeSpan Cat# LS-C347-1000, RRID:AB_1271963)
   Experimental Models: Cell Lines
   Sentences	Resources
   Then, HeLa cells were rinsed with PBS without Ca2+ and Mg2+ and were incubated with goat anti-mouse-488 1:500 in 1% BSA in PBS1X without Ca2+ and Mg2+, or with goat anti-rabbit-488 diluted 1:500 in 1% BSA in PBS1X without Ca2+ and Mg2+.	HeLa suggested: None
   Software and Algorithms
   Sentences	Resources
   Whole pancreata were processed following standardized procedures at University of Pisa.	Pisa suggested: (PISA, RRID:SCR_015749)
   The Salmon software version 0.13.2 (35) was used to re-analyse our original RNA-seq data (32–34) by mapping the sequenced reads to the human reference transcriptome from GENCODE version 31 (GRCh38) (36) using the quasi-alignment model.	Salmon suggested: (Salmon, RRID:SCR_017036) GENCODE suggested: (GENCODE, RRID:SCR_014966)
   Differentially expressed genes were identified with DESeq2 version 1.24.0 (37).	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   The raw data obtained were analyzed using the Biopharma Finder 2.1 software from ThermoFisher Scientific.	Biopharma Finder suggested: None
   , trypsin as digestion enzyme and eventual modifications (carbamidomethylation, oxidation, etc.). 2.16 ACE2 promoter transcription factors (TF) binding motifs analysis: ACE2 proximal promoter sequence was retrieved from Ensembl Genome browser database (http://www.ensembl.org/index.html)	Ensembl Genome browser suggested: (Ensembl Genome Browser, RRID:SCR_013367)
   In TRAP, ACE2 promoter sequence was analysed by using both TRANSFAC.2010.1 and JASPAR vertebrate databases and human_promoters as background model.	JASPAR suggested: (JASPAR, RRID:SCR_003030)

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   Results from OddPub: Thank you for sharing your data.

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

   Of note, such datasets rely on β- or α-cells bulk RNA-seq and do not suffer from common limitations present in single-cell RNA seq (65), which usually detects only 1,000-3,000 genes/cell, thus representing a minor fraction (25-30%) of the total number of genes identified by bulk cells RNA sequencing (>20,000 genes). Another recent study analyzed SARS-CoV-2 host receptors expression using two different methods (microarray and bulk RNA-seq) and further confirmed that ACE2 is indeed expressed in human pancreatic islets, and also demonstrated that ACE2 expression was higher in sorted pancreatic β-cells relative to other endocrine cells (66). Additional evidence of ACE2 expression in endocrine pancreas and in β-cells derive also from mouse studies, which demonstrated ACE2 expression in insulin-producing cells as well as its critical role in the regulation of β-cell phenotype and function (67–70). Collectively, our in-situ expression data alongside with multiple published datasets and reports both in human and mouse show that ACE2 is expressed in pancreatic islets, albeit at relatively low levels. ACE2 expression in human β-cells may render these cells sensitive to SARS-CoV-2 entry (23). Such hypothesis is consistent with the known sensitivity of these cells to infection by several enterovirus serotypes. Indeed, multiple evidence from our and other groups (71–74) showed that enteroviruses are capable to competently infect β-cells but less so α-cells (75,76); these viruses are thus ...

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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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3. 

   Version published to 10.1101/2020.07.23.208041 on bioRxiv

   Jul 23, 2020