Disrupting ACE2 Dimerization Mitigates the Infection by SARS-COV-2

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1. 

   ScreenIT

   Apr 15, 2022

   SciScore for 10.1101/2022.04.09.487739: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   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 membrane was incubated with ACE-2 Antibody (1:2,000, Novus Biologicals, CO, USA), Myc-Tag (9B11	Myc-Tag suggested: None
   The membrane was then incubated with secondary HRP-linked, Anti-rabbit IgG (1:10,000, Cell Signaling Technology, MA, USA) and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (1:2,000, Thermo Fisher Scientific, MA, USA) for 1 h at room temperature.	Anti-rabbit IgG suggested: None anti-Mouse IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resour…

   SciScore for 10.1101/2022.04.09.487739: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   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 membrane was incubated with ACE-2 Antibody (1:2,000, Novus Biologicals, CO, USA), Myc-Tag (9B11	Myc-Tag suggested: None
   The membrane was then incubated with secondary HRP-linked, Anti-rabbit IgG (1:10,000, Cell Signaling Technology, MA, USA) and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP (1:2,000, Thermo Fisher Scientific, MA, USA) for 1 h at room temperature.	Anti-rabbit IgG suggested: None anti-Mouse IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   HEK293T cells were cultured in FP medium (DMEM containing 10% FBS, 2 mM GlutaMAX™ Supplement, 0.1 mM MEM Non-Essential Amino Acids, 50 U/mL and 50 μg/mL Penicillin-Streptomycin).	HEK293T suggested: None
   To establish ACE2-expressed cell line (ACE2-HEK293T cells), HEK293T cells were infected with ACE2-expressing lentivirus and ACE2-positive cells were selected by 2 ug/mL of puromycin.	ACE2-HEK293T suggested: None
   Recombinant DNA
   Sentences	Resources
   For protein expression, the cell membrane penetrating peptide (TAT), red fluorescence protein (DsRed) and NK-NT or NKN1 fragments were cloned into pET6xHN-N Vector (Takara, CA, USA)	pET6xHN-N suggested: None
   For ACE2-expressing lentivirus packaging, pscALPSpuro-HsACE2 (human) (Addgene, MA, USA) were co-transfected with psPAX2 and pCMV-VSV-G packaging plasmids into HEK293T cells using FuGENE 6 (Promega, WI, USA)	pCMV-VSV-G suggested: RRID:Addgene_8454)
   For doxycycline (Dox) inducible, Spike protein pseudotyped luciferase-expressing lentivirus preparation, HEK293T cells were transfected with FUW-RLuc-T2A-PuroR(Kanarek et al., 2018) (Addgene, MA), psPAX2 and pUNO1-SARS2-S (D614G) (InvivoGen, CA) packaging plasmids using FuGENE 6.	psPAX2 suggested: RRID:Addgene_12260)
   In competition BiFC Assay, HEK293T cells were co-transfected with 0.5 μg of each construct expressed in pBiFC-VN155 (I152L) and 0.5 μg pBiFC-VC155 vectors, together with and 5 μg competitor constructs with stop codon in pBiFC-VN155 (I152L) vector.	pBiFC-VC155 suggested: RRID:Addgene_22011) pBiFC-VN155 suggested: None
   Software and Algorithms
   Sentences	Resources
   Fluorescence images were taken at 24 h and 48 h after transfection using a Nikon fluorescence microscope and fluorescence intensity was quantified by Image J.	Image J suggested: (ImageJ, RRID:SCR_003070)

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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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2. 

   Version published to 10.1101/2022.04.09.487739v2 on bioRxiv

   Apr 12, 2022
3. 

   Version published to 10.1101/2022.04.09.487739v1 on bioRxiv

   Apr 11, 2022