Impairment of SARS-CoV-2 spike glycoprotein maturation and fusion activity by nitazoxanide: an effect independent of spike variants emergence

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1. Version published to 10.1007/s00018-022-04246-w

   Apr 7, 2022
2. 

   ScreenIT

   Apr 15, 2021

   SciScore for 10.1101/2021.04.12.439201: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	Images shown in all figures are representative of at least three random fields (scale-bars are indicated).
   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
   Membranes were incubated with the selected antibodies, followed by incubation with peroxidase-labeled anti-rabbit or anti-mouse IgG.	anti-rabbit suggested: None anti-mouse IgG suggested: None
   Cells were fixed, permeabilized and processed for immunofluorescence as described (72) using anti-FLAG antibodies, followed by decoration with Alexa Fluor 488-or 555-conjugated …

   SciScore for 10.1101/2021.04.12.439201: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	Images shown in all figures are representative of at least three random fields (scale-bars are indicated).
   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
   Membranes were incubated with the selected antibodies, followed by incubation with peroxidase-labeled anti-rabbit or anti-mouse IgG.	anti-rabbit suggested: None anti-mouse IgG suggested: None
   Cells were fixed, permeabilized and processed for immunofluorescence as described (72) using anti-FLAG antibodies, followed by decoration with Alexa Fluor 488-or 555-conjugated antibodies (Molecular Probes, Invitrogen).	anti-FLAG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture, treatments, plasmids and transfections: Human alveolar type II-like epithelial A549 cells, airway epithelial Calu-3 cells and MRC-5 lung fibroblasts, colorectal adenocarcinoma HCT116 cells, colon carcinoma Caco-2 cells, embryonic kidney 293T cells, and African green monkey kidney Vero E6 cells, murine L929 fibroblasts and bat lung epithelial Tb-1 Lu cells were obtained from ATCC (Manassas, VA, USA).	MRC-5 lung fibroblasts suggested: None HCT116 suggested: None
   , DMEM (Euroclone; Vero E6, Huh7, 293T, HTC116 and Caco-2 cells), EMEM (ATCC; MRC-5 and Calu-3 cells) (Lonza; Tb-1 Lu cells) or MEM (Gibco; L929 cells) medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and antibiotics.	Huh7 suggested: None HTC116 suggested: None MRC-5 suggested: None L929 suggested: None
   In the first protocol, Vero E6, Huh7, A549 and A549 hACE2 cells grown in 8-well chamber slides were transfected with Flag-tagged SARS-CoV-2 (SARS-2 S-CF wild type or mutated, pCAGGS-SARS2-S-D614, pCAGGS-SARS2-S-G614) or SARS-CoV spike plasmids or empty vector and, after 4h, were treated with NTZ or vehicle.	A549 suggested: None A549 hACE2 suggested: None
   After 4h, cells were treated with NTZ or vehicle. 293T cells transfected with the GFP-encoding plasmid were used as negative control.	293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   Pseudovirus entry assays: Vero E6, Calu-3, Caco-2, Caco-2 hACE2 and 293T hACE2 cells were plated at a density of 2x104 cells/well in 96 well white clear-bottom plates (Costar).	Calu-3 suggested: KCLB Cat# 30055, RRID:CVCL_0609) Caco-2 suggested: None 293T hACE2 suggested: None
   Antiviral activity assay: To determine the antiviral activity of tizoxanide, Vero E6 cells were infected with SARS-CoV-2 for 2h, and then treated with a three-fold serial dilution of compound ranging from 20 µM to 0.01 µM.	Vero E6 suggested: None
   Software and Algorithms
   Sentences	Resources
   For confocal microscopy, images were acquired on Olympus FluoView FV-1000 confocal laser scanning system (Olympus America Inc., Center Valley, PA) and analyzed using Imaris (v6.2) software (Bitplane, Zurich, Switzerland)	Imaris suggested: (Imaris, RRID:SCR_007370)
   Syncytia images were captured using a ZEISS Axio Observer inverted microscope and analyzed using ZEN 3.1 (blue edition) software.	ZEN suggested: None
   To measure the extent of cell–cell fusion, the GFP area was delimited, measured using ImageJ software (53), divided by the total cell area and expressed as percentage relative to control.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Statistical analysis: Statistical analysis was performed using the Student’s t test for unpaired data or one-way ANOVA test (Prism 5.0 software, GraphPad).	Prism suggested: (PRISM, RRID:SCR_005375) 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: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04343248	Active, not recruiting	Trial to Evaluate the Efficacy and Safety of Nitazoxanide (N…

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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: 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 10.1101/2021.04.12.439201 on bioRxiv

   Apr 12, 2021