Melatonin drugs inhibit SARS-CoV-2 entry into the brain and virus-induced damage of cerebral small vessels

Version published to 10.1007/s00018-022-04390-3

Jun 13, 2022

SciScore for 10.1101/2021.12.30.474561: (What is this?)

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

Table 1: Rigor

Ethics	Field Sample Permit: All animal experiments were approved by the ANSES/EnvA/UPEC Ethics Committee (CE2A-16) and authorized by the French ministry of Research under the number APAFIS#25384-2020041515287655 v6, in accordance with the French and European regulations. IRB: All animal experiments were approved by the ANSES/EnvA/UPEC Ethics Committee (CE2A-16) and authorized by the French ministry of Research under the number APAFIS#25384-2020041515287655 v6, in accordance with the French and European regulations. Euthanasia Agents: SARS-CoV-2 virus infection: At day of infection (day post-infection DPI-0), mice were infected via intra-nasal inoculation of SARS-CoV-2 (10 µL each nostril, 104 TCID50 in total) in Dulbecco’s modified Eagle medium, under isoflurane anesthesia. IACUC: The score for clinical symptoms was attributed following an IACUC approved clinical scoring system, and included the following criteria: body weight, posture/fur, activity/ mobility, eye closure, respiratory rate.
Sex as a biological variable	Animals: K18-hACE2 C57BL/6 transgenic mice (males, 10-week-old), which expresses human ACE2 driven by a human cytokeratin 18 (K18) promoter (Jackson Laboratory, https://www.jax.org/strain/034860) were housed in an animal facility of biosafety level 3 (BSL3) at the French National Veterinary School in Maisons–Alfort, with water and food ad libitum.
Randomization	K18-hACE2 transgenic mice were randomly divided into the following groups (6 mice/group): vehicle, melatonin 10mg/kg (MLT10)
Blinding	For all analyses, we imaged four fields from at least two sections per mouse and analysis was performed blinded using ImageJ.
Power Analysis	Sample sizes were designed to give statistical power while minimizing animal use.
Cell Line Authentication	Contamination: Cell lines were checked regularly for any mycoplasma contamination. hCMEC/D3 cells: For Mpro-induced cell death assay, hCMEC/D3 cells were cultivated and transfected as described previously 29.

Table 2: Resources

Antibodies
Sentences	Resources
Mouse BD Fc Block™) for 15 min at 37°C before incubation with primary antibodies Alexa Fluor 647 Rat Anti-mouse CD31 (BD Pharmigen cat:563608) for CD31 cell labeling and Alexa Fluor 647 Rat IgG2a,k Isotype control (BD Pharmigen cat:557690)	Anti-mouse CD31 suggested: (Bio-Rad Cat# MCA2388P647, RRID:AB_871974)
Sections were blocked with 3% BSA in PBS containing 0.1% Triton X-100 for 6h at room temperature, and incubation with primary antibodies (collagen IV: Bio-Rad, #134001, 1:200; caveolin-1: Cell Signaling Technology, #3267, 1:400) was performed at 4 °C overnight, while incubation with secondary antibodies was performed in blocking solution at room temperature for 2h.	1:200; caveolin-1: Cell Signaling Technology , #3267 suggested: None
After RNAscope® assays, a classical immunofluorescence was performed using the Anti-Collagen IV primary antibody (Anti-Collagen IV, Abcam: ab6586, 1:500°) and a secondary Alexa488 Fluor-conjugated antibody (1:500; Molecular Probes, Invitrogen) and DAPI (ACDBio) was used to stain the nuclei.	Anti-Collagen IV suggested: (Abcam Cat# ab6586, RRID:AB_305584)
Staining was performed as described above using primary antibodies against GFP (Abcam, #ab13970, 1:2000) and DAPI (1:2000), and imaged using a fluorescence microscope (DMI6000B, Leica).	GFP suggested: (Abcam Cat# ab13970, RRID:AB_300798)
Briefly, Lumi4-Tb-labelled ACE2 cells were incubated with d2-labelled anti-FLAG tag antibody (2 μg/mL, 1h at room temperature; 61FG2DLF, Cisbio Bioassays), followed by addition of non-labelled RBD (5 nM) or melatonin (100 µM) and TR-FRET signal was immediately read during 1h.	anti-FLAG suggested: None melatonin suggested: (Novus Cat# NB100-62745, RRID:AB_964468)
Experimental Models: Cell Lines
Sentences	Resources
Cell culture and transfection: HEK293T cells: HEK293T (RRID:CVCL 0063) cells were obtained from Sigma-Aldrich and authenticated by the provider.	HEK293T suggested: None
SNAP-tagged ACE2, expressed in HEK293 cells, were fluorescently labelled by incubating cells with a SNAP suicide substrate conjugated to the long-lived fluorophore Terbium cryptate (Tb;	HEK293 suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
Briefly, Lumi4-Tb-labelled ACE2 cells were incubated with d2-labelled anti-FLAG tag antibody (2 μg/mL, 1h at room temperature; 61FG2DLF, Cisbio Bioassays), followed by addition of non-labelled RBD (5 nM) or melatonin (100 µM) and TR-FRET signal was immediately read during 1h.	ACE2 suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Animals: K18-hACE2 C57BL/6 transgenic mice (males, 10-week-old), which expresses human ACE2 driven by a human cytokeratin 18 (K18) promoter (Jackson Laboratory, https://www.jax.org/strain/034860) were housed in an animal facility of biosafety level 3 (BSL3) at the French National Veterinary School in Maisons–Alfort, with water and food ad libitum.	C57BL/6 suggested: None
K18-hACE2 transgenic mice were randomly divided into the following groups (6 mice/group): vehicle, melatonin 10mg/kg (MLT10)	K18-hACE2 suggested: RRID:IMSR_GPT:T037657)
Recombinant DNA
Sentences	Resources
Briefly, after withdrawing heparin from the medium, we transfected the cells using Lipofectamine 3000 (Thermo Fisher Scientific) and the following plasmids: pCAG-GFP, pCAG-p.Cys145Ala-Mpro-HA or pCAG-Mpro-HA (100 ng per well on 96-well plates).	pCAG-GFP suggested: RRID:Addgene_11150) pCAG-p.Cys145Ala-Mpro-HA suggested: None pCAG-Mpro-HA suggested: None
Software and Algorithms
Sentences	Resources
Analysis was performed blinded using ImageJ.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
Molecular dynamics simulations were computed with GROMACS 2020.5 67.	GROMACS suggested: (GROMACS, RRID:SCR_014565)
All statistical comparisons were performed using Prism 9 (GraphPad).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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).

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.

Results from TrialIdentifier: No clinical trial numbers were referenced.

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).

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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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.

Results from scite Reference Check: We found no unreliable references.

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Melatonin drugs inhibit SARS-CoV-2 entry into the brain and virus-induced damage of cerebral small vessels

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