Recognition and inhibition of SARS-CoV-2 by humoral innate immunity pattern recognition molecules
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SciScore for 10.1101/2021.06.07.21258350: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: The collection of bronchial epithelial cells and their study to investigate airway epithelium physiopathology were specifically approved by the Ethics Committee of the Istituto Giannina Gaslini following the guidelines of the Italian Ministry of Health (registration number: ANTECER, 042-09/07/2018).
IRB: The collection of bronchial epithelial cells and their study to investigate airway epithelium physiopathology were specifically approved by the Ethics Committee of the Istituto Giannina Gaslini following the guidelines of the Italian Ministry of Health (registration number: ANTECER, 042-09/07/2018).
Consent: Each patient provided informed consent to the study using a …SciScore for 10.1101/2021.06.07.21258350: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Field Sample Permit: The collection of bronchial epithelial cells and their study to investigate airway epithelium physiopathology were specifically approved by the Ethics Committee of the Istituto Giannina Gaslini following the guidelines of the Italian Ministry of Health (registration number: ANTECER, 042-09/07/2018).
IRB: The collection of bronchial epithelial cells and their study to investigate airway epithelium physiopathology were specifically approved by the Ethics Committee of the Istituto Giannina Gaslini following the guidelines of the Italian Ministry of Health (registration number: ANTECER, 042-09/07/2018).
Consent: Each patient provided informed consent to the study using a form that was also approved by the Ethics Committee.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 Rabbit anti-PTX3 antibody was produced in house (Bottazzi et al., 1997), rabbit anti-MBL Ab was purchased from Abcam. anti-PTX3suggested: NoneAnti-C1q polyclonal antibody was purchased from Dako. Anti-C1qsuggested: (Agilent Cat# F0254, RRID:AB_2335713)Anti-CRP and anti-SAP antibodies were from Merck. Anti-CRPsuggested: Noneanti-SAPsuggested: NoneBound MBL was detected by incubation with rabbit anti-MBL antibody, followed by HRP-conjugated secondary antibody and TMB development, as described above. anti-MBLsuggested: NoneAfter washing, C5b-9 deposition was assayed by incubation for 1 h at 37°C with rabbit anti-sC5b-9 antibody (ComplemenTech Inc., anti-sC5b-9suggested: NoneCells were then incubated for 2 h in blocking buffer with the following primary antibodies: mouse anti-cytokeratin 14 (Krt14) (#LL002; 1µg/ml; cat. anti-cytokeratinsuggested: (Leica Biosystems Cat# RTU-LL002, RRID:AB_563793)Krt14suggested: NoneAfter washing with PBS and 0.05% Tween, cells were incubated for 1 h with the following species- specific cross-adsorbed secondary antibodies form Invitrogen-ThermoFisher Scientific: donkey anti-rabbit IgG Alexa Fluor® 488 (1µg/ml; cat. N° A-21206); donkey anti-rat IgG Alexa Fluor® 594 (1µg/ml; cat. N° A-21209); donkey anti-mouse IgG Alexa Fluor® 647 (1µg/ml; cat. N° A-31571). 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) was used for nucleus staining. anti-rabbit IgGsuggested: (Thermo Fisher Scientific Cat# A-21206, RRID:AB_2535792)anti-rat IgGsuggested: (Thermo Fisher Scientific Cat# A-21209, RRID:AB_2535795)anti-mouse IgGsuggested: (Molecular Probes Cat# A-31571, RRID:AB_162542)Experimental Models: Cell Lines Sentences Resources Recombinant human PTX3 and its domains from CHO cells were produced in house, as previously described (Bottazzi et al., 1997). CHOsuggested: NoneRecombinant RBD and trimeric Spike were produced in EXPI293 cells and purified as reported (De Gasparo et al., 2021). EXPI293suggested: RRID:CVCL_D615)Pseudotyped virus production: Human embryonic kidney 293T cells were transfected with a lentiviral vector expressing the Green Fluorescent Protein (GFP) under the control of a human Phosphoglycerate Kinase promoter (PGK) (Cesana et al., 2014) and a pCMV expressing vector containing the SARS-CoV-2 Spike sequence (accession number MN908947) that was codon- optimized for human expression and contained a deletion at the 3’ end aimed at deleting 19 amino acid residues at the C-terminus. 293Tsuggested: NoneSecondary viral stocks were generated by infection of adherent Vero E6 cells seeded in a 25 cm2 tissue culture flask with 0.5 ml of the primary isolate diluted in 5 ml of complete medium. Vero E6suggested: NoneAfter 48 and 72 h post-infection (PI), cell culture supernatants were collected and stored at – 80 °C until determination of the viral titers by a plaque-forming assay in Vero cells. Verosuggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)Briefly, virus incubation with MBL was performed as described above whereas Calu-3 cells were incubated with 10-fold serial dilutions of MBL (from 0.01 to 10 µg/ml – 0.034-34 nM). Calu-3suggested: NoneRecombinant DNA Sentences Resources Pseudotyped virus production: Human embryonic kidney 293T cells were transfected with a lentiviral vector expressing the Green Fluorescent Protein (GFP) under the control of a human Phosphoglycerate Kinase promoter (PGK) (Cesana et al., 2014) and a pCMV expressing vector containing the SARS-CoV-2 Spike sequence (accession number MN908947) that was codon- optimized for human expression and contained a deletion at the 3’ end aimed at deleting 19 amino acid residues at the C-terminus. pCMVsuggested: RRID:Addgene_20783)Software and Algorithms Sentences Resources Reference distances (∼ 40 Å) between mannose molecules were computed in PYMOL. PYMOLsuggested: (PyMOL, RRID:SCR_000305)The human lung epithelial Calu-3 cell line was obtained from NovusPharma. NovusPharmasuggested: NoneSTED images were de-convolved with Huygens Professional software (Scientific Volume Imaging B. V.; version 19.10) and presented as MIP. Huygens Professionalsuggested: NoneNext, we accurately checked cases and controls for solving within-Italian relationships and for testing the possible existence of population stratification within and across batches: to this aim, we performed principal component analysis (PCA), using a LD-pruned subset of SNPs across chromosome 10 and the Plink v. Plinksuggested: (PLINK, RRID:SCR_001757)Statistical analysis: Prism GraphPad software v. Prism GraphPadsuggested: None1.07 (Purcell et al., 2007); ii) by an unsupervised approach by means of the Beagle software v3.3 (http://faculty.washington.edu/browning/beagle/b3.html), which uses the method described by Browning & Browning (Browning and Browning, 2007) for inferring haplotype phase. Beaglesuggested: (BEAGLE, RRID:SCR_001789)Results from OddPub: Thank you for sharing your data.
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 did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 26. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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