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Jan 2022
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https://medrxiv.org/cgi/content/10.1101/2022.01.25.22269808

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1. sciscore 31 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.25.22269808: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: These studies were reviewed and approved by the Mount Sinai Hospital Institutional Review Board (IRB-20-03374,IRB-16-00791).<br>Consent: All participants signed written consent forms prior to sample and data collection.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After three washes with PBS-T, 50 μl/well of the pre-diluted secondary anti-human IgG (Fab-specific) horseradish peroxidase antibody (produced in goat; Sigma-Aldrich) diluted 1:3,000 in PBS-T containing 1% milk powder were added and plates were incubated for one hour incubation at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>the pre-diluted secondary anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The biotinylated mAb 1C7C7, a mouse anti-SARS nucleoprotein monoclonal antibody generated at the Center for Therapeutic Antibody Development at the Icahn School of Medicine at Mount Sinai ISMMS (Millipore Sigma), was used for NP staining at a concentration of 1μg/ml in PBS, 1% BSA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were incubated with an anti-SARS-CoV spike primary antibody directly conjugated to Alexafluor-647 (CR3022-AF647) for up to 4 hours at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following this, 50 μL per well of 1X MSD SULFO-TAG Anti-Human IgG detection antibodies were added and incubated for 1 hour.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Human IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, residues 1-1208 of the spike protein (Wuhan-1 strain numbering) were codon optimized with proline substitutions at residues 986 and 987, the furin cleavage site modified to “GSAS”, and a T4 fibritin trimerization motif and a 8X HIS tag were added on the C-terminus and the construct was cloned into pCDNA3.4. 293F cells were transfected with plasmid using PEIMax in FreeStyle 293 Expression Medium (Fisher) and cultured for three days at 32°C, 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293F</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero.E6 cells were seeded in 96-well high binding cell culture plates (Costar) at a density of 20,000 cells/well in complete Dulbecco’s modified Eagle medium (cDMEM) a day before infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero.E6</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibody-virus mixture was then added to Vero.E6-TMPRSS2 cells and incubated at 37°C for 1 hour.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero.E6-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequences for the proteins were cloned into a mammalian expression vector, pCAGGS, as previously described and proteins were purified after transient transfections with each respective plasmid (23, 26).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_127347)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, residues 1-1208 of the spike protein (Wuhan-1 strain numbering) were codon optimized with proline substitutions at residues 986 and 987, the furin cleavage site modified to “GSAS”, and a T4 fibritin trimerization motif and a 8X HIS tag were added on the C-terminus and the construct was cloned into pCDNA3.4. 293F cells were transfected with plasmid using PEIMax in FreeStyle 293 Expression Medium (Fisher) and cultured for three days at 32°C, 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDNA3.4</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Area under the curve (AUC) values were calculated and plotted using Prism 9 software (GraphPad).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed using GraphPad Prism 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All the analyses were performed using Prism 7 software (GraphPad).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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: We detected the following sentences addressing limitations in the study:
  Our study has several limitations. We include a limited number of samples, and our dataset comes from a comparison of imperfectly matched groups from a clinical trial in Thailand and an observational cohort study in New York City. Strengths of the study include the use of authentic SARS-CoV-2 for neutralization assays and that the findings could be replicated with different methods in a different, blinded, and independent laboratory. In summary, we show that a vaccine candidate which can be produced locally in LMICs at low cost induces neutralizing antibody titers to SARS-CoV-2 comparable to those observed in cohorts having received mRNA-based COVID-19 vaccines. The NDV-HXP-S vaccine candidate induces a strong RBD focused immune response resulting in a high proportion of neutralizing antibodies, which are associated with protection from infection and severe disease (20, 24, 25).
  Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04871737</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Study of a Live rNDV Based Vaccine Against COVID-19</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT05181709</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Live Recombinant Newcastle Disease Virus-vectored COVID-19…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04830800</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Phase 1/2 Safety and Immunogenicity Trial of COVID-19 Vacc…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04764422</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Assess the Safety and Immunogenicity of NDV-HXP-S Vaccine in…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04993209</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Clinical Trial of the COVID-19 Vaccine (Recombinant, Inactiv…</td></tr></table>
  Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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Tagaholic - I Am Machine Tag And So Can You

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https://biorxiv.org/cgi/content/10.1101/2022.01.25.477753

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1. sciscore 30 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.25.477753: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: The absence of mycoplasma contamination in cells was routinely tested.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blots were probed with the following antibodies: rabbit anti-eIF4E2/4EHP (Proteintech, 12227-1-AP), rabbit anti-DDX6 (Proteintech, 14632-1-AP), rabbit anti-CNOT9 (Proteintech, 22503-1-AP), mouse anti-Flag M2 (Sigma-Aldrich, F1804), rabbit anti-GIGYF2 (Proteintech, 24790-1-AP)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-eIF4E2/4EHP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-DDX6</div><div>suggested: (Proteintech Cat# 14632-1-AP, RRID:AB_2091264)</div></div><div style="margin-bottom:8px"><div>anti-CNOT9</div><div>suggested: (Proteintech Cat# 22503-1-AP, RRID:AB_11232413)</div></div><div style="margin-bottom:8px"><div>anti-Flag</div><div>suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)</div></div><div style="margin-bottom:8px"><div>F1804</div><div>suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)</div></div><div style="margin-bottom:8px"><div>anti-GIGYF2</div><div>suggested: (Proteintech Cat# 24790-1-AP, RRID:AB_2879727)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flp-In T-REx 293 cells (Thermo Fisher Scientific) were grown in similar conditions supplemented with 100μg/ml zeocin and 15μg/ml blasticidin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Flp-In T-REx 293</div><div>suggested: RRID:CVCL_U427)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The full-length cDNA encompassing the coding region of human 4EHP, obtained by RT-PCR using total RNA from HEK293T cells, were cloned into the pCI-Neo vector (Promega) at the XhoI-NotI sites in frame with a sequence encoding a V5 tag inserted at the NheI-XhoI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CRISPR-Cas9-mediated genome editing of HEK293 cells was performed according to Ran et al. (53).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cell line inducibly expressing Flag-NSP2 was generated by co-transfecting pcDNA5-FRT-TO-FH-Nsp2 (Addgene plasmid 157683) and pOG44 (Thermo Fisher Scientific) with a 1:10 ratio.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA5-FRT-TO-FH-Nsp2</div><div>suggested: RRID:Addgene_157683)</div></div><div style="margin-bottom:8px"><div>pOG44</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Truncated versions of NSP2 were generated by replacing the sequence encoding full-length NSP2 by PCR-amplified fragments at the BamHI-XhoI sites of the pcDNA5-FRT-TO-FH vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA5-FRT-TO-FH</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mCherry2-NSP2 expressing plasmids were created by cloning a PCR-amplified NSP2 fragment into the mCherry2-C1 (Addgene plasmid 54563) and mCherry2-N1 vectors (Addgene plasmid 54517) using EcoRI and BamHI restriction enzymes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>mCherry2-NSP2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>mCherry2-C1</div><div>suggested: RRID:Addgene_54563)</div></div><div style="margin-bottom:8px"><div>mCherry2-N1</div><div>suggested: RRID:Addgene_54517)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate the pCI-λNV5-GIGYF2 vector, a fragment containing the GIGYF2 sequence was obtained by PCR from the pcDNA4/TO/GFP-GIGYF2 vector (Addgene plasmid 141189) (34) and inserted into the pCI-λNV5 at the XhoI-NotI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-λNV5-GIGYF2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA4/TO/GFP-GIGYF2</div><div>suggested: RRID:Addgene_141189)</div></div><div style="margin-bottom:8px"><div>pCI-λNV5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A similar strategy was used to generate the V5-tagged fragments of GIGYF2 using primers to isolate the following domains encompassing residues: 1-267; 257-495; 485-752; 742-1,085; 1,075-1,320. pCI-eGFP-GIGYF2 was generated by inserting the eGFP sequence, PCR-amplified from pEGFP-C1 (Clontech), at the NheI-XhoI sites of the pCI-Neo vector and then by adding the fragment encoding GIGYF2 at the XhoI-NotI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-eGFP-GIGYF2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pEGFP-C1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The full-length cDNA encompassing the coding region of human 4EHP, obtained by RT-PCR using total RNA from HEK293T cells, were cloned into the pCI-Neo vector (Promega) at the XhoI-NotI sites in frame with a sequence encoding a V5 tag inserted at the NheI-XhoI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-Neo</div><div>suggested: RRID:Addgene_16574)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate the pCI-λNV5-GW182SD vector, a fragment encoding the residues 1382-1690 was cloned by PCR as a XhoI-NotI fragment from the pFRT/TO/FLAG/HA-DEST TNRC6C vector (Addgene plasmid 19885) (52) and inserted into the pCI-λNV5 at the XhoI-NotI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-λNV5-GW182SD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFRT/TO/FLAG/HA-DEST TNRC6C</div><div>suggested: RRID:Addgene_19885)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For recombinant GST-fused protein expression, full-length 4EHP and GIGYF2742-1,085 coding sequences contained in PCR-amplified BamHI-NotI and XhoI-NotI fragments, respectively, were cloned into a pGEX-6P-1 vector (Amersham) in frame with the GST coding sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGEX-6P-1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pET-28b vector (EMD Biosciences) was used to express NSP2 as a recombinant protein fused to an His6 tag at the N-terminus.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-28b</div><div>suggested: RRID:Addgene_47327)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate KO HEK293 cells, we transfected 700,000 cells with the pSpCas9(BB)-2A-Puro plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pSpCas9 ( BB)-2A-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the reporter containing the 3’UTR of Ifnb1, 20 ng of psiCHECK2-RLuc-Ifnb1 3’ UTR reporter (31), or empty psiCHECK2 (Promega), was added along with 100 ng of vectors encoding Flag-NSP2 or Flag (empty vector).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psiCHECK2</div><div>suggested: RRID:Addgene_40763)</div></div></td></tr></table>
  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:
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2022.01.25.477753
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2022.01.25.477789

1
1. sciscore 30 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.25.477789: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: The mouse and hamster studies were carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals under the approval of the Institutional Animal Care and Use Committee of Xiamen University.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Rhesus monkey immunizations: Ten rhesus monkeys were allocated randomly into two groups (three females and two males per group).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Rhesus monkey immunizations: Ten rhesus monkeys were allocated randomly into two groups (three females and two males per group).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">Microscopic evaluation of pathological lung lesions was performed blindly by pathologists following a semiquantitative scoring system with the inclusion of three indicators (49): (i) alveolar septum thickening and consolidation; (ii) hemorrhage, exudation, pulmonary edema, and mucous; and (iii) recruitment and infiltration of inflammatory immune cells.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two weeks after boosting, serum samples were collected for antibody analyses, including measurement of anti-spike IgG, anti-RBD IgG, pseudovirus neutralizing antibody, and authentic neutralizing antibody titers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-spike IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-RBD IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, single-cell suspensions were obtained from mouse spleen (106 cells per well) through grinding in 70 μm cell strainers and were seeded in anti-mouse IFN-γ antibody precoated ELISpot plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IFN-γ</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mixtures (150 μL per well) were added to a monolayer of Vero cells in a 96-well plate and incubated at 37 °C supplying with 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Experimental animals: BALB/c and C57BL/6 mice were purchased from Shanghai SLAC Laboratory Animal Co.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mouse immunizations: Six to eight-week-old BALB/c mice (n=6 per group) were immunized with STFK vaccines at 0.01, 0.1, 1, or 10 μg per dose in 150 μL through intramuscular injection following a two-dose schedule at weeks 0 and 3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The tag-free STFK and STFK variants were constructed into a pGS01b vector, modified from the pCGS3 vector containing glutamine synthetase (GS) selection marker (Sigma Aldrich).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGS01b</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCGS3</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were acquired using the SerialEM software on an FEI Tecnai F30 transmission electron microscope (ThermoFisher Scientific) operated at 300 kV and equipped with a Gatan K3 direct detector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SerialEM</div><div>suggested: (SerialEM, RRID:SCR_017293)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing and 3D reconstruction: Drift and beam-induced motion correction were performed with MotionCor2 (38) to produce a micrograph from each movie.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MotionCor2</div><div>suggested: (MotionCor2, RRID:SCR_016499)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We initially fitted the templates into the corresponding final cryo-EM maps using Chimera (43), and further corrected and adjusted them manually by real-space refinement in Coot (44).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting models were then refined with phenix.real_space_refine in PHENIX (45).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The final atomic models were validated with Molprobity (46, 47).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Molprobity</div><div>suggested: (MolProbity, RRID:SCR_014226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical analyses were conducted in GraphPad Prism 8 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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sciscore

URL

biorxiv.org/cgi/content/10.1101/2022.01.25.477789
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2022.01.26.477860

1
1. sciscore 30 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.26.477860: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">A pool of DNA constructs containing a randomized 18 bp barcode sequence (N18) was cloned into the NotI and AscI sites upstream of the LexA promoter sequence via restriction digestion, ligation and transformation into chemically competent E. coli.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">15 µg of total cellular protein was resolved by SDS-PAGE, transferred to a PVDF membrane, and probed using an anti-his antibody (ref).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-his</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">W303 cells were transformed with the p416LexA-UbMpro(C145A)-his construct and the resulting yeast cells were grown to exponential phase in SD-ura media at 30°C. 125 nM β-estradiol was added when indicated and cells were grown for an additional eight hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>W303</div><div>suggested: Coriell Cat# GM03889, RRID:CVCL_W303)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Gal4, Gal80 and Pdr5 genes were disrupted to create the following strain: W303 HO::Gal1-GFP-v5-His3; gal4::trp1; gal80::leu2 pdr5::natMX.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gal4 , Gal80</div><div>suggested: RRID:BDSC_51280)</div></div><div style="margin-bottom:8px"><div>HO::Gal1-GFP-v5-His3; gal4::trp1; gal80::leu2 pdr5::natMX</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Construction of WT Ub-Mpro vector (p416LexA_UbMpro(WT)_B112): The Ubiqutin-Mpro gene fusion was constructed using overlapping PCR of the yeast ubiquitin gene and SARS-CoV-2 Mpro gene (Jin, Du et al. 2020) and was inserted into the pRS416 vector after digestion with SpeI and BamHI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pRS416</div><div>suggested: RRID:Addgene_158144)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(Addgene plasmid # 58434; http://n2t.net/addgene:58434 ; RRID:Addgene_58434))(Ottoz, Rudolf et al. 2014) and inserted between the SacI and SpeI sites upstream of the ubiquitin-Mpro gene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_58434)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Addgene plasmid # 58437; http://n2t.net/addgene:58437; RRID:Addgene_58437))(Ottoz, Rudolf et al. 2014) and inserted into the KpnI site.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_58437)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Barcode association: To associate barcodes with Mpro variants, we digested the p416-UbMpro(lib)-B112 plasmid upstream of the N18 sequence and downstream of the Mpro sequence with NotI and SalI enzymes (NEB).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416-UbMpro(lib)-B112</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The randomized 18 bp barcode sequence (N18) was cloned between the NotI and AscI sites upstream of the LexA promoter sequence in the p416LexA-Ub-Mpro(WT)-B112 vector with the goal of the WT sequence being represented by approximately 100 barcodes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416LexA-Ub-Mpro(WT)-B112</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting DBD-MproCS-AD fusion gene was inserted between the EcoRI and SacI sites downstream of the CUP promoter in the integrative bidirectional pDK-ATC plasmid (kindly provided by D. Kaganovich)(Amen and Kaganovich 2017).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDK-ATC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mCherry gene was subsequently cloned into the XhoI/BamHI sites downstream of the TEF promoter in the opposite orientation to create the plasmid pDK-CUP-DBD-MproCS-AD-TEF-mCherry.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDK-CUP-DBD-MproCS-AD-TEF-mCherry</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bulk Split transcription factor (TF) competition experiment: Barcoded WT UbMpro (p416LexA-UbMpro(WT)-N18) plasmid was mixed with the barcoded UbMpro library (p416LexA-UbMpro(lib)-N18) at a ratio of 20-fold WT to the average library variant.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416LexA-UbMpro(WT)-N18</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The CyPet gene was amplified by PCR from the pCyPet-His vector (pCyPet-His was a gift from Patrick Daugherty (Addgene plasmid # 14030 ; http://n2t.net/addgene:14030 ; RRID:Addgene_14030)) with a forward primer containing the BamHI site (BamHI_F) and a reverse primer containing the extending MproCS overhang sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_14030)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The YPet gene was amplified by PCR from the pYPet-His vector (pYPet-His was a gift from Patrick Daugherty (Addgene plasmid # 14031 ; http://n2t.net/addgene:14031 ; RRID:Addgene_14031)) with a forward primer containing the extending MproCS overhang sequence and a reverse primer containing the XhoI site (XhoI_R).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div></div><div>detected: RRID:Addgene_14031)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting CyPet-MproCS-YPet gene was inserted between the BamHI and XhoI sites downstream of the TEF promoter in the integrative bidirectional pDK-ATG plasmid (kindly provided by D. Kaganovich)(Amen and Kaganovich 2017).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDK-ATG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis of Mpro expression and Ubiquitin removal by Western Blot: To facilitate analysis of expression levels of Mpro and examine effective removal of Ubiquitin, a his tag was fused to the C-terminus of Mpro to create the plasmid p416LexA-UbMpro-his6-B112.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416LexA-UbMpro-his6-B112</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">W303 cells were transformed with the p416LexA-UbMpro(C145A)-his construct and the resulting yeast cells were grown to exponential phase in SD-ura media at 30°C. 125 nM β-estradiol was added when indicated and cells were grown for an additional eight hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>p416LexA-UbMpro(C145A)-his</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PacBio circular consensus sequences (CCS) were generated from the raw reads using SMRTLink v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SMRTLink</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Illumina sequence reads were filtered for Phred scores > 10 and strict matching of the sequence to the expected template and identifier sequence.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phred</div><div>suggested: (Phred, RRID:SCR_001017)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The figures were generated using Matplotlib (Hunter 2007), PyMOL and GraphPad Prism version 9.3.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Matplotlib</div><div>suggested: (MatPlotLib, RRID:SCR_008624)</div></div><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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: We detected the following sentences addressing limitations in the study:
  However, there are a couple of caveats that should be kept in mind when utilizing these data sets. For example, we do not fully understand how Mpro’s biochemical function relates to viral fitness. Having some Mpro function is essential to the virus, so mutations that destroy Mpro function will form non-functional viruses. Function-fitness relationships tend to be non-linear (Heinrich and Rapoport 1974, Kacser and Fell 1995, Jiang, Mishra et al. 2013) and it may be likely that Mpro function must be decreased by a large amount in order to cause measurable changes in viral replication efficiency. This relationship between Mpro function and SARS-CoV-2 fitness would need to be determined in order to translate our functional scores to fitness scores. Additionally, our TF and FRET screens quantify cleavage at one defined site (Nsp4/5) and it may be important to analyze all sites in order to fully understand the selection pressures acting on Mpro. Another important caveat is that our fitness landscape captures single amino acid changes and therefore does not provide information on the potential interdependence or epistasis between double and higher order mutations. Information regarding epistasis will be important for accurately predicting the impacts of multiple mutations on fitness. Despite these caveats, the similarity in fitness landscapes for the TF and FRET screens with the yeast growth screen suggests that all three capture fundamental and general aspects of Mpro selection. In...
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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URL

biorxiv.org/content/10.1101/2022.01.26.477860v2
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2022.01.26.477856

1
1. sciscore 30 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.26.477856: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After incubation for 1h at room temperature and washing, the ACE-2 mFc Tag was revealed using a peroxidase-conjugated anti-mouse secondary antibody (1:1000 dilution).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 clinical isolates (D614G variant; GenBank accession number MW322968) , Alpha (GenBank accession number MW633280) , Beta (GenBank accession number MW580244) , Gamma (Gene accession number pending), Delta (Gene accession number pending) and Omicron (Gene accession number pending) were isolated from SARS-CoV-2 RT-PCR confirmed patients by inoculating Vero cells with sputum sample or nasopharyngeal swabs in the biosafety level-3 (BSL-3) facility of the Pitié-Salpêtrière University Hospital.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Hundred μl of a Vero E6 cell suspension (3 × 105 cells/ml) were then added to the mixture and incubated at 37°C under an atmosphere containing 5% CO2 until microscopy examination on day 4 to assess cytopathogenic effect (CPE), as previously described (Vanhove et al</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IC50s were analyzed by nonlinear regression using a four-parameter dosage-response variable slope model with the GraphPad Prism 8.0.2 software (GraphPad Software, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr style="background-color:#FF0000"><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT0492830</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Trial number did not resolve on clinicaltrials.gov. Is the number correct?</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NA</td></tr></table>
  Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2022.01.26.477856
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https://biorxiv.org/cgi/content/10.1101/2022.01.24.477625

1
1. sciscore 29 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.24.477625: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Library Design: A library of antibodies directed against SARS-CoV-2 Spike (S) protein was developed using paired antibody sequences, meaning antibody sequences for which the heavy and light chain are both known, from the Coronavirus Antibody Database, CoV-AbDab57.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CoV-AbDab57</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CrossMAb, antibody, hFc-ACE2, and Lentiviral plasmids were maxi prepped in the same fashion.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>hFc-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two microliters of mouse anti-hexa His antibody (BioLegend) were added to the 10 mL sample and incubated for 1 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-hexa</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral transfections were done in HEK293T cells using calcium phosphate transfection reagent.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resultant 10mL was added to plated HEK cells from which the medium had been removed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization: The target cells used for infection in viral neutralization assays were from a HeLa cell line stably overexpressing the SARS-CoV-2 receptor, ACE2, as well as the protease known to process SARS-CoV-2, TMPRSS2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa</div><div>suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2/TMPRSS2/HeLa cells were plated one day prior to infection at 5,000 cells per well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2/TMPRSS2/HeLa</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Equimolar aliquots of each scFv plasmid were pooled and the resultant pool was amplified using primers which annealed to the hexa-his Tag (reverse primer) or signal peptide (forward primer) and had a 50bp overlap with the pPNL6 vector digested with NheI and BamHI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>scFv</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Yeast were resuspended in electroporation buffer (10 mM Tris Base, 250 mM sucrose, 2mM MgCl2) containing the gel extracted library amplification and digested pPNL6 vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pPNL6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Light chain (LC) and LC-ACE2 fusion proteins: Antibody sequences were cloned into the CMV/R plasmid backbone for expression under a CMV promoter.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CMV/R</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">hCoV spike proteins constructs: Spike proteins from six hCoVs were cloned into a pADD2 vector between the rBeta-globin intron and β-globin poly(A).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pADD2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lentivirus plasmids: Plasmids encoding the full-length spike proteins with native signal peptides were cloned into the background of the HDM-SARS2-Spike-delta21 plasmid (Addgene Plasmid #155130).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HDM-SARS2-Spike-delta21</div><div>suggested: RRID:Addgene_155130)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">They are: pHAGE-Luc2- IRS-ZsGreen (NR-52516), HDM-Hgpm2 (NR-52517), pRC-CMV-Rev1b (NR-52519), and HDM- tat1b (NR-52518).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHAGE-Luc2- IRS-ZsGreen</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The EtOH solution was then added to a geneJET plasmid miniprep column and the regular wash steps and elution steps were followed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>geneJET</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Digested FAbs were purified by SP/AKTA using 50 mM NaOAc, pH5 with gradient NaCl elution (using 50 mM NaOAc + 1M NaCl, pH5).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pH5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plasmids were added to D10 medium in the following ratios: 10 µg pHAGE-Luc2-IRS-ZsGreen, 3.4 µg FL Spike, 2.2 µg HDM-Hgpm2, 2.2 µg HDM-Tat1b, 2.2 µg pRC-CMV-Rev1b in a final volume of 1000 µL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pRC-CMV-Rev1b</div><div>suggested: RRID:Addgene_164443)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequence alignment was again produced using MUSCLE31 and sequence identity was calculated using Geneious</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Geneious</div><div>suggested: (Geneious, RRID:SCR_010519)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequences used were: 6VXX, UniRef90_U5NJG5, _L7UP8, _A0A7U3W1C7, _K9N5Q8, _A0A2I6PIW5, _A0A3Q8AKM0, _U5WHZ7, _A0A5H2WTJ3, _A0A0U1WJY8, _A0A166ZND9, _A0A678TRJ7, _A0A2R4KP93, _A0A2Z4EVK1, _A0A7R6WCE7, _E0ZN36, _A0A6M3G9R1, _F1DAZ9, _A0A0U1UYX4, _A0A2R3SUW7, _A0A2Z4EVN5, _A0A2Z4EVN2, _U5LMM7, _A0A5Q0TVR4, _E0XIZ3, _A0A023Y9K3, _A0A2R4KP86, _A0A088DJY6, _A0A7G6UAJ9, _S4X276, _A0A4Y6GL90, _A3EXG6, _F1BYL9, _E0ZN60, _A0A0K1Z054, _A0A0U1WHI2, and NCBI accession numbers: YP_009047204.1, QLR06867.1, AAK32191.1, AGZ48828.1, AAT84362.1, QHR63300.2, ABD75513.1, YP_003767.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NCBI</div><div>suggested: (NCBI, RRID:SCR_006472)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequences were first aligned using the MUSCLE algorithm, and then two phylogenetic trees were made, both using PhyML 3.3.20180621.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MUSCLE</div><div>suggested: (MUSCLE, RRID:SCR_011812)</div></div><div style="margin-bottom:8px"><div>PhyML</div><div>suggested: (PhyML, RRID:SCR_014629)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequences were then analyzed by sequence alignment using SnapGene software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SnapGene</div><div>suggested: (SnapGene, RRID:SCR_015052)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">FAb Production from IgGs: 1/10 volume of 1M Tris, pH 8 was added to IgGs at ∼2 mg/mL in PBS. 2 µL of a 1 mg/mL stock of Lys-C (stock stored at -70C) was added for each mg of human IgG1 and digested for 1 hour at 37 °C with moderate rotation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgGs</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Final data analysis was done in Prism.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
  </footer>
Visit annotations in context

Annotators

sciscore

URL

biorxiv.org/cgi/content/10.1101/2022.01.24.477625
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2022.01.24.477037

1
1. sciscore 29 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.24.477037: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The 293 cells were co-transfected with split GFP vectors, and fluorescence intensity was determined by 5 random areas with the same exposure, followed by ImageJ analysis.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies against GSK-3β (610202, BD Transduction Laboratories), pS9-GSK-3β (9323S, Cell Signaling), pY216-GSK-3β (612312, BD Transduction Laboratories), Flag (F-3165, Sigma), Snail (L70G2, Cell Signaling), β-catenin (610154, BD Transduction Laboratories), and Tubulin (LF-PA0146, AbFrontier) were obtained from the commercial vendors</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pS9-GSK-3β ( 9323S , Cell Signaling) , pY216-GSK-3β ( 612312</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Snail ( L70G2 , Cell Signaling) ,</div><div>suggested: (Cell Signaling Technology Cat# 3895, RRID:AB_2191759)</div></div><div style="margin-bottom:8px"><div>β-catenin</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Tubulin ( LF-PA0146 , AbFrontier )</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Phosphorylation status of N protein was determined by anti-flag antibody and mobility shift on a Phos-tag gel (Wako) as described previously (38).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-flag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were then washed three times with PBS containing 0.1% Tween 20 followed by incubation with anti-mouse-Alexa Fluor-488 (for green) or anti-rabbit-Alexa Fluor-594 (for red) secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse-Alexa</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit-Alexa</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 293 cells were co-transfected with split GFP vectors, and fluorescence intensity was determined by 5 random areas with the same exposure, followed by ImageJ analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293</div><div>suggested: NCI-DTP Cat# NCI-293TT, RRID:CVCL_1D85)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The expression vector pGBW-m4134490 (plasmid number 152580) having codon optimized N of SARS-CoV, pGBW-m4134909 (plasmid number 151901) having N of human coronavirus 229E,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGBW-m4134490</div><div>suggested: RRID:Addgene_152580)</div></div><div style="margin-bottom:8px"><div>pGBW-m4134909</div><div>suggested: RRID:Addgene_151901)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">pGBW-m4134899 (plasmid number 151902) having N of human coronavirus OC43 and pGBW-m4134901 (plasmid number 151922) having N of human coronavirus HKU1 229E were obtained from Addgene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGBW-m4134899</div><div>suggested: RRID:Addgene_151902)</div></div><div style="margin-bottom:8px"><div>pGBW-m4134901</div><div>suggested: RRID:Addgene_151922)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Those N expression vectors were subcloned into pcDNA3.1 with C-terminal flag or EGFP tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies against GSK-3β (610202, BD Transduction Laboratories), pS9-GSK-3β (9323S, Cell Signaling), pY216-GSK-3β (612312, BD Transduction Laboratories), Flag (F-3165, Sigma), Snail (L70G2, Cell Signaling), β-catenin (610154, BD Transduction Laboratories), and Tubulin (LF-PA0146, AbFrontier) were obtained from the commercial vendors</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pS9-GSK-3β</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">TCF/LEF and E-cadherin reporter assay: For TCF/LEF and E-cadherin reporter assay, the cells were transfected with 50 ng of the Super-Top or E-cad(−108)-Luc reporter vector (30,39) and 1 ng of pSV40-Renilla expression vector in combination with N or N-mutant as indicated.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>E-cad(−108)-Luc reporter</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pSV40-Renilla</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 293 cells were co-transfected with split GFP vectors, and fluorescence intensity was determined by 5 random areas with the same exposure, followed by ImageJ analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">: Amino acid sequences of N for SARS-CoV-2 (P0DTC9), SARS-CoV (P59595), HKU1 (Q0ZME3), HCoV-OC43 (P33469), HCoV-229E (PP15130), HCoV-NL63 (Q6Q1R8) were obtained from UniProt (https://www.uniprot.org/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>UniProt</div><div>suggested: (UniProtKB, RRID:SCR_004426)</div></div><div style="margin-bottom:8px"><div>https://www.uniprot.org/</div><div>suggested: (Universal Protein Resource, RRID:SCR_002380)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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  About SciScore
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https://biorxiv.org/cgi/content/10.1101/2022.01.21.477288

1
1. sciscore 29 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.21.477288: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The study protocol was approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00010350)</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two hours after infection, the cells were washed 5 times with DMEM and grown in DMEM supplemented with 10% FBS and 1% PenStrep along with an anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-VSV-G</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK-293T cells and HEK-293T cells stably expressing the human ACE2 receptor (HEK-ACE2)38 were grown in DMEM supplemented with 10% FBS and 1% PenStrep at 37°C and 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero cells stably expressing the human protease TMPRSS2 (Vero-TMPRSS2) were grown in DMEM supplemented with 10% FBS, 1% PenStrep, and 8 µg/mL puromycin at 37°C and 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant protein expression and purification: To produce the SARS-CoV-2 spike ectodomain containing the E406W mutation, 125 mL of Expi293 cells were grown to density of 2.5 × 106 cells per mL and transfected with 125 µg of DNA using PEI MAX diluted in Opti-MEM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization assays with vaccine-elicited sera and monoclonal antibodies: For neutralization assays using vaccine-elicited sera, HEK-ACE2 cells were seeded in 96-well poly-D-lysine coated plates at a density of 30,000 cells per well and grown overnight until they reached approximately 80% confluency.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralizations were conducted as described above with one modification: prior to the addition of the virus-antibody mixture, Vero-TMPRSS2 cells were washed 3 times with DMEM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The spike ectodomain was codon optimized, stabilized with the hexapro mutations39 and mutation of the furin cleavage site (682RRAR685 to 682GSAS685), and inserted into the pCDNA3.1 vector containing a C-terminal foldon followed by an avi tag and an octa-histidine tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The construct encoding the E406W RBD was generated by performing around-the-horn mutagenesis using a pCMVR vector encoding the wildtype SARS-CoV-2 RBD containing an N-terminal mu-phosphatase signal peptide and a C-terminal avi tag and octa-histidine tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMVR</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Reference-free 2D classification was performed using cryoSPARC to select for well-defined particle images42.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These selected particles were then used for 3D classification with 50 iterations (angular sampling 7.5° for 25 iterations followed by 1.8° with local search for 25 iterations) using Relion and a previously reported closed model for the SARS-CoV-2 spike ectodomain (PBD: 6VXX) as the initial model without imposing any symmetry.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Relion</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Luminescence readings from the neutralization assays were normalized and analyzed using GraphPad Prism 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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'Magic dirt': How the internet fueled, and defeated, the pandemic's weirdest company

1
1. tylercastro 28 Jan 2022
  
  in Public
  
  BOO, which “can be taken by anyone at any age, as well as animals,” according to the company, claims many benefits and uses, including improved brain function and heart health, and ridding the body of so-called toxins that include heavy metals, pesticides and parasites.
  
  At this point in the article the information that the audience has been met with the introduction that the black liquid being referenced can be used for faces and feet, overhyping jargon, and $110 price tag for what is essentially dirt.
  
  What kind of proof is available about not only the improved brain function, heart health, and toxin eliminator, but also that it can simply work for both all-ages humans and animals?
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Home · stared/tag-graph-map-of-stackexchange Wiki

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1. kael 27 Jan 2022
  
  in Public
  
  viz stackoverflow tagging
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Map7D2 and Map7D1 facilitate microtubule stabilization through distinct mechanisms to control cell motility and neurite outgrowth.

2
1. EMBOpress 27 Jan 2022
  
  in Review Commons
  
  Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
  
  Learn more at Review Commons
  
  Reply to the reviewers
  
  Manuscript number: RC-2021-01129
  
  Corresponding author(s): Koji Kikuchi
  
  Reviewer #1
  
  Evidence, reproducibility and clarity (Required):
  
  In this manuscript, Kikuchi et al describe the characterization of MAP7D2 and MAP7D1, two MAP7 family members in mouse with specific expression patterns. Focusing mostly on MAP7D2, they assess its expression pattern across the body and find that it is mostly expressed in certain neuronal subsets. They then characterize the MT-related properties of MAP7D2 based on previous knowledge of other MAP7 family members. They show that MAP7D2 binds MTs (via the N-terminus), determine the binding affinity, and show that it can stimulate MT polymerization (or stabilization) both in vitro and in vivo. Using a specific antibody, they localize MAP7D2 to centrosomes, midbody and neurites in N1-E115 cells. Functionally, they show that loss of MAP7D1/2 mildly affects microtubule stability as judged by acetyl-tubulin staining, and properties of these cells that rely on cytoskeletal elements such as cell migration and neurite growth. Interestingly, there might be a feedback loop regulating MAP7D1/2 expression, as knockdown of MAP7D1 upregulates MAP7D2.
  
  Overall, the experiments and conclusions are very solid and convincing, such that I would not ask for further experiments. This is in part because the experiments are largely based on previous characterizations of other MAP7 family members, which are largely confirmed. The presentation of the data is also very clear.
  
  Significance (Required):
  
  I see the value of the study in the fact that it provides solid and specific research tools for MAP7D1/2 which could be very useful for the microtubule/neuronal cytoskeleton community.
  
  Response: We thank the reviewer very much for appreciating the content of our manuscript.
  
  \*Referees cross-commenting***
  
  Reviewers 2 and 3 criticize that the evidence for an effect of MAP7D1/2 on MT dynamics is weak. I would agree in that ac-tub stainings and in vitro experiments are rather indirect. The experiments suggested by reviewer 2 should clarify this (esp. nocodazole should be easy). I also agree that an experiment addressing the potential involvement of kinesin-1 would help, the involvement of which seems to have been omitted by the authors. A kinesin-binding deficient mutant would add another MAP7D1/2 tool and increase the value for the community.
  
  Response: As for the reviewer’s suggestions listed above, please refer to our responses to the comments of Reviewer #2.
  
  Reviewer #2
  
  Evidence, reproducibility and clarity (Required):
  
  In this study, the authors investigate 2 members from the MAP7 family Map7D2 and Map7D1. They first address the tissue distribution of Map7D2, by northern blotting using a variety of rat tissues. To complement their analysis, they also raised an antibody to look at the protein distribution. From their studies, they concluded that Map7D2 is abundantly expressed in the brain and testis. The authors went on to perform a series of functional assays. First, they biochemically demonstrated that rat Map7D2 directly binds to MTs by MT co-sedimentation assay. The MT binding domain was mapped to the N-terminal half. They performed MT turbidity assay to demonstrate enhanced MT polymerisation in the presence of Map7D2, suggesting that this Map stabilises MTs. The authors went on to characterise in detail the subcellular localisation of Map7D2 which was predominantly present in the centrosome and partially localised to MTs including within neurites from N1-E115 cells. Kikuchi et al. further revealed the overlap in expression between Map7D2 and another family member, Map7D1. The authors continued these studies by a series of functional studies in N1-E115 cells where they performed single or combined knock-downs of Map7D2 and Map7D1 and studied the levels of acetylated and detyrosinated tubulins and the effect of the knock-downs on migration and neurite extension. The main conclusion from this work was that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms which are important in controlling cell motility and neurite outgrowth. Map7D2 is proposed to stabilise MTs by direct binding whereas Map7D1 does it indirectly by affecting acetylation.
  
  Major comments:
  
  The main conclusion from this work that Map7D2 and Map7D1 facilitate MT stabilization and that this is necessary for correct migration and neurite extension has not been convincingly demonstrated. In my opinion, a more detailed study of MT properties to demonstrate a role in MT stabilisation would greatly benefit the work, eg. experiments using MT destabilising agents such as nocodazole. In addition, a series of experiments aiming to study MT dynamics would help to understand the function of these MT regulators. The authors proposed an elevation in microtubule dynamics to explain the increase in migration and neurite extension but no experimental proof was provided.
  
  Response: According to the reviewer’s suggestion, we plan to assess the role of MT stabilization in greater detail by analyzing the sensitivity to the MT-destabilizing agent, nocodazole.
  
  To study MT dynamics, methods such as analyzing the velocity and direction of an EB1-GFP comet are commonly used. We have previously analyzed the roles of Map7 and Map7D1 in MT dynamics using HeLa cells stably expressing EB1-GFP (Kikuchi et al., EMBO Rep., 2018). However, no such tools have been developed for analyzing MT dynamics in N1-E115 cells, which were used in this study. In addition, it is difficult to analyze MT dynamics by transient expression of EB1-GFP because of the low plasmid transfection efficiency. Therefore, we instead plan to assess the effect on MT dynamics by measuring the EB1 comet length by immunofluorescence, referring to Fig. 7D in EMBO J. 32:1293–1306, 2013.
  
  Moreover, considering the possibility that the Map7D2 dynamics are altered when MT stability is changed, e.g., before and after differentiation induction, we analyzed the Map7D2 dynamics at the centrosome by fluorescence recovery after photobleaching (FRAP) using N1-E115 cells stably expressing EGFP-rMap7D2. We found that the dynamics were altered between the proliferative and differentiated states (see the figure below). Compared to the proliferative state, the recovery rate of EGFP-Map7D2 was reduced (lower left panel), and the immobile fraction of Map7D2 was increased in the differentiated state (lower right panel). As these data suggest that the increase in immobile Map7D2 may enhance MT stabilization, we will present them in a new figure in our manuscript along with the results of the above two experiments.
  
  It has been previously demonstrated that loss of MAP7D2 leads to a decrease in axonal cargo entry to axons resulting in defects in axon development and neuronal migration. The C-terminus is necessary for this function as it mediates interaction with Kinesin-1 (Pan et al., 2019). Such mechanisms could also explain the defects in migration and neurite growth that the authors observed. This possibility has not been considered but instead, the subtle changes in total α-tubulin led to suggest MT stabilisation as a key function without proof of causation. Could the authors provide some further experimental evidence to demonstrate that stability is the main contributor to the phenotypes observed? Eg. by rescuing migration and neurite phenotypes with a variant of MAP7D2 which cannot bind kinesin1.
  
  Response: The reviewer states “Such mechanisms could also explain the defects in migration and neurite growth that the authors observed;” however, our results showed that loss of Map7D2 elevated the rates of both cell motility and neurite outgrowth (original Fig. 5). In contrast, it has been reported in several papers that when Kinesin-1 function is impaired, both cell motility and neurite outgrowth are reduced (Curr. Biol., 23: 1018–1023, 2013; Mol. Cell. Biol., 39: e00109–19, 2019; etc.). Therefore, it is likely that the phenotypes we observed are independent of the functions associated with Kinesin-1 in N1-E115 cells. It is indeed possible that the experiment suggested by the reviewer may reveal relationships between Map7D2 and kinesin-1 in terms of cell motility and neurite outgrowth, however, it is difficult to conduct such an experiment because transient expression of Map7D2 induces MT bundling, as shown in original Fig. 2F. Based on the above, we plan to add a discussion of the relationship between Map7D2 and Kinesin-1.
  
  A key conclusion proposed by the authors is that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms. Such different roles in MT stabilisation are important in controlling cell motility and neurite outgrowth. In my opinion, their data does not fully support this statement and the findings using MT readouts do not match the defects in migration and neurite growth. Loss of Map7D2 leads to a very subtle phenotype on α-tubulin, while Map7D1 decreases both α-tubulin and acetylated tubulin, but Map7D1 seems to have a milder or similar effect on migration and neurite growth than Map7D2. Furthermore, it would be expected that the combined loss of function would lead to a stronger phenotype in cell migration when compared to the single loss of functions due to their distinct roles on MT stability, however, this seems not to be the case.
  
  Response: The fact that no stronger phenotype was observed may be because, besides Map7D2 and Map7D1, other molecules are involved in MT stabilization. Another possible explanation is that the increases in both cell motility and neurite outgrowth caused by decreased MT stabilization are offset by Kinesin-1 dysfunction. We plan to add a discussion of the above two possibilities.
  
  Minor comments:
  
  1) In the first result section, the author refers to Fig. S3 to suggest the expression of MAP7D2 in the cerebral cortex, however, there are no transcripts in the cerebral cortex according to the figure. Similarly, the immunofluorescence analysis done by the authors shows marginal expression of MAP7D2 in the cerebral cortex.
  
  Response: According to the reviewer’s comment, we have changed the order of the data shown in Fig. 1C, top panels. The data from the olfactory bulb, cerebellum, and hippocampus, in which Map7D2 expression was detected in the database, were arranged in the top three rows, and the data from the cerebral cortex, in which Map7D2 expression was not detected in the database, were moved to the bottom row as a negative control. In addition, we have revised the relevant part of the Results section as follows: “Based on RNA-seq CAGE, RNA-Seq, and SILAC database analysis (Expression Atlas, https://www.ebi.ac.uk/gxa/home/), Map7D2 expression was detected in the cerebellum, hippocampus, and olfactory bulb, and not in the cerebral cortex (Fig. S3). We further confirmed Map7D2 expression in the above four brain tissue regions of postnatal day 0 mice by immunofluorescence. Among these regions, Map7D2 was the most highly expressed in the Map2-negative area of the olfactory bulb, i.e., the glomerular layer (Fig. 1C). Weak signals were detected in the cerebellum, and marginal signals were observed in the hippocampus and cerebral cortex (Fig. 1C).” (page 5, lines 4–11)
  
  2) The authors use γ-Tubulin as a housekeeping gene in Fig. 3D, since Map7D2 is enriched in centrosomes this may not be the most appropriate choice.
  
  Response: γ-Tubulin is abundant in both the cytosol and the nuclear compartments of cells (Sig. Transduct. Target Ther. 3: 24, 2018). As it has been used for similar purposes in several other studies (Cancer Res., 61: 7713–7718, 2001; J. Biol. Chem., 291: 23112–23125, 2016; etc.), we considered it acceptable for use as a loading control for immunoblotting.
  
  3) According to the authors, knockdown of Map7D2 leads to a decrease in the intensity of α-tubulin and Map7D1 (Fig. 4C and D). This data doesn't agree with the previous statement made by the authors where they show that Map7D2 knockdown or knockout did not affect Map7D1 expression by Western Blot Analysis (Fig. S2C and S5B)
  
  Response: The immunoblotting results indicate that the total amount of Map7D1 in the cells is not affected by loss of Map7D2. In contrast, the immunofluorescence results indicate that the amount (distribution) of Map7D1 localized around the centrosome is decreased by loss of Map7D2, presumably due to a reduction in the number of MT structures that can serve as scaffolds for Map7D1. We plan to add this interpretation in the Results section.
  
  4) Line 6 page 7 "Endogenous Map7D2 expression is suppressed in N1-E115 cells stably expressing EGFP-rMap7D2 and was restored by specific knock-down of EGFP-rMap7D2 using gfp siRNA (Fig. 3D)". No quantifications and stats are shown. Also, endogenous Map7D2 after knock-down of EGFP-rMap7D2 is not comparable to the control.
  
  Response: According to the reviewer’s suggestion, we have quantified the amount of endogenous Map7D2 or EGFP-rMap7D2, normalized it to the amount of γ-tubulin, and calculated relative values to endogenous Map7D2 in the parental control. The amount of endogenous Map7D2 was decreased to 53% in N1-E115 cells stably expressing EGFP-rMap7D2, suggesting that EGFP-rMap7D2 expression suppressed endogenous Map7D2 expression. In this cell line, the total amount of Map7D2 (EGFP-rMap7D2 + endogenous Map7D2) was increased, however, when EGFP-rMap7D2 was depleted using sigfp in this cell line, endogenous Map7D2 was expressed to the same level as EGFP-rMap7D2 before knock-down. Together with the finding that Map7d1 knock-down increased the amount of Map7D2, these findings indicate that the amount of Map7D2 in the cells is regulated in response to the amount of Map7D1 and exogenous Map7D2. We have added this interpretation in the Results section. (page 7, lines 8–15)
  
  In addition, we have changed the legend of the original Fig. 3D to clarify the quantification method, as follows: “(D) Generation of N1-E115 cells stably expressing EGFP-rMap7D2. To check the expression level of EGFP-rMap7D2, lysates derived from the indicated cells were probed with anti-GFP (top panel) and anti-Map7D2 (middle panel) antibodies. The blot was reprobed for γ-tubulin as a loading control (bottom panel). The amount of endogenous Map7D2 or EGFP-rMap7D2 was normalized to the amount of γ-tubulin, and the value relative to endogenous Map7D2 in the parental control was calculated.” (page 22, lines 18–20)
  
  5) Line 8 page 7 "These results suggest that the expression of Map7D2 was influenced by changes in that of Map7D1" This statement seems in the wrong place, after the Map7D2 and EGFP-rMap7D2 experiment. Instead for clarity, it would be better placed after line 5 where the authors explain the effect of Map7D1 knock-down on the levels of Map7D2.
  
  Response: According to the reviewer’s suggestion, we have rephrased the relevant sentence as “Interestingly, Map7d1 knock-down upregulated Map7D2 expression, as confirmed with three different siRNAs (Fig. S2C), suggesting that Map7D2 expression is affected by changes in Map7D1 expression, not by off-target effects of a particular siRNA.” (page 7, lines 7, 8)
  
  6) Line 8 page 8 "Although the physiological role of the C-terminal region of Map7D2 is currently unknown..." This statement seems not adequate as there are several studies reporting the role of the C-terminal region of Map7D2 in Kinesin1- mediated transport. The authors mention such studies in the discussion.
  
  Response: According to the reviewer’s suggestion, we plan to add a discussion of the relationship between Map7D2 and kinesin-1.
  
  7) Line 6 page 9 " Further, the knock-down of either resulted in a comparable reduction of MT intensity (Fig. 4C and D) ..." This is not visible and/or justified by the images provided and would benefit from some sort of quantification at other regions such as neurites.
  
  Response: Considering the cell motility, quantification of α-tubulin/Ace-tubulin/Map7D1/Map7D2 intensities in neurites is not appropriate. Instead, we have added arrowheads indicating α-tubulin/Ace-tubulin/Map7D1/Map7D2 in Fig. 4C, for better understanding.
  
  8) In Fig. 2B, a band corresponding to his6-rMAP7D2 of molecular weight >97 kDa co-sedimented with the microtubules. However, the cloned rMAP7D2 had a molecular weight of 84.82 kDa and the addition of 6XHis-Tag would add another 2-3 kDa, therefore, the final protein band observed should be less than 90 kDa. It would be beneficial if the authors could specify the molecular weight of the purified protein after the addition of the V5-his tag and/or if there was addition of amino acids due to cloning strategy.
  
  Response: In Fig. 2B, we used full-length GST-tagged rMap7D2, like in Fig. 2E and D; therefore, we have corrected His6-rMap7D2 as GST-rMap7D2. We apologize for the mistake.
  
  9) In Fig. 2C, there is misalignment of the western blot with the panel or text underneath.
  
  Response: We thank the reviewer for pointing this out; we have corrected the misalignment of the CBB staining in Fig. 2C.
  
  10) In Fig. 3C the inset from the first panel seems to correspond to a different focal plane than the main image.
  
  Response: We have revised the relevant part of the figure legend as follows: “In C, images of differentiated cells were captured by z-sectioning, because the focal planes of the centrosome and neurites are different. Each inset shows an enlarged image of the region indicated with a white box at each focal plane. Arrowheads indicate the centrosomal localization of Map7D2.”
  
  11) In Fig. 4A, the cell type is not specified and is referred as "indicated cells", also the material and methods section seems to omit the specific cells used.
  
  Response: We have added “in N1-E115 cells treated with each siRNA” in the legend of Fig. 4A.
  
  12) Fig. S6 is not mentioned in the results.
  
  Response: We apologize for having referred to Fig. S6 only in the Discussion section in the original manuscript. We plan to describe the findings shown in the original Fig. S6 to the Results section and renumber the figures accordingly.
  
  Significance (Required):
  
  MTs play essential roles in practically every cellular process. Their precise regulation is therefore crucial for cellular function and viability. MAPs are specialised proteins that interact with MTs and regulate their behaviour in different manners. Understanding their precise function in different cellular contexts is of utmost importance for many biological and biomedical fields.
  
  MAPs are well known for their ability to promote MT polymerization, bundling and stabilisation in vitro (Bodakuntla et al., 2019). Several members of the Map7 family have been shown to regulate microtubule stability. For instance, MAP7 can prevent nocodazole-induced MT depolymerization and maintain stable microtubules at branch points in DRG neurons (Tymanskyj & Ma, 2019). Ensconsin, the Drosophila Map, is required for MT growth in mitotic neuroblasts by regulating the mean rate of MT polymerization (Gallaud et al., 2014). However, this family of Maps seems to have diverse functions encompassing a variety of mechanisms, as exemplified by a series of studies demonstrating the involvement of MAP7 family proteins in the recruitment and activation of kinesin1 (Hooikaas et al., 2019; Pan et al., 2019) and in microtubule remodelling and Wnt5a signalling (Kikuchi et al., 2018). Further understanding of this family of Maps and how its members differ in their function is important and will help to advance the field.
  
  Response: We appreciate the reviewer’s comments. We believe that our revision plan will greatly improve the quality of our manuscript.
  
  Reviewer #3
  
  Evidence, reproducibility and clarity (Required):
  
  Summary:
  
  Microtubule Associated Proteins (MAPs) are important regulators of microtubule dynamics, microtubule organization and vesicular transport by modulating motor protein recruitment and processivity. In the current manuscript the authors have characterized 2 members of the MAP7 protein family, MAP7D1 and MAP7D2. The authors characterized MAP7D2 expression pattern in the brain and its microtubule binding properties in vitro and in cells. In cells both proteins localize to the centrosome and to microtubules and upon depletion centrosome localized microtubules seem reduced, and cell migration and neurite outgrowth are increased. Surprisingly, they find that microtube acetylation (a common marker for stable microtubules) is reduced upon MAP7D1 depletion but not MAP7D2 depletion. Based on this finding the authors conclude that these proteins have a distinct mechanism in stabilizing MTs to affect cell migration and neurite outgrowth; MAP7D2 stabilizes by binding to MTs, whereas MAP7D1 stabilizes MTs by acetylation.
  
  Main comments:
  
  - Both MAP7 proteins show strong localization to the centrosome and to a lesser degree to MTs. Knockdown of either protein leads to reduced MTs around the centrosome, which lead the authors to conclude the MAP7s are stabilizing the MTs. However, the effect could just as well be an indirect effect due to a function of these MAPs at the centrosome. To address this authors could e.g. quantify microtubule properties in postmitotic cells. In addition, antibody specificity should be tested using knockdown of knockout cells, as this centrosome localization was not observed in Hela cells (Hooikaas, 2019; Kikuchi, 2018). Maybe this localization is specific to rat MAP7s or to the cell line used.
  
  Response: We think that this comment partly overlaps with the comments raised by Reviewer #2. We plan to assess the role of MT stabilization in greater detail by analyzing the sensitivity to the MT-destabilizing agent, nocodazole, and the effect on MT dynamics by measuring the EB1 comet length by immunofluorescence.
  
  Regarding the reviewer’s concern about antibody specificity, we had carefully confirmed the antibody specificity, as shown in Fig. S2 of the original manuscript. Subsequently, Map7D2 localization was confirmed in N1-E115 cells stably expressing EGFP-rMap7D2, as shown in Fig. 3D, E of the original manuscript. In addition, we are currently conducting analyses using Map7d1-egfp knock-in mice, which confirmed that Map7D1 localizes around the centrosome in cortical neurons, as shown below (we would like to disclose these unpublished data to the reviewers only). Therefore, it is thought that the localization pattern of Map7D2 and Map7D1 differs depending on the cell type and cell line. We plan to add this interpretation to the Results section.
  
  - Centrosome nucleated microtubules are typically highly dynamic and little modified. Therefore is the Ac-tub staining at the centrosome really MTs? I cannot identify MTs in the fluorescent images in 4C. Maybe authors could consider ac-tub/alpha-tub ratio in non centrosomal region (e.g. neurites). Moreover, as both Acetylation and detyrosination are associated with long-lived/stable MTs, it is surprising that only acetylated tubulin goes down on WB. Does this suggest that long-lived MTs are still present to normal level? If so, can one still argue that the loss of acetylation is the cause of the lower MT levels? This should at least be discussed.
  
  Response: As for the reviewer’s statement “Centrosome nucleated microtubules are typically highly dynamic and little modified. Therefore is the Ac-tub staining at the centrosome really MTs?”, it has been previously reported that tubulin acetylation is observed around the centrosome in some cell lines (J. Neurosci., 30: 7215–7226, 2010; PLoS One, 13: e0190717, 2018; etc.). N1-E115 is one of the cell lines in which tubulin acetylation is observed around the centrosome.
  
  It is not surprising that “only acetylated tubulin goes down on WB,” as it has been previously reported that acetylated and detyrosinated tubulins are sometimes not synchronous (J. Neurosci., 23: 10662–10671, 2003; J. Neurosci., 30: 7215–7226, 2010; J. Cell Sci., 132: jcs225805, 2019., etc.). For instance, Montagnac et al. (Nature, 502: 567–570, 2013) showed that defects in the α-tubulin acetyltransferase αTAT1-clathrin-dependent endocytosis axis reduce only tubulin acetylation, resulting in a shift from directional to random cell migration. Although the details of the molecular function of Map7D1 are beyond the main purpose of this study, we plan to add a discussion of the reduced tubulin acetylation by Map7d1 knock-down based on the above.
  
  - MAP7D1 and MAP7D2 depletion leads to subtle defect in cell migration and neurite outgrowth, which the author suggest is caused by reduced MT stability. However, MAP7 proteins have well characterized functions in kinesin-1 transport, and thus the phenotypes may well be caused by defects in kinesin-1 transport. Ideally the authors would do rescue experiments with FL or just the MT binding N-termini to separate these functions. Moreover this is needed to substantiate the claim of the authors that MAP7D1 effect on MT stability is not mediated by direct binding.
  
  Response: As this comment largely overlaps with the comments raised by Reviewer #2, please refer to our responses to the comments of Reviewer #2.
  
  - The authors do not refer well to published work. Several papers have published very similar work (especially to Fig1+2) and it would help the reader much if this would be discussed/compared along the results section and not briefly mention these in the results section. In addition, authors overstate the novelty of their results e.g. page 3: these proteins are not "functionally uncharacterized" nor are their expression patter and biochemical properties analyzed for the first time in this manuscript; page 8 "Although the physiological role of the C-terminal region of Map7D2 is currently unknow, ..." There is a clear function for the C-terminus for the recruitment/activation of kinesin-1.
  
  Response: According to the reviewer’s suggestion, we plan to add a comparison with data on the Map7 family members presented in previous papers in the Results section and rephrase the relevant part regarding the physiological role of the C-terminal region of Map7D2.
  
  Minor comments
  
  - P6 Map7D3 also binds with its N-terminus to MTs, like other MAP7s (Yadav et al)
  
  Response: According to the reviewer’s comment, we have revised this as “Map7D3 binds through a conserved region on not only the N-terminal side, but also the C-terminal side (Sun, 2011; Yadav et al., 2014).” (page 6, lines 4, 5)
  
  - P7 "As Map7D2 has the potential to functionally compensate for Map7D1 loss" where is this based on?
  
  Response: For clarity, we have rephrased this as “As Ma7D2 expression was upregulated upon suppression of Map7D1 expression, Map7D2 has the potential to functionally compensate for Map7D1 loss.” (page 7, line 17, 18)
  
  - Fig2F quality of black-white images is low potentially due to conversion issues
  
  Response: We thank the reviewer for pointing out these conversion issues, and we have made the necessary corrections.
  
  Significance (Required):
  
  At this stage the conceptual advance is limited. Part of the findings are not novel. The finding that MAP7s depletion have a different effect on MTs acetylation may be interesting to cytoskeleton researchers, although the potential mechanism has not been addressed experimentally or textually.
  
  However, their conclusion that this leads to reduced MTs and then to cellar migration and neurite formation defects is not sufficiently supported by experimental evidence.
  
  Response: We appreciate the reviewer’s comments. We believe that our revision plan will greatly improve the quality of our manuscript.
  
  \*Referees cross-commenting***
  
  I completely agree with reviewer #2: At this stage the paper's conclusions are not sufficiently supported by the data. Important will be to further characterize the effect om the MTs (do they really have a different effect) and to look at the possible involvement of the motor recruitment. Maybe that a 3 to 6 months revision time would have been more accurate.
  
  Response: Please refer to our responses to the comments of Reviewer #2.
  
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  Referee #2
  
  Evidence, reproducibility and clarity
  
  In this study, the authors investigate 2 members from the MAP7 family Map7D2 and Map7D1. They first address the tissue distribution of Map7D2, by northern blotting using a variety of rat tissues. To complement their analysis, they also raised an antibody to look at the protein distribution. From their studies, they concluded that Map7D2 is abundantly expressed in the brain and testis. The authors went on to perform a series of functional assays . First, they biochemically demonstrated that rat Map7D2 directly binds to MTs by MT co-sedimentation assay. The MT binding domain was mapped to the N-terminal half. They performed MT turbidity assay to demonstrate enhanced MT polymerisation in the presence of Map7D2, suggesting that this Map stabilises MTs. The authors went on to characterise in detail the subcellular localisation of Map7D2 which was predominantly present in the centrosome and partially localised to MTs including within neurites from N1-E115 cells. Kikuchi et al. further revealed the overlap in expression between Map7D2 and another family member, Map7D1. The authors continued these studies by a series of functional studies in N1-E115 cells where they performed single or combined knock-downs of Map7D2 and Map7D1 and studied the levels of acetylated and detyrosinated tubulins and the effect of the knock-downs on migration and neurite extension. The main conclusion from this work was that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms which are important in controlling cell motility and neurite outgrowth. Map7D2 is proposed to stabilise MTs by direct binding whereas Map7D1 does it indirectly by affecting acetylation.
  
  Major comments:
  
  The main conclusion from this work that Map7D2 and Map7D1 facilitate MT stabilization and that this is necessary for correct migration and neurite extension has not been convincingly demonstrated. In my opinion, a more detailed study of MT properties to demonstrate a role in MT stabilisation would greatly benefit the work, eg. experiments using MT destabilising agents such as nocodazole. In addition, a series of experiments aiming to study MT dynamics would help to understand the function of these MT regulators. The authors proposed an elevation in microtubule dynamics to explain the increase in migration and neurite extension but no experimental proof was provided.
  
  It has been previously demonstrated that loss of MAP7D2 leads to a decrease in axonal cargo entry to axons resulting in defects in axon development and neuronal migration. The C-terminus is necessary for this function as it mediates interaction with Kinesin-1 (Pan et al., 2019). Such mechanisms could also explain the defects in migration and neurite growth that the authors observed. This possibility has not been considered but instead, the subtle changes in total -tubulin led to suggest MT stabilisation as a key function without proof of causation. Could the authors provide some further experimental evidence to demonstrate that stability is the main contributor to the phenotypes observed? Eg. by rescuing migration and neurite phenotypes with a variant of MAP7D2 which cannot bind kinesin1.
  
  A key conclusion proposed by the authors is that Map7D2 and Map7D1 facilitate MT stabilization through distinct mechanisms. Such different roles in MT stabilisation are important in controlling cell motility and neurite outgrowth. In my opinion, their data does not fully support this statement and the findings using MT readouts do not match the defects in migration and neurite growth. Loss of Map7D2 leads to a very subtle phenotype on -tubulin, while Map7D1 decreases both -tubulin and acetylated tubulin, but Map7D1 seems to have a milder or similar effect on migration and neurite growth than Map7D2. Furthermore, it would be expected that the combined loss of function would lead to a stronger phenotype in cell migration when compared to the single loss of functions due to their distinct roles on MT stability, however, this seems not to be the case.
  
  Minor comments:
  
  In the first result section, the author refers to Fig. S3 to suggest the expression of MAP7D2 in the cerebral cortex, however, there are no transcripts in the cerebral cortex according to the figure. Similarly, the immunofluorescence analysis done by the authors shows marginal expression of MAP7D2 in the cerebral cortex.
  
  The authors use -Tubulin as a housekeeping gene in Fig. 3D, since Map7D2 is enriched in centrosomes this may not be the most appropriate choice.
  
  According to the authors, knockdown of Map7D2 leads to a decrease in the intensity of -tubulin and Map7D1 (Fig. 4C and D). This data doesn't agree with the previous statement made by the authors where they show that Map7D2 knockdown or knockout did not affect Map7D1 expression by Western Blot Analysis (Fig. S2C and S5B)
  
  Line 6 page 7 "Endogenous Map7D2 expression is suppressed in N1-E115 cells stably expressing EGFP-rMap7D2 and was restored by specific knock-down of EGFP-rMap7D2 using gfp siRNA (Fig. 3D)". No quantifications and stats are shown. Also, endogenous Map7D2 after knock-down of EGFP-rMap7D2 is not comparable to the control.
  
  Line 8 page 7 "These results suggest that the expression of Map7D2 was influenced by changes in that of Map7D1" This statement seems in the wrong place, after the Map7D2 and EGFP-rMap7D2 experiment. Instead for clarity, it would be better placed after line 5 where the authors explain the effect of Map7D1 knock-down on the levels of Map7D2.
  
  Line 8 page 8 "Although the physiological role of the C-terminal region of Map7D2 is currently unknown..." This statement seems not adequate as there are several studies reporting the role of the C-terminal region of Map7D2 in Kinesin1- mediated transport. The authors mention such studies in the discussion.
  
  Line 6 page 9 " Further, the knock-down of either resulted in a comparable reduction of MT intensity (Fig. 4C and D) ..." This is not visible and/or justified by the images provided and would benefit from some sort of quantification at other regions such as neurites.
  
  In Fig. 2B, a band corresponding to his6-rMAP7D2 of molecular weight >97 kDa co-sedimented with the microtubules. However, the cloned rMAP7D2 had a molecular weight of 84.82 kDa and the addition of 6XHis-Tag would add another 2-3 kDa, therefore, the final protein band observed should be less than 90 kDa. It would be beneficial if the authors could specify the molecular weight of the purified protein after the addition of the V5-his tag and/or if there was addition of amino acids due to cloning strategy.
  
  In Fig. 2C, there is misalignment of the western blot with the panel or text underneath.
  
  In Fig. 3C the inset from the first panel seems to correspond to a different focal plane than the main image.
  
  In Fig. 4A, the cell type is not specified and is referred as "indicated cells", also the material and methods section seems to omit the specific cells used.
  
  Fig. S6 is not mentioned in the results.
  
  Significance
  
  MTs play essential roles in practically every cellular process. Their precise regulation is therefore crucial for cellular function and viability. MAPs are specialised proteins that interact with MTs and regulate their behaviour in different manners. Understanding their precise function in different cellular contexts is of utmost importance for many biological and biomedical fields.
  
  MAPs are well known for their ability to promote MT polymerization, bundling and stabilisation in vitro (Bodakuntla et al., 2019). Several members of the Map7 family have been shown to regulate microtubule stability. For instance, MAP7 can prevent nocodazole-induced MT depolymerization and maintain stable microtubules at branch points in DRG neurons (Tymanskyj & Ma, 2019). Ensconsin, the Drosophila Map, is required for MT growth in mitotic neuroblasts by regulating the mean rate of MT polymerization (Gallaud et al., 2014). However, this family of Maps seems to have diverse functions encompassing a variety of mechanisms, as exemplified by a series of studies demonstrating the involvement of MAP7 family proteins in the recruitment and activation of kinesin1 (Hooikaas et al., 2019; Pan et al., 2019) and in microtubule remodelling and Wnt5a signalling (Kikuchi et al., 2018). Further understanding of this family of Maps and how its members differ in their function is important and will help to advance the field.
  
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L'ontologie NiceTag : les tags en tant que graphes nommés

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1. kael 26 Jan 2022
  
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  Abstract : Current tag modelling does not fully take into account the rich and diverse nature tags, as signs, can take on. We propose an ontology of tags in which tags are modelled as named graphs. These named graphs are made of a resource linked to a “sign” which can be any resource reachable on the Web (an ontology concept, an image, etc.). The purpose of our model is to be able to describe tags in a very general manner, and as an immediate conse- quence, to describe tags as modelled by other tag models (SCOT, CommonTag, etc.).
  
  nicetag rdf tagging
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Nice Tag Ontology : modeling tags as RDF named graphs

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Eco1-dependent cohesin acetylation anchors chromatin loops and cohesion to define functional meiotic chromosome domains

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1. Public_Reviews 26 Jan 2022
  
  in eLife
  
  Author Response:
  
  Reviewer #2 (Public Review):
  
  This manuscript by Barton and colleagues explores the roles of the conserved Eco1 transacetylase in modulating cohesin function in meiosis in budding yeast. Numerous studies in mitotically dividing cells have shown that the Eco1 family of transacetylases acetylate the Smc3 subunit of cohesin and that this acetylation renders cohesin on chromosomes resistant to removal by the Wapl (Wpl1 in budding yeast) family of proteins. Cohesins play critical roles in both sister chromatid cohesion and chromatin organization (through the formation of intrachromosomal loops). How cohesins are regulated by Eco1 in meiosis to accommodate meiotic chromosome structures such as the synaptonemal complex, chromatin domains around centromeres, repair of programmed meiotic double strand DNA breaks in prophase, and sequential removal of cohesins - first at arms in meiosis I and centromeres at meiosis II - is largely unexplored. Thus, this manuscript is exploring important new areas.
  
  The authors show that Eco1 persists thru prophase I (longer than it does in vegetative cell cycles), that it is not necessary for cohesin loading at centromeres but is needed to counteract Wpl1 to protect centromeric cohesion, that it is critical for the establishment of chromatin loops on meiotic chromosome arms and that it is critical for protection of the arm cohesin from removal by Wpl1. The authors also provide evidence that, in meiosis, Wpl1 exhibits underappreciated functions in cohesin loading or cohesion establishment in addition to its recognized role in cohesin removal.
  
  The experiments demonstrate that Eco1 is necessary for sharp cohesin boundaries that flank the centromeres and suggest this might be a replication-independent function of Eco1 (the boundaries form in clb5, clb6 cells with no DNA replication phase) but it is unclear if the detectable, but diminished, boundaries in clb5,clb6 cells were formed in the replication-free meiosis or presist from the S-phase associated loading and cohesion establishment from the preceding mitotic cycle.
  
  Entry into meiosis occurs from G1 when there is no cohesin on the chromosomes and boundaries are not present, therefore this would only be a concern if there were persistent mitotic cells in G2 (i.e. after DNA replication). Our flow cytometry shows that the cells used in the experiment were unreplicated, so even if mitotic cells were present, they would not have been through S phase.
  
  Nevertheless, we addressed this point by analysis of pre-S phase meiotic cells (ime1/ime4 block) and by anchoring away Eco1 in unreplicated cells.
  
  Immunofluorescence imaging assays are used to observe the behavior of sister chromatids in meiosis I and meiosis II as a function of Eco1 activity. In wild-type cells sister chromatids co-orient in meiosis I and move to the same pole of the spindle. In mammalian cells and fission yeast this co-orientation requires cohesin while studies in budding yeast have suggested the co-orientation is cohesin-independent. Here, the authors show that when Eco1 is depleted, the sisters often move to opposite poles at meiosis I, and suggest that cohesin (and Eco1) is indeed required for sister co-orientation. An alternate possibility is that the sisters have lost their association in meiotic prophase (due to cohesin failures) before attaching to microtubles and segregating randomly - often to opposite poles.
  
  We agree with this point, but would argue that the “alternative possibility” (which our data support) still leads to the conclusion that cohesin and Eco1 are required for sister co-orientation. A prior study (Monje-Casas et al., 2007) had suggested that monopolin could link sister kinetochores even without cohesin. We now show that this is not the case, which we believe to be an important conclusion.
  
  Our results indicate that establishment of monoorientation requires the cohesin that is localized at centromeres. WPL1 deletion in eco1-aa rescues centromeric cohesion (Figure 2F, Figure 8E), but not chromosome arm cohesion (Figure 2H) or sister chromatid segregation in meiosis II (Figure 8F), indicating that pericentromeric cohesion must still be defective.
  
  For clarity, please note that the relevant data is not immunofluorescence, but live cell imaging (now shown in Figure 8) so these conclusions are based on observation of single chromosomes in individual live cells from prophase I until anaphase II.
  
  In summary the authors show that Eco1 has distinct roles on chromosome arms and centromeres and probably in both replication-linked and replication-independent events, acts to modulate cohesin location and function in meiosis.
  
  Reviewer #3 (Public Review):
  
  This paper investigates the meiotic roles of two regulators of cohesin, the cohesin destabilizer Wpl1 and the cohesin acetyltransferase Eco1. The authors provide evidence that Eco1 antagonizes Wpl1 to allow stabilization of centromeric cohesin, which is important to establish meiotic chromosome segregation patterns. In addition, Eco1 regulates the stable anchoring of cohesin at boundaries to promote defined chromosome loop formation in meiotic prophase.
  
  The study uses a combination of calibrated ChIP-seq analysis, and chromosome conformation capture techniques to convincingly show that loop formation is altered in wpl1 depletion and eco1 depletion mutants. Well-established cytological techniques are used to demonstrate different effects on chromosome cohesion along arms and at centromeres, and to show that Eco1 is important for establishing the meiotic segregation pattern. The paper is well written and the data largely support the conclusions. As such, this paper is expected to be of substantial interest to the field.
  
  One notable weakness is the poor definition of the eco1 anchor-away allele (eco1-aa), on which much of the eco1 phenotypic analysis is based. The presented data indicate that addition of the FRB-GFP tag alone causes most of the phenotypes, regardless of nuclear depletion. It is well possible that the tag creates a meiosis-specific loss-of-function allele, although it is surprising that the tag does not have mitotic defects even though Eco1 presumably has the same substrate (the cohesin subunit Smc3) in both situations. Encouragingly, some of the phenotypes could be confirmed using a non-acetylatable smc3 mutant. However, the tag may also create neomorphic effects that may contribute to the Wpl1-independent effects and the apparent stronger defects of the eco1-aa allele compared to the non-acetylatable smc3 mutant.
  
  Available evidence suggests that eco1-aa is a loss of function allele.
  
  scietyType:AuthorResponse
2. Public_Reviews 26 Jan 2022
  
  in eLife
  
  Reviewer #3 (Public Review):
  
  This paper investigates the meiotic roles of two regulators of cohesin, the cohesin destabilizer Wpl1 and the cohesin acetyltransferase Eco1. The authors provide evidence that Eco1 antagonizes Wpl1 to allow stabilization of centromeric cohesin, which is important to establish meiotic chromosome segregation patterns. In addition, Eco1 regulates the stable anchoring of cohesin at boundaries to promote defined chromosome loop formation in meiotic prophase.
  
  The study uses a combination of calibrated ChIP-seq analysis, and chromosome conformation capture techniques to convincingly show that loop formation is altered in wpl1 depletion and eco1 depletion mutants. Well-established cytological techniques are used to demonstrate different effects on chromosome cohesion along arms and at centromeres, and to show that Eco1 is important for establishing the meiotic segregation pattern. The paper is well written and the data largely support the conclusions. As such, this paper is expected to be of substantial interest to the field.
  
  One notable weakness is the poor definition of the eco1 anchor-away allele (eco1-aa), on which much of the eco1 phenotypic analysis is based. The presented data indicate that addition of the FRB-GFP tag alone causes most of the phenotypes, regardless of nuclear depletion. It is well possible that the tag creates a meiosis-specific loss-of-function allele, although it is surprising that the tag does not have mitotic defects even though Eco1 presumably has the same substrate (the cohesin subunit Smc3) in both situations. Encouragingly, some of the phenotypes could be confirmed using a non-acetylatable smc3 mutant. However, the tag may also create neomorphic effects that may contribute to the Wpl1-independent effects and the apparent stronger defects of the eco1-aa allele compared to the non-acetylatable smc3 mutant.
  
  peerReview
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https://biorxiv.org/cgi/content/10.1101/2022.01.20.477107

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1. sciscore 26 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.20.477107: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: EXPERIMENTAL MODEL AND SUBJECT DETAILS: Blood, PBMCs and HLA typing: Whole blood from COVID-19 convalescent donors, healthy donors, or vaccine recipients was collected under protocol approved by the UMass Chan Medical School Institutional Review Board of the University of Massachusetts and informed consent was obtained from all subjects.<br>Consent: EXPERIMENTAL MODEL AND SUBJECT DETAILS: Blood, PBMCs and HLA typing: Whole blood from COVID-19 convalescent donors, healthy donors, or vaccine recipients was collected under protocol approved by the UMass Chan Medical School Institutional Review Board of the University of Massachusetts and informed consent was obtained from all subjects.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For blocking of antigen-stimulation assays, in-house produced antibodies to HLA-DR (LB3.1), HLA-DQ (SPVL-3), HLA-DP (B7/21), or HLA-ABC (w6/32), were added at a final concentration of 10 μg/mL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HLA-DR</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HLA-DP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HLA-ABC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were collected, washed, and stained using a standard protocol which included: staining for dead cells with Live/Dead Fixable Aqua Dead Cell Stain Kit™ (Life Technologies, Thermo Fisher Scientific, Waltham, MA); blocking of Fc receptors with human Ig (Sigma-Aldrich, St. Louis, MO); staining with the mix of DP4-PE and APC tetramers (final concentration of 2-4 μg/mL each) at 37°C for 2 hours; surface staining antibodies CD3-APC-H7, CD4-PerCP-Cy5.5, CD8-APC-R700, CD14-BV510, CD19-BV510, CD56-BV510 were added for the last 20 minutes of incubation, followed by washes and resuspension in buffer for data acquisition.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD19-BV510</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD56-BV510</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Partially-match HLA cell lines: EBV-transformed LG2 cell line (10984, IPD-IMGT/HLA), 9068 cell line (BM9, IHWG), and mouse DP4-transfected MN605 cell line (M12C3-DPA1*0103/DPB1*0401; (Williams et al., 2018); kindly provided by Dr. S. Kent, UMMS), were maintained in RPMI 1640 medium supplemented with L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and 10% FBS at 37°C/5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MN605</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 6 hours incubation, cells were collected, washed, and stained using a standard protocol, which included: staining for dead cells with Live/Dead Fixable Aqua Dead Cell Stain Kit™ (Life Technologies, Thermo Fisher Scientific, Waltham, MA); blocking of Fc receptors with human Ig (Sigma-Aldrich, St. Louis, MO); surface staining with mouse anti-human CD3-APC-H7 (SK7), CD4-PerCPCy5.5 (RPA-T4), CD8-APC-R700 (RPA-T8), CD14-BV510 (MϕP9), CD19-BV510 (SJ25C1), CD56-BV510 (NCAM16.2); fixation and permeabilization using BD Cytofix/Cytoperm™; and intracellular staining with mouse anti-human IFN-γ-V450 (B27), TNF-α-PE-Cy7 (MAb11), IL-2-BV650 (5344.111), (all from BD Biosciences, San Jose, CA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD Cytofix/Cytoperm™</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were acquired using a BD LRSII flow cytometer equipped with BD FACSDiva software (BD Biosciences, San Jose, CA) and analyzed using FlowJo v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD FACSDiva</div><div>suggested: (BD FACSDiva Software, RRID:SCR_001456)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Polyfunctional analysis was performed in FlowJo, defining Boolean combinatorial gates for all the markers in the CD3+/CD4+/CD8-population.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These results were visualized in SPICE software v6.0 (Roederer et al., 2011). t-SNE analysis was done in concatenated samples (control, SARS-CoV-2, peptide 163 and peptide 164) from 3 donors using the available plugin in FlowJo.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPICE</div><div>suggested: (SPICE, RRID:SCR_016603)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All primers sequences shown in STAR Methods.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>STAR</div><div>suggested: (STAR, RRID:SCR_004463)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These files were processed using MIGEC v1.2.9 pipeline: Checkout-batch for de-multiplexing and UMI tag extraction, Histogram for MIG (molecular identifier groups) statistics, and Assemble-batch to perform UMI-guided assembly (Shugay et al., 2014); followed by MiXCR v3.0.13: analyze amplicon pipeline, to align, assemble and export clonotypes (Bolotin et al., 2015).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MIGEC</div><div>suggested: (migec, RRID:SCR_016337)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HLA-peptide binding prediction was performed with NetMHCIIpan v4.0 server (Reynisson et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NetMHCIIpan</div><div>suggested: None</div></div></td></tr></table>
  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: We detected the following sentences addressing limitations in the study:
  There are several limitations of our study. We mapped the specificity of the cross-reactive response by following IFN-γ-secreting cells, but non-IFN-γ-secreting populations could also contribute to the response. In expanded T cell lines we observed higher frequencies of T cells staining with DP4-163/164 tetramer than responding to the same peptide in IFN-γ ELISPot essays, indicating that some T cells can recognize the epitope but not secrete IFN-γ. We observed the cross-reactive T cell response to involve mostly CD4+ T cells. This might be due to in vitro culture conditions that favor CD4+ over CD8+ T cell populations, or an intrinsic bias of cross-reactive T cells because of the different patterns of pMHC-TCR interaction for MHC-I and MHC-II proteins. We studied a relatively small group of 27 individuals exposed to SARS-CoV-2 antigens by infection or vaccination, mostly over 40 years of age. Younger individuals with more frequent previous exposures to HCoVs might show a different pattern of response. Our initial screen for immunodominant epitopes involved only three donors, all of whom recognized 163/164, but other immudominant cross-reactive epitopes might have escaped our attention, including those recognized by other MHC proteins. For all of the donors, previous HCoV infection was inferred but not observed, and we did not attempt to determine which donors were exposed previously to which of the HCoVs. In conclusion, we identified a pan-coronavirus epitope that dominates t...
  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 pages 32 and 36. 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.
  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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Standpunkt von Marcos Buser: Endlager für die Ewigkeit: Ein finnischer Traum

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1. rafo 26 Jan 2022
  
  in Public
  
  In Deutschland, im ehemaligen Versuchsendlager Asse für schwach- und mittelaktive Abfälle, rieselt das Grundwasser ins alte Bergwerk: 12 000 Liter pro Tag, seit Jahrzehnten. Die Sanierungskosten werden heute auf rund zehn Milliarden Euro veranschlagt. Das geht wie immer zulasten der SteuerzahlerInnen und wurde bei den deutschen Atomruinen wie Kalkar, Hamm-Uentrop oder Wackersdorf schon vorexerziert. Ennet der ehemaligen ostdeutschen Grenze muss die Bundesregierung Milliarden in das zweite havarierte Endlager Morsleben pumpen, um dieses zu stützen und zu verschliessen.
  
  kosten endlager
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bluetooth keyboard keeps disconnecting - Apple Community

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1. johnsidi 25 Jan 2022
  
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https://biorxiv.org/cgi/content/10.1101/2022.01.20.477056

1
1. sciscore 25 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.20.477056: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Ethical statement: The study was approved by the ethical review board in Gothenburg (Dnr: 2021-02252).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Levels of human anti-SARS-CoV-2 IgG antibodies in convalescent serum samples: Serum samples from SARS-CoV-2 convalescent individuals (n=24) were obtained from the department of Clinical Microbiology, Sahlgrenska University Hospital, Gothenburg, Sweden.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-SARS-CoV-2 antibody reactivity assay: The antibody reactivity towards glycosidase treated proteins were assessed using an enzyme-linked immunosorbent assay (ELISA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The comparison between anti-RBD IgG levels and antibody reactivity towards recombinant RBD was done with Pearson correlation coefficient, assuming normal distribution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lec3.2.8.1 cells (a mutated CHO cell line kindly received from Prof. P Stanley (Chen & Stanley, 2003)) were cultured under the same conditions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The HEK293 derivate HEK293F cell line (Cat nr R79007, Thermo Fisher Scientific) were cultured in Freestyle 293 medium.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293F</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression of recombinant S protein constructs: The receptor-binding domain of the SARS-CoV-2 spike protein (amino acids 319-541) was produced in three cell lines using an expression vector obtained through BEI Resources, NIAID, NIH, which is vector pCAGGS containing the SARS-CoV-2, Wuhan-Hu-1 spike glycoprotein gene RBD with C-terminal Hexa-Histidine tag (NR-52309) (Table EV1).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_127347)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Glycan database search and data processing: The acquired data were analyzed using Proteome Discoverer version 2.4 (Thermo Fisher Scientific).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Proteome Discoverer</div><div>suggested: (Proteome Discoverer, RRID:SCR_014477)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 IgG II Quant assay is a chemiluminescent microparticle immunoassay (CMIA) used for quantitative determination of IgG antibodies to SARS-CoV-2 in human serum and plasma on the ARCHITECT System (Abbott Laboratories, Chicago, IL).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Abbott Laboratories</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical analyses were performed using the Graphpad Prism software version 9.3.1</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(GraphPad Software Inc, San Diego, CA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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biorxiv.org/cgi/content/10.1101/2022.01.20.477056
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https://biorxiv.org/cgi/content/10.1101/2022.01.19.476940

1
1. sciscore 24 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.19.476940: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: To generate HEK-293T cells that transiently expressed wild type and mutated spike protein (ATCC; mycoplasma-free low passage stock), the cells were transfected with Spike protein expressing pLP plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol and stained for their spike protein expression 72 hours after the transfection as described in Staining and Flow cytometry Analysis.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2 CAR construct: CAR constructs consisting of CD8 alpha signal peptide, extracellular domain of ACE2 molecule or single chain variable fragment (scFv) of anti-CD19 or anti-Spike protein antibodies, CD8 hinge domain, CD8 transmembrane domain, 4-1BB (CD137) intracellular domain and CD3ζ domain were designed with Snapgene and synthesized via Genscript.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-CD19</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Spike protein</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD8 transmembrane domain, 4-1BB (CD137</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-CD3 antibody single-chain variable fragment, His-Tag, and Hemagglutinin (HA) Tag sequences were obtained from Addgene plasmid #85437.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-CD3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>His-Tag</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Hemagglutinin (HA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Activation of CAR CD8 T cells was determined with CD25 staining using CD25-APC antibody (Biolegend).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD25-APC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CAR expressions of ACE2 CAR and anti-Spike CAR and ACE2 expression of ACE2-293 cells were determined with SARS-CoV-2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) incubation followed with APC Goat anti-mouse IgG2a Fc Antibody (Invitrogen) staining and RFP expression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Spike CAR</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Mouse IgG2a</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CAR expression of anti-CD19 CAR was determined with Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity) (Acro) followed by a secondary staining with APC conjugated anti-human IgG Fc Antibody (Biolegend) and RFP expression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-CD19 CAR</div><div>suggested: (Miltenyi Biotec Cat# 130-115-965, RRID:AB_2811310)</div></div><div style="margin-bottom:8px"><div>Human CD19 (20-291) Protein, Fc Tag, low endotoxin (Super affinity)</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For Spike protein flow cytometry analysis, the cells were stained with Biotinylated Human ACE2 / ACEH Protein, Fc,Avitag (Acro Biosystems), then stained with APC anti-human IgG Fc Antibody clone HP6017 (Biolegend).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Detection reagent was prepared using Human CD3 epsilon Protein, Mouse IgG2a Fc Tag (Acro) and Phycoerythrin-conjugated Goat anti-Mouse IgG2a Cross-Adsorbed Secondary Antibody (Fisher) for ACE2-Bite and APC anti-human IgG Fc Antibody clone HP6017 (Biolegend) for ACE2-Fc were added to the wells and incubated for another hour at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Human CD3 epsilon Protein, Mouse IgG2a Fc Tag (Acro)</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VSVG and Spike Protein pseudotyped lentivirus production: The lentiviruses pseudotyped with vesicular stomatitis virus G protein envelope were generated with HEK293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To measure viral titers of VSV-G pseudotyped lentiviruses, virus preparations were serially diluted on Jurkat cells and 3 days post-infection, infected cells were measured using flow cytometry and the number of cells transduced with 1 mL of virus supernatant was calculated as infectious units per milliliter.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat</div><div>suggested: KCB Cat# KCB 94018YJ, RRID:CVCL_0065)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate HEK-293T cells that transiently expressed wild type and mutated spike protein (ATCC; mycoplasma-free low passage stock), the cells were transfected with Spike protein expressing pLP plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol and stained for their spike protein expression 72 hours after the transfection as described in Staining and Flow cytometry Analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All engineered and wild-type HEK-293 and T2 cells were cultured in complete RPMI 1640 medium (RPMI 1640 supplemented with 10% FBS; Atlanta Biologicals, Lawrenceville, GA), 8% GlutaMAX (Life Technologies), 8% sodium pyruvate, 8% MEM vitamins, 8% MEM nonessential amino acid, and 1% penicillin/streptomycin (all from Corning Cellgro).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>T2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate T2s and 293s with stable Spike overexpression, wild-type T2 and 293 cells were transduced with 3 MOI of Spike protein overexpressing VSVG lentivirus and proliferated.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293</div><div>suggested: NCI-DTP Cat# NCI-293TT, RRID:CVCL_1D85)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CAR expressions of ACE2 CAR and anti-Spike CAR and ACE2 expression of ACE2-293 cells were determined with SARS-CoV-2 S1 protein, Mouse IgG2a Fc Tag (Acro Biosystems) incubation followed with APC Goat anti-mouse IgG2a Fc Antibody (Invitrogen) staining and RFP expression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2-293</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Spike protein constructs: Human codon optimized wild-type full-length SARS-CoV-2 Spike protein sequence was synthesized by MolecularCloud (MC_0101081) and then cloned into pLP/VSVG plasmid from Thermo Fisher under CMV promoter after removing the VSVG sequence via EcoRI-EcoRI restriction digestion. 5’-ACGACGGAATTCATGTTCGTCTTCCTGGTCCTG-3’ and 5’-ACGACGGAATTCTTAACAGCAGGAGCCACAGC-3’ primers were used to generate wild-type SARS-CoV-2 Spike protein sequence without the Endoplasmic Reticulum Retention Signal (ERRS, last 19 amino acids of Spike) (Ou et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLP/VSVG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">E484K and N501Y mutated spike protein sequences without ERRS domain were obtained from VectorBuilder plasmids pRP[Exp]-CMV-human beta globin intron>S(E484K,deltaC19)/3xFLAG and pRP[Exp]-CMV-human beta globin intron>S(N501Y,deltaC19)/3xFLAG, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pRP[Exp]-CMV-human</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Since wild-type Spike protein did not have the FLAG tag and efficiently incorporated into the lentiviruses, FLAG Tags in each construct were removed to have the same amino acid sequences among all Spike constructs with the exception of the necessary mutations, via PCR amplification with 5’-ACGACGGAATTCATGTTCGTTTTCCTTGTTCTGTTGC-3’ and 5’-ACGACGGAATTCTTAGCAACATGATCCGCAAGAGCA-3’ primers and cloned into the same pLP expression plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLP</div><div>suggested: RRID:Addgene_22310)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the lentivector plasmids containing the constructs were co-transfected with vesicular stomatitis virus G protein, pLP1, and pLP2 plasmids into HEK293T cells at 80–90% confluency using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLP1</div><div>suggested: RRID:Addgene_22614)</div></div><div style="margin-bottom:8px"><div>pLP2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2 extracellular domain, CD8a signal peptide, CD8 hinge, CD8 transmembrane domain, 4-1BB intracellular domain and CD3ζ domain sequences were obtained from Ensembl Gene Browser and codon optimized with SnapGene by removing the restriction enzyme recognition sites that are necessary for subsequent molecular cloning steps, while preserving the amino acid sequences.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Ensembl Gene Browser</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-CD19 and anti-Spike scFv amino acid sequences were obtained from Addgene plasmids #79125 and #155364, respectively, reverse translated to DNA sequences and codon optimized with Snapgene 5.2.4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Snapgene</div><div>suggested: (SnapGene, RRID:SCR_015052)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Samples were acquired on a BD FACSymphony A5 analyzer and data were analyzed using FlowJo (BD Biosciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Geometric means of PE fluorescence in different titrations were used to generate the titration curve and the area under the curve was calculated using GraphPad Prism 9.0 software (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical Analyses and Reproducibility: All statistical analyses were performed and graphs were prepared using GraphPad Prism V9 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 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 pages 37, 38, 39, 41 and 42. 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.
  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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  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2022.01.19.476940
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Feature suggestion: error handling API · Issue #1096 · sveltejs/svelte

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1. TylerRick 21 Jan 2022
  
  in Public
  
  Additionally, if you're writing a notification display in every single component, wrapped in a <svelte:error> tag, that's the very definition of boilerplate.
  
  In other words, adding a svelte:error tag wouldn't help much.
  
  good point
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github.com/sveltejs/svelte/issues/1096
stackoverflow.com stackoverflow.com

403 Forbidden vs 401 Unauthorized HTTP responses

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1. TylerRick 20 Jan 2022
  
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  Possibly there are authorizations that will permit access to the resource, possibly there are not, but let's give it a try and see what happens.
  
  annotation meta: may need new tag
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stackoverflow.com/questions/3297048/403-forbidden-vs-401-unauthorized-http-responses
auth0.com auth0.com

Forbidden (403), Unauthorized (401), or What Else?

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1. TylerRick 20 Jan 2022
  
  in Public
  
  In the spirit of mutual collaboration between the client and the API, the response must include a hint on how to obtain such authorization.
  
  annotation meta: may need new tag: client/server cooperation?
  
  annotation meta: may need new tag
2. TylerRick 20 Jan 2022
  
  in Public
  
  If the client request does not include any access token, demonstrating that it wasn't aware that the API is protected, the API's response should not include any other information.
  
  annotation meta: may need new tag: demonstrating....
  
  annotation meta: may need new tag
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RECALLING AND GENERATING IMPORTANT INFORMATION USING THE CAPER STAR METHOD: UPDATING CLASSICAL MNEMONICS FOR THE MODERN CLASSROOM

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1. chrisaldrich 19 Jan 2022
  
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  If we follow the caper star clockwise, starting with “checklist” and signifying just the facts as they are presented, we have ready at hand a way to begin rethinking the types of inquiry proper to certain areas of thought [FIGURE 7]. On the first of the five points then, “checklist,” let us hang journalism, objective accounts, and the raw data of scientific research. On the second, concerning “characters” and their relations, let us place psychology, sociology, anthropology, and politics. The third, at the bottom left, concerning “words,” let us imagine linguistics, philology, rhetoric, and dialectic. The fourth point, “questions,” accommodates philosophy broadly speaking, and the generating of topics and concepts, as well as modes of inquiry, whether inductive or deductive, proper to law and medical research. And finally, the top point, concerning “U” (a tag which stands for “you” as well as the first letter of “universal”), let us place ethics, religion, theology, and practices conducive to reflection and self realization—any means of understanding your place in the world and your stake in the matter under consideration. As the crossing lines of the five-pointed star indicate, all points are interrelated. As for the center, whatever one wants to place there can be illuminated by the five categories broadly conceived as just outlined.
  
  Caper Star cross-disciplinary studies tools for thought
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[[@neil/chemical pollution has passed safe limit for humanity]] - anagora.org

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1. mayaland 18 Jan 2022
  
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  Doesn't sound good.
  
  Reminds me of that really long series of things on a contamination theory of obesity.
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https://medrxiv.org/cgi/content/10.1101/2022.01.14.22268980

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1. sciscore 18 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.14.22268980: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Whole blood samples: The 60 samples used for figures 6, S1, S2 and S3 were routine care residues from patients of the Toulouse hospital, where all patients give, by default, their consent for any biological material left over to be used for research purposes after all the clinical tests requested by doctors have been duly completed.<br>Field Sample Permit: Those were anonymized within 24 hours of collection, transferred from the hospital to the research lab, and kept at room temperature until being used for HAT assays within 24 hours (i.e. less than 48 hours after blood samples were collected).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Authentication: Ethical statement: RBCs from O-blood donors were obtained from the Toulouse branch of the Etablissement Français du Sang (EFS), with whom the project was validated under agreement n° 21PLER2020-025.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Polyclonal anti-human Igs secondary antibodies, all conjugated to Alexa-488, were from Jackson laboratories, and purchased from Ozyme (France)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human Igs secondary</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti RBD monoclonal antibodies: CR3022 (ter Meulen et al. 2006) and EY6A (Zhou et al. 2020) were obtained using antibody-expression plasmids, as previously described (Townsend et al. 2021).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti RBD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>antibody-expression</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The activity of those aliquots of working stocks was then evaluated at regular intervals by performing titrations of the IH4-RBD reagent against either the CR3022 monoclonal antibody diluted to a final concentration of 100 ng/ml, or various immune sera diluted to give similar endpoint to those obtained with CR3022.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CR3022</div><div>suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For titration experiments, we prepared stocks containing RBCs and the appropriate amount of the reagent to be kept constant (either the IH4-RBD reagent at 1 µg/ml or IH4 alone at 0.5 µg/ml for plasma titrations, or the appropriate antibody dilution for IH4-RBD titrations).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IH4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">One drop (i.e. ca. 30 µl) of either the pan-specific anti-Ig-GAM secondary fluorescent antibodies, as well as anti-IgG, -IgA or -IgM, all diluted 1/200 in PFN was added to each of the four wells for each sample, and the cells resuspended by gentle shaking of the plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Ig-GAM</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">One drop (i.e. ca. 30 µl) of anti-human secondary antibody conjugated to alexa-488, diluted 1/200 in PFN was added to each of the wells, and the cells resuspended by gentle shaking of the plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human secondary</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>alexa-488</div><div>suggested: (Bioss Cat# bsm-4579M-Alexa488, RRID:AB_11071160)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Covid-19 sera 186, 197 and 203 were obtained from the virology department of the Toulouse hospital; plasmas 79 and 206 were from whole blood samples used in our previous paper describing the Jurkat-S&R-flow test (Maurel Ribes et al. 2021) IH4-RBD (Wuhan and Delta) and IH4 alone were produced by transient transfection of HEK-293T cells, and purified from the supernatant by HIS-tag affinity purification, as previously described (Townsend et al. 2021)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Jurkat-S&R-flow: Briefly, Jurkat-S and Jurkat-R cell lines, obtained and grown as previously described (Maurel Ribes et al. 2021), were resuspended in their own tissue culture medium at a concentration of 2.2 106 cells/ mL before pooling equal volumes of the two.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat-R</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The values used as results are those for specific staining, i.e. the difference between the GMFI (geometric mean fluorescent index) measured on the Jurkat cells expressing the SARS-CoV-2 spike protein and the control Jurkat cells expressing the mCherry fluorescent protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat</div><div>suggested: KCB Cat# KCB 94018YJ, RRID:CVCL_0065)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Post-acquisition analysis of all the samples was performed using the Flowjo software (version 10.7.1).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Flowjo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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  About SciScore
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Communications Intern, Careers At The Aspen Institute

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1. SonuParagon 18 Jan 2022
  
  in Public
  
  Basic Function The Communications intern will provide support to the Communications and Marketing department in its work to promote the Institute’s programs, content, and overall brand to external audiences. Essential Duties and Responsibilities: Support a wide range of projects and focus areas including strategy execution, writing, production, media, design, social media and content creation. Create social media content for Institute channels, including creating visual graphics and selecting images for use on Twitter, Facebook, LinkedIn, and Instagram. Find and store stock and editorial images for later use on our digital channels. Organize, tag, and archive Institute event photos. Write and stage content contributed by Institute staff and partners in a Wordpress CMS, adding art, hyperlinks, and copy editing. Support senior managers on time sensitive projects and program initiatives and events. Support project management efforts as needed. Provide administrative and logistical support for the communications team in support of Institute events. Other duties as needed.
  
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https://biorxiv.org/cgi/content/10.1101/2022.01.11.475922

1
1. sciscore 16 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.11.475922: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Western blot: Western blot was performed using an anti-SARS-COV-2 S antibody following a protocol described previously (56).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-COV-2 S</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Alkaline phosphatase conjugated anti-Rabbit IgG (1:5000) (Sigma-Aldrich, St. Louis, MO) was used as a secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To measure binding of a full-length S protein to monoclonal antibodies, the antibody was immobilized to anti-human IgG Fc Capture (AHC) biosensor (ForteBio, Fremont, CA) following a protocol recommended by the manufacturer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Control sensors with no ACE2 or antibody were also dipped in the S protein solutions and the running buffer as references.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, Expi293F cells transfected with monomeric ACE2 or dimeric ACE2 expression construct and the supernatant of the cell culture was collected.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293F</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, to produce S-expressing cells, HEK293T cells were transfected by polyethylenimine (PEI; 80 μg) with either 5 or 10 μg of the full-length SARS-CoV2 (G614</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To prepare for infection, 7.5×103 of HEK 293 cells, stably transfected with a full-length human ACE2 expression construct, in 15 μl culture medium were plated into a 384-well white-clear plate coated with poly-D-Lysine to enhance the cell attachment.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped virus particles were produced in 293T/17 cells (ATCC) by co-transfection of plasmids encoding codon-optimized SARS-CoV-2 full-length S constructs, packaging plasmid pCMV DR8.2, and luciferase reporter plasmid pHR’ CMV-Luc.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T/17</div><div>suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 293T cell line stably overexpressing the human ACE2 cell surface receptor protein was kindly provided by Drs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The S gene was fused with a C-terminal twin Strep tag (SGGGSAWSHPQFEKGGGSGGGSGGSSAWSHPQFEK) and cloned into a mammalian cell expression vector pCMV-IRES-puro (Codex BioSolutions, Inc, Gaithersburg, MD).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-IRES-puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped virus particles were produced in 293T/17 cells (ATCC) by co-transfection of plasmids encoding codon-optimized SARS-CoV-2 full-length S constructs, packaging plasmid pCMV DR8.2, and luciferase reporter plasmid pHR’ CMV-Luc.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV DR8.2, and luciferase reporter</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pHR’</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Automated data collection was carried out using SerialEM version 3.8.6 (59) at a nominal magnification of 105,000× and the K3 detector in counting mode (calibrated pixel size, 0.83 Å) at an exposure rate of 13.362 electrons per pixel per second.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SerialEM</div><div>suggested: (SerialEM, RRID:SCR_017293)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing and 3D reconstructions: Drift correction for cryo-EM images was performed using MotionCor2 (60), and contrast transfer function (CTF) was estimated by Gctf (61) using motion-corrected sums without dose-weighting.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MotionCor2</div><div>suggested: (MotionCor2, RRID:SCR_016499)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Density maps were corrected from the modulation transfer function of the K3 detector and sharpened by applying a temperature factor that was estimated using post-processing in RELION.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RELION</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Several rounds of manual building were performed in Coot (64).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr></table>
  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 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 pages 32, 26, 28 and 30. 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.
  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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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https://biorxiv.org/cgi/content/10.1101/2022.01.11.475901

1
1. sciscore 16 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.11.475901: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The use of HEK293 cells in this study was approved by the University of Waterloo Research Ethics Board (ORE#42425). 2.2 Plasmids, transfections, and treatments: Plasmids expressing ORF8 protein (YP_009724396.1) tagged at the C-terminus with 3 x DYKDDDK tag (Ex-NV229-M14), ORF10 protein (YP_009725255.1) tagged at the C-terminus with 3 x hemagglutinin tag (Ex-NV231-M07), and M protein (YP_009724393.1) tagged at the C-terminus with green fluorescent protein (Ex-NV225-M03) were from GeneCopoeia (Rockland, MD, U.S.A).<br>Field Sample Permit: All work was performed in accordance with a Health Canada approved Cannabis Tracking and Licencing System Research License held by the University of Waterloo (PI: Dr. Robin Duncan).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK293 cells were seeded (1 × 104 cells) in 96-well plates and transfected with the respective plasmids after 24 h, then treated a few hours after transfection with either CBD or vehicle for 24 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The control plasmid was pCMV-3Tag-3A (pCMV) (Agilent Technologies, Santa Clara, CA, U.S.A.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV</div><div>suggested: RRID:Addgene_16459)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Live cells were maintained at 37° C while fluorescence was recorded at 469/525 nm for the detection of pSIVA and at 531/647 nm for the detection of PI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pSIVA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were grown in 24 well plates and transfected with either pCMV-3Tag-3A, or plasmids expressing ORF8, ORF10, or M protein, and then treated with either 2 μM CBD or vehicle overnight for 14 h, so that analyses were performed prior to measures of effects on cell number and apoptosis markers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-3Tag-3A</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analyses were performed using Prism GraphPad 9 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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:
  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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置身事内中国政府与经济发展【复旦经院“毕业课”解读经济生活背后的政府角色。罗永浩、刘格菘、张军、周黎安、王烁联袂推荐】

1
1. songyt 15 Jan 2022
  
  in Public
  
  有让我们骄傲的繁华，也有让我们梦碎的房价
  
  牛了牛了终于实现pdf带tag批注自由
  
  #新大陆
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cleverpdf.com/87476/1_Zhi Shen Shi Nei Zhong Guo Zhe - Lan Xiao Huan.pdf
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https://biorxiv.org/cgi/content/10.1101/2022.01.10.475532

1
1. sciscore 14 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.10.475532: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the constructs were transiently transfected into HEK293F cells using polyethylenimine (PEI).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293F</div><div>suggested: RRID:CVCL_6642)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All MAbs were 4-fold serially diluted and tested by pseudovirus neutralization assay with human ACE2-overexpressing HEK 293T cells (293T-Hace2) following our previous protocol44.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">) S glycoprotein ectodomain (residues M1-Q1208) with proline substitutions at K986 and V987, a “GSAS” substitution at the furin cleavage site (R682–R685) was cloned into vector pcDNA 3.1+.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA 3.1+</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A gene encoding human ACE2 PD domain (Q18-D615) with an N-terminal interleukin-10 (IL-10) signal peptide and a C-terminal His tag was cloned into vector pcDNA 3.436.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA 3.436</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK 293T cells in 10-cm dish were co-transfected using PEI (polysciences) with 10 μg of Pcmv-Dr8.91 packaging plasmid, 10 μg of recombinant Plvx-IRES-ZsGreen1 plasmid containing luciferase reporter gene, and 2 μg of recombinant Pvax1 plasmids encoding SARS-CoV-2 S proteins.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Plvx-IRES-ZsGreen1</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then, the S trimer proteins were biotinylated using the EZ-Link™ Sulfo-NHS-LC-LC-Biotin kit (Thermo Fisher) and then purified by Zeba™ spin desalting columns (Thermo Fisher)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Zeba™</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed by non-linear regression using GraphPad Prism 8 to calculate half inhibitory concentration (IC50).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cryo-EM 3D reconstruction: For each dataset, the motion correction of image stack was performed using the embedded module of Motioncor2 in Relion 3.136,52,53 and CTF parameters were determined using CTFFIND454 before further data processing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Motioncor2</div><div>suggested: (MotionCor2, RRID:SCR_016499)</div></div><div style="margin-bottom:8px"><div>Relion</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In addition, after obtaining the 3.5-Å-resolution map, we performed focused 3D classification on the S3H3-SD1 region of protomer 2 (highlighted by dotted orange ellipsoid), leading to a dataset of 101,192 particles, which was further local refined on the S3H3-SD1 region in cryoSPARC, deducing a 3.61-Å-resolution map of this region.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The atomic models were validated using Phenix.molprobity command in Phenix.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phenix</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div><div style="margin-bottom:8px"><div>molprobity</div><div>suggested: (MolProbity, RRID:SCR_014226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Interaction interface analyses were conducted through PISA server58.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PISA</div><div>suggested: (PISA, RRID:SCR_015749)</div></div></td></tr></table>
  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 pages 33, 34 and 31. 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.
  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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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Open Natural Products Research: Curation and Dissemination of Biological Occurrences of Chemical Structures through Wikidata

1
1. Public_Reviews 13 Jan 2022
  
  in eLife
  
  Author Response
  
  Reviewer #1 (Public Review):
  
  This manuscript addresses a major issue facing consumers of structure-organism pair data: the landscape of databases is very difficult to navigate due to the way data is made available (many resources do not have structured data dumps) and the way data is standardized (many resources' structured data dumps do not standardize their nomenclature or use stable entity identifiers). The solution presented is a carefully constructed pipeline (see Figure 1) for importing data, harmonizing/cleaning it, automating decisions about exclusions, and reducing redundancy. The results are disseminated through Wikidata to enable downstream consumption via SPARQL and other standard access methods as well as through a bespoke website constructed to address the needs of the natural products community. The supplemental section of the manuscript provides a library of excellent example queries for potential users. The authors suggest that users may be motivated to make improvements through manual curations on Wikidata, through semi-automated and automated interaction with Wikidata mediated by bots, or by addition of importer modules to the LOTUS codebase itself.
  
  Despite the potential impact of the paper and excellent summary of the current landscape of related tools, it suffers from a few omissions and tangents:
  
  It does not cite specific examples of downstream usages of structure-organism pairs, such as an illustration on how this information in both higher quantity and quality is useful for drug discovery, agriculture, artificial intelligence, etc. These would provide a much more satisfying bookend to both the introduction and conclusion.
  
  Thank you for this remark. We deliberately decided not to insist too heavily on the application examples of the LOTUS outputs. Indeed we are somehow biased by our main investigation field, natural products chemistry, and expect that the dissemination of specialized metabolites occurrences will benefit a wide range of scientific disciplines (ecology, drug discovery, chemical ecology, ethnopharmacology, etc.)
  
  However, Figure 5 was established to illustrate how the information available through LOTUS is quantitatively (size) and qualitatively (color classes) superior to what is available through single natural products resources.
  
  As added in the introduction, one of the downstream usages of those pairs is for example to perform taxonomically informed scoring as described in https://doi.org/10.3389/fpls.2019.01329. Obtaining an open database of natural products’ occurrences to fuel such taxonomically informed metabolite annotation tools was the initial impulse for us to build LOTUS. These metabolite annotation strategies, tailored for specialized metabolites, have been shown to offer appreciable performance improvements for current state-of-the-art computational metabolite annotation tools. Since metabolite annotation is still regularly cited as “the major bottleneck” in metabolomics in the scientific literature over the last 15 years (https://europepmc.org/article/med/15663322, https://doi.org/10.1021/acs.analchem.1c00238), any tangible improvement in this field is welcome. With LOTUS we offer a reliable and reusable structures-organisms data source that can be exploited by the community to tackle such issues of importance.
  
  Other possible usages are suggested in the conclusion, but benchmarking or even exemplifying such uses is clearly out of the scope of this paper, each one of them being an article per se.
  
  The additional queries are written in our first answer (see “essential revisions”) and demonstrate the impact of LOTUS on accelerating the initial bibliographic survey of chemical structures occurrences over the tree of life.
  
  This query (https://w.wiki/4VGC) can be compared to a literature review work, such as https://doi.org/10.1016/j.micres.2021.126708. In seconds, it allows retrieving a table listing compounds reported in given taxa and limits the search by years.
  
  The mentions of recently popular buzzwords FAIR and TRUST should be better qualified and be positioned as a motivation for the work, rather than a box to be checked in the modern publishing climate.
  
  It is true that the modern publishing system certainly suffers from some drawbacks (also critically mentioned within the paper). However, after consultation of all authors, we believe that because LOTUS checks both boxes of FAIR and TRUST, we would rather stick to these two terms. In our view, rules 1 (Don’t reinvent the wheel) and 5 (put yourself in your user’s shoes) of https://doi.org/10.1371/journal.pcbi.1005128 apply here. Both terms are indeed commonly (mis-)used but we felt that redefining other complicated terms would not help the reader/user.
  
  The current database landscape really is bad; and the authors should feel emboldened to emphasize this in order to accentuate the value of the work, with more specific examples on some of the unmaintained databases
  
  We perfectly agree with this statement and it is the central motivation of the LOTUS initiative to improve this landscape. It was a deliberate choice not to emphasize how bad the actual landscape is, but rather to focus on better habits for the future. We do not want to start devaluing other resources and elevate our initiative at the cost of others. We also believe that an attentive look at the complexity of the LOTUS gathering, harmonization, and curation speaks for itself and describes the huge efforts required to access properly formatted natural products occurrence data.
  
  If the reviewer and editors insist, although not in our scope, we are happy to list a series of specific (but anonymized) examples of badly formatted entries, of wrong structures-organisms associations, or poorly accessible resources.
  
  While the introduction and supplemental tables provide a thorough review of the existing databases, it eschews an important more general discussion about data stewardship and maintenance. Many databases in this list have been abandoned immediately following publication, have been discontinued after a single or limited number of updates, or have been decommissioned/taken down. This happens for a variety of reasons, from the maintainer leaving the original institution, from funding ending, from original plans to just publish then move on, etc. The authors should reflect on this and give more context for why this domain is in this situation, and if it is different from others.
  
  We do agree with the reviewer and added a “status” column in the table https://github.com/lotusnprod/lotus-processor/blob/main/docs/dataset.csv We chose 4 possible statuses:
  
  Maintained (self-explanatory)
  
  Unmaintained: the database did not see any update in the last year.
  
  Retired: the authors stated they will not maintain the database anymore.
  
  Defunct: the database is not accessible anymore
  
  As for question 3 above, we decided not to focus too heavily on the negative points and resume the current situation in the previous table. Reasons for the databases publishing being in this situation are multiple, and we think they are well summarized in https://doi.org/10.1371/journal.pcbi.1005128 (Rule 10: Maintain, update, or retire), already cited in the manuscript introduction.
  
  Related to data stewardship: the LOTUS Initiative has ingested several databases that are no longer maintained as well as several databases with either no license or a more restrictive license than the CC0 under which LOTUS and Wikidata are distributed. These facts are misrepresented in Supplementary Table 1 (Data Sources List), which links to notes in one of the version controlled LOTUS repositories that actually describes the license. For example, https://gitlab.com/lotus7/lotus-processor/-/blob/8b60015210ea476350b36a6e734ad6b66f2948bc/docs/licenses/biofacquim.md states that the dataset has no license information. First, the links should be written with exactly what the licenses are, if available, and explicitly state if no license is available. There should be a meaningful and transparent reflection in the manuscript on whether this is legally and/or scientifically okay to do - especially given the light that many of these resources are obviously abandoned.
  
  This point is a very important one. We did our best to be as transparent as possible in our initial table. Following the reviewer’s suggestion, we updated it to better reflect the licensing status of each resource (https://github.com/lotusnprod/lotus-processor/blob/main/docs/dataset.csv). Therefore, we removed the generic “license” header, which could indeed be misleading, and replaced it with ”licensing status”, filled with the attributed license type and hyperlink to its content). It remains challenging since some resources changed their copyright in the meantime. We remain at the editor and reviewers’ disposal for any further improvement.
  
  Moreover, as stated in the manuscript, we took care of collecting all licenses and contacted authors of resources whose license was not perfectly explicit to us, therefore accomplishing our due diligence. Additionally, we contacted legal offices in our University and explained our situation. We did everything that we had been advised.
  
  1) To the best of our knowledge, the dissemination of the LOTUS initiative data falls under the Right to quote for scientific articles, as we do not share the whole information, but only a very small part.
  
  2) We do not redistribute original content. What comes out of LOTUS has undergone several curation and validation steps, adding value to the original data. The 500 random test entries, provided in their original form for the sake of reproducibility and testing, are the only exception.
  
  Many scientific authors forget about the importance of proper licensing. While it might be deliberate to restrict the use, inappropriate license choice (or omission) is too often due to a lack of information on its implication.
  
  All authors of the utilized resources can freely benefit from our curation. We are sharing with the community the results of our work, while always citing the original reference.
  
  Concerning the possible evolution of licensing, it remains a real challenge. While we tried to “freeze” the license status when we accessed the data, some resources updated their licensing since then. This can be tracked in the git history of the table (https://github.com/lotusnprod/lotus-processor/blob/main/docs/dataset.csv). Discrepancies between our frozen licensing (at the time of gathering) and actual license can therefore occur. Initiatives such as https://archive.org/web could help solving this issue, coming with other legal challenges.
  
  The order of sections of the manuscript results in several duplicated, but not further substantiated explanations. Most importantly, the methods should be much more specific throughout and the results/discussion should more heavily cross-link to it, as a reader who examines the paper from top to bottom will be left with large holes of misunderstanding throughout.
  
  As our paper focuses a lot on the methods, the barrier between results & methods becomes thinner. We took into account the reviewers’ suggestions and added some additional cross-links for the reader to be able to quickly access related methods.
  
  The work presented was done in a variety of programming languages across a variety of repositories (and even version control systems), making it difficult to give a proper code review. It could be argued that the most popular language in computational science at the moment is Python, with languages like R, Bash, and in some domains, still, Java maintaining relevance. The usage of more esoteric languages (again, with respect to the domain) such as Kotlin hampers the ability for others to deeply understand the work presented. Further, as the authors suggest additional importers may implemented in the future, this restricts what external authors may be able to contribute.
  
  Scientific software has indeed always been written in multiple languages. To this day, scientists have used all kinds of languages adapted both to their needs and their knowledge. Numpy uses Fortran libraries and many projects published in biology and chemistry recently are in Java, R, Python, C#, PHP, Groovy, Scala… We understand that some authors are more comfortable with one language or another. But R syntax is for example much more distant from Python's syntax than Kotlin can be. We needed a highly performant language for some parts of the pipeline and R, Bash, or Python were not sufficient. We decided to use Kotlin as it provides an easier syntax than Java while staying 100% compatible with it.
  
  The advantage of the way LOTUS is designed is that importers are language-agnostic. As long as the program can produce a file or write to the DB in the accepted format, it can be integrated into the pipeline. This was our goal from the beginning, to have a pipeline that can have its various parts replaced without breaking any of the processes.
  
  As a follow up to the woes of point 4., 5., and 7., the manuscript fails to reflect on the longevity of the LOTUS Initiative. Like many, will the project effectively end upon publication? If not, what institutions will be maintaining it for how long, how actively, and with what funding source? If these things are not clear, it only seems fair to inform the reader and potential user.
  
  LOTUS is an initiative that aims to improve knowledge management and sharing in natural products research. Our first project, which is the object of the current manuscript, is to provide a free and open resource of natural products occurrences for the scientific community. Its purpose is not to be a database by itself, but instead to provide through Wikidata and associated tools a way to access natural products knowledge. The objective was not to create yet another database (https://doi.org/10.1371/journal.pcbi.1005128), but instead to remove this need and give our community the tools and the power to act on its knowledge. This way, as everything is on Wikidata, the initiative is not “like many”. This also means that this project should not be considered and evaluated exactly like a classical DB. Once the initial curation, harmonization, and dissemination jobs have been done, they should ideally not be run again. The community should switch to Wikidata as a point of access, curation, and addition of data. If viewed with such arguments in mind, yes, LOTUS can live long!
  
  Wikimedia is a public not-for-profit organization, whose financial development appears to indicate solid health https://en.wikipedia.org/wiki/Wikimedia_Foundation#Finances.
  
  In terms of funding sources, we would like to refer to https://elifesciences.org/articles/52614#sa2 , which stated the following in response to a similar question: "Wikidata is sustained by funding streams that are different from the vast majority of biomedical resources (which are mostly funded by the NIH). Insulation from the 4-5 year funding cycles that are typical of NIH-funded biomedical resources does make Wikidata quite unique." The core of the Wikidata funding streams are donations to the Wikipedia ecosystem. These donations - with a contributor base of millions of donors from almost any country in the world, chipping in at an average order of magnitude of around 10 dollars - are likely to continue as long as that ecosystem is useful to the community of its users. See <https://wikimediafoundation.org/about/financial-reports for details>.
  
  Overall, there were many opportunities for introspection on the shortcomings of the work (e.g., the stringent validation pipeline could use improvement). Because this work is already quite impactful, I don't think the authors will be opening themselves to unfair criticism by including more thoughtful introspection, at minimum, in the conclusions section.
  
  We agree with the reviewer and therefore, list again the major limitations of our processing pipeline:
  
  First, our processing pipeline is heavy. It includes many dependencies and requires a lot of time for understanding. We are aware of this issue and tried to simplify it as much as possible while keeping what we considered necessary to ensure high data quality. Second, it can sometimes induce errors. Those errors, ranging from unnecessary discarded correct entries to more problematic ones can be attributed to various parameters, reflecting the variety of our input. We will therefore try listing them, keeping in mind that the list won’t be exhaustive. For each detected issue, we tried fixing it at best, knowing it will not lead to an ideal result, but hopefully increase data quality gradually.
  
  ● Compounds
  
  ○ Sanitization (the three steps below are performed automatically since we observed a higher ratio of incorrect salts, charged or dimerized compounds. However, this also means that true salts, charged or dimeric compounds were erroneously “sanitized”.)
  
  ■ Salt removals
  
  ■ Charged molecules
  
  ■ Dimers
  
  ○ Translation (both processes below are pretty error-prone)
  
  ■ Name to structure
  
  ■ Structure to name
  
  ● Biological organisms
  
  ○ Synonymy
  
  ■ Lotus (https://www.wikidata.org/wiki/Q3645698, https://www.wikidata.org/wiki/Q16528).
  
  This is also one of the reasons why we decided to call the resource Lotus, as it illustrates part of the problem.
  
  ■ Iris (https://www.wikidata.org/wiki/Q156901, https://www.wikidata.org/wiki/Q2260419)
  
  ■ Ficus variegata (https://www.wikidata.org/wiki/Q502030, https://www.wikidata.org/wiki/Q5446649)
  
  ○ External and internal dictionaries are not exhaustive, impacting translation
  
  ○ Some botanical names we use might not be the accepted ones anymore because of the tools we use and the pace taxonomy is renaming taxa.
  
  ● References
  
  ○ The tool we favored, Crossref, returns a hit whatever the input. This generates noise and incorrect translations, which is why our filtering rules focus on reference types.
  
  ● Filtering rules:
  
  ○ Limited validation set, requires manual validation
  
  ○ Validates some incorrect entries (False positives)
  
  ○ Does not validate some correct entries (False negatives)
  
  Again, our processing pipeline removes entries we do not yet know how to process properly.
  
  Our restrictive filters but substantial contribution to Wikidata in terms of structure-organisms pairs data upload should hopefully incentivize the community to contribute by further adding its human validated data.
  
  We updated the conclusion part of the manuscript accordingly. See https://github.com/lotusnprod/lotus-manuscript/commit/a866a01bad10dfd8b3af90e2f30bb3ae51dd7b9e.
  
  Reviewer #2 (Public Review):
  
  Rutz et al. introduce a new open-source database that links natural products structures with the organisms they are present in (structure-organism pairs). LOTUS contains over 700,000 referenced structure-organism pairs, and their web portal (https://lotus.naturalproducts.net/) provides a powerful platform for mining literature for published data on structure-organism pairs. Lotus is built within the computer-readable Wikidata framework, which allows researchers to easily contribute, edit and reuse data within a clear and open CC0 license. In addition to depositing the database into Wikidata, the authors provide many domain-specific resources, including structure-based database searches and taxon-oriented searches.
  
  Strengths:
  
  The Lotus database presented in this study represents a cutting-edge resource that has a lot of potentials to benefit the scientific community. Lotus contains more data than previous databases, combines multiple resources into a single resource.
  
  Moreover, they provide many useful tools for mining the data and visualizing it. The authors were thoughtful in thinking about the ways that researchers could/would use this resource and generating tools to make it ways to use. For example, their inclusion of structure-based searches and multiple taxonomy classification schemes is very useful.
  
  Overall the authors seem conscientious in designing a resource that is updatable and that can grow as more data become available.
  
  Weaknesses/Questions:
  
  1) Overall, I would like to know to what degree LOTUS represents a comprehensive database. LOTUS is clearly, the best database to date, but has it reached a point where it is truly comprehensive, and can thus be used for a metanalysis or as a data source for research questions. Can it truly replace doing a manual literature search/review?
  
  As highlighted by the reviewer, even if LOTUS might be the most comprehensive natural products occurrences ressources at the moment, TRUE or FULL comprehensive quality of such resource will always be limited to the available data in the litterature. And the community is far from fully describing the metabolome of living beings. We however hope that the LOTUS infrastructure will offer a good place to start this ambitious and systematic description process.
  
  1) Yes it can serve as data source for research questions, as exemplified in the query table
  
  2) No, it cannot and must not replace manual literature search. Manual literature search is the best but at an enormous cost. If the outcome of such search can be made available to the whole community (eg. via Wikidata), the value of such would be even bigger. However, LOTUS can expedite a decent part of a manual litterature search and liberate time to complement this search. See our comment to the editors “To further showcase the possibilities opened by LOTUS, and also answer the remark on the comprehensiveness of our resource, we established an additional query (https://w.wiki/4VGC).This query is comparable to a literature review work, such as: https://doi.org/10.1016/j.micres.2021.126708. In seconds, it allows retrieving a table listing compounds reported in given taxa and limits the search by years.”
  
  We added these examples in the manuscript (see https://github.com/lotusnprod/lotus-manuscript/commit/a6ee135b83e56e8e2041d09d7ce2d5b913c1029d)
  
  2) Data Cleaning & Validation. The manuscript could be improved by adding more details about how and why data were excluding or included in the final upload. Why did only 30% of the initial 2.5 million get uploaded? Was it mostly due to redundant data or does the data mining approach result in lots of missed data?
  
  The reason for this “low” yield is that we highly favored quality over quantity (as in the F-score equation, ß being equal to 0.5, so more importance is given to the precision than the recall). Of course there is redundancy, but the rejected entries are mostly because of too low confidence level according to our developed rules. It is not fully discarded data as we keep it for further curation (ideally including the community) before uploading to Wikidata. We adapted the text accordingly.
  
  3) Similarly, more information about the accuracy of the data mining is needed. The authors report that the test dataset (420 referenced structure-organisms pairs) resulted in 97% true positives, what about false negatives? Also, how do we know that 420 references are sufficiently large to build a model for 2.5M datapoints? Is the training data set is sufficiently large to accurately capture the complexities of such a large dataset?
  
  False negatives are 3%, which is, in our opinion, a fair amount of “loss” given the quality of the data. We actually manually checked 500+ documented pairs, which is more or less the equivalent of a literature review. We were careful in sampling the entries in the right proportions, but we cannot (and did not) state they are enough. We cannot model it either, since the 2.5M+ points have absolutely different distributions, in terms of databases, quality, etc. Only “hint” is the similar behaviour among all subsets. (the 420 + 100 entries) were divided between 3 authors, which obtained similar results.
  
  4) Data Addition and Evolution: The authors have outlined several mechanisms for how the LOTUS database will evolve in the future. I would like to know if/how their scripts for data mining will be maintained if they will continue to acquire new data for the database. To what extent does the future of LOTUS depend on the larger natural products community being aware of the resource and voluntarily uploading to it? Are there mechanisms in place such as those associated with sequencing data and NCBI?
  
  Programs have been not only maintained but also updated with new possibilities (as, for example: the addition of a “manual mode” allowing user to run the LOTUS processing pipeline on a set of their own entries and make them Wikidata-ready (https://github.com/lotusnprod/lotus-processor/commit/f49e4e2b3814766d5497f9380bfe141692f13f23). We will of course do our best to keep on maintaining it, but as no one in academia can state he/she will maintain programs forever. However the LOTUS initiative hopefully embraces a new way of considering database dynamics. If the repository and website of the LOTUS initiative shut down tomorrow, all the work done will still be available to anyone on Wikidata. Of course, future data addition strongly relies on community involvement. We have already started to advocate for the community to start taking part of it, in the form of direct upload to Wikidata, ideally. At the time, there are no mechanisms in place to push publishing of the pairs on Wikidata (as for sequencing, mass spec data), but we will be engaged in pushing forward this direction. The initiative needs stronger involvement of the publishing sector (also reviewers) to help change those habits.
  
  5) Quality of chemical structure accuracy in the database. I would imagine that one of the largest sources of error in the LOTUS database would be due to variation in the quality of chemical structures available. Are all structure-organism pairs based on fully resolved NMR-based structures are they based on mass spectral data with no confirmational information? At what point is a structural annotation accurate enough to be included in the database. More and more metabolomics studies are coming out and many of these contain compound annotations that could be included in the database, but what level (in silico, exact mass database search, or relative to a known standard) are required.
  
  This is a very interesting point and some databases have this “tag” (NMR, cristal, etc.). We basically rely on original published articles, included in specialized databases. If poorly reported structures have been accepted for publication, labelled as “identified” (and not “annotated”) and the authors publishing the specialized databases overlooked it, we might end up with such structures.
  
  Here, the Evidence Ontology (http://obofoundry.org/ontology/eco.html) might be a good direction to look at and further characterize the occurrences links in the LOTUS dataset.
  
  Reviewer #3 (Public Review):
  
  Due to missing or incomplete documentation of the LOTUS processes and software, a full review could not be completed.
  
  Some parts of LOTUS were indeed not sufficiently described and we improved both our documentation and accessibility to external users a lot. We thank the reviewer for insisting on this point as it will surely improve the adoption of our tool by the community.
  
  scietyType:AuthorResponse
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1. trbrt 11 Jan 2022
  
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  let's start with tag notes so i'm going to create a new note called tag note and a tag note takes
  
  My tagnote structure for my tag doesnt work on repeated trying 1248hrs
  
  obsidian/plugin/query
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Choose your IMAP email client settings for Gmail - Gmail Help

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1. TylerRick 10 Jan 2022
  
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  In the "When sending message, automatically" section, uncheck "Place a copy in."
  
  This seems inconsistent (since we do specify a folder for Drafts), but I confirmed that it still gets stored in Sent even after unchecking this. I guess because Gmail adds the Sent tag on the server, whereas with a draft, it is initiated client-side so the client has to be responsible for adding that tag.
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1. TylerRick 10 Jan 2022
  
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  So if you subscribe to both Inbox and All mail you are in practise downloaded your mails twice. If you then delete a mail in inbox it doesnt go away in All mail. It just get the trash-tag. In my opinion you should never subscribe to All mail. You never need to see it. All you need to see are INBOX, TRASH, SENT and the folders YOU created in your gmail-account.
  
  caveat Gmail
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1. mcwork 09 Jan 2022
  
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  如果文本中本身就存在关键字，那么我会使用双链，如果没有，我会使用标签。当然无论是双链还是标签，对我来说都是一个左右：创建一个主题。 [[关键词]] 的实质是 [[#关键词]]，也是 #关键词。
  
  .imp inline tag和双链在我的认知里面第一次完成了联合
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https://biorxiv.org/cgi/content/10.1101/2022.01.05.475037

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1. sciscore 08 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.05.475037: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Written informed consent was obtained from all study subjects.<br>IRB: This study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster (Ref No. UW 21-120 452).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Briefly, 6-10-week-old male and female hamsters were obtained from the Chinese University of Hong Kong Laboratory Animal Service Centre through the HKU Centre for Comparative Medicine Research.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The hamsters were randomized from different litters into experimental groups.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Serially diluted plasma from healthy individuals or previously published monoclonal antibodies against SARS-CoV-2 (B8) were used as negative controls.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two consecutive staining steps were conducted: the first one used an antibody and spike cocktail incubation of 30 min at 4 °C; the second staining involved staining with anti-His-APC and anti-His-FITC antibodies (Abcam) at 4 °C for 30 min to detect the His tag of the RBD.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His-APC</div><div>suggested: (Miltenyi Biotec Cat# 130-101-322, RRID:AB_2800415)</div></div><div style="margin-bottom:8px"><div>anti-His-FITC</div><div>suggested: (Miltenyi Biotec Cat# 130-092-675, RRID:AB_1103226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were further permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit sera anti-SARS-CoV-2-N for 1 hour at RT before adding an Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2-N</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines: HEK293T cells, HEK293T-hACE2 cells and Vero-E6-TMPRSS2 cells were maintained in DMEM containing 10% FBS, 2 mM L-glutamine, 100 U/mL/mL penicillin and incubated at 37 □in a 5% CO2 setting 51</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HEK293T-hACE2</div><div>suggested: RRID:CVCL_A7UK)</div></div><div style="margin-bottom:8px"><div>Vero-E6-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, The pseudovirus was generated by co-transfection of 293T cells with pVax-1-S-COVID19 and pNL4-3Luc_Env_Vpr, carrying the optimized spike (S) gene (QHR63250) and a human immunodeficiency virus type 1 backbone, respectively 54</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibody-virus mixtures were subsequently added to pre-seeded HEK 293T-ACE2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293T-ACE2</div><div>suggested: RRID:CVCL_A7UK)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mixtures were then transferred to 96-well plates pre-seeded with 1×104/well Vero E6 cells and incubated at 37°C for 24 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, The pseudovirus was generated by co-transfection of 293T cells with pVax-1-S-COVID19 and pNL4-3Luc_Env_Vpr, carrying the optimized spike (S) gene (QHR63250) and a human immunodeficiency virus type 1 backbone, respectively 54</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVax-1-S-COVID19</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pNL4-3Luc_Env_Vpr</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">https://www.ncbi.nlm.nih.gov/igblast/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>https://www.ncbi.nlm.nih.gov/igblast/</div><div>suggested: (IgBLAST, RRID:SCR_002873)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequences were aligned using Clustal W in the BioEdit sequence analysis package (Version 7.2)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BioEdit</div><div>suggested: (BioEdit, RRID:SCR_007361)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Half-maximal (IC50) or 90% (IC90) inhibitory concentrations of the evaluated antibody were determined by inhibitor vs. normalized response --4 Variable slope using GraphPad Prism 8 or later (GraphPad Software Inc.)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The fluorescence density of SARS-CoV-2 infected cells were scanned using a Sapphire Biomolecular Imager (Azure Biosystems) and the neutralization effects were then quantified using Fiji software (NIH)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Fiji</div><div>suggested: (Fiji, RRID:SCR_002285)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The structure alignment, cartoon representations, labeling of amino acids in RBD (from PDB 7K45) were generated by PyMOL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Quantification and statistical analysis: Statistical analysis was performed using PRISM 8.0 or later.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PRISM</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr></table>
  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: We detected the following sentences addressing limitations in the study:
  Limitations of the study: ZCB11 probably represents the broadest breadth among bNAbs reported thus far with comparable potency against all current SARS-CoV-2 VOCs including Omicron and OmicronR346K. We are still in the process in determining its mode of action by solving structures of the RBD-ZCB11 Fab complex. Such information will be useful to guide vaccine design as mentioned because the frequency of elite vaccine remains low (2/34 in this study). To understand the frequency of ZCB11-like bNAb among BNT162b2-vaccinees, we need to investigate other elite responders who show equally potent bNAb responses. More ZCB11-like bNAbs should be also discovered to improve current antibody-based cocktail immunotherapy. For animal challenge experiments, we have done a single dose efficacy experiment. Different doses and routes of administration or antibody combination will be tested in future experiments to provide useful information to support clinical development of ZCB11 and ZCB11-like bNAb.
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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https://biorxiv.org/cgi/content/10.1101/2022.01.04.474979

1
1. sciscore 07 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.04.474979: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: Cells were frequently tested for mycoplasma contamination and consistently tested negative.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 15 additional minutes, cells were incubated in blocking buffer (permeabilization buffer supplemented with 1 % BSA), and further incubated in blocking buffer containing anti-Strep mouse antibody (1:1000 dilution), and anti-Sirt5 rabbit antibody (1:1000 dilution).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Strep</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Sirt5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The next day, the cells were washed with PBS three times and incubated in the blocking buffer containing anti-mouse IgG donkey antibody conjugated with Alexa 488 (1:500 dilution, Thermo Fisher)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">anti-rabbit IgG donkey antibody conjugated with Alexa 555 (1:500 dilution, Thermo Fisher), and for counter-staining, DAPI (1 μg/ml</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit IgG donkey antibody</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A549 cells stably expressing ACE2 (A549-ACE2) were a gift from O. Schwartz (Pasteur Institute, Paris).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Early passage HEK293T cells were transfected with 1.5 μg of dCas9-KRAB-MeCP2 repressor plasmid (Addgene #110824) and 0.5 μg of Piggyback Transposase (gift from Maxim Greenberg), using PEI 25K transfection reagent (Polysciences Inc, Cat. 23966-1), according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We combined 10 pmol of Streptococcus pyogenes NLS-Sp.Cas9-NLS (SpCas9) nuclease (Aldevron, USA, Cat. 9212) with 30 pmol of total synthetic sgRNA (10 pmol each sgRNA) to form ribonucleoproteins (RNPs) in 20 μL of total volume with SE Buffer for A549-ACE2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Transfection, Strep affinity purification, and Flag immunoprecipitation in HEK-293T cells: HEK-293T cells were plated in six-well plates or 10-cm dishes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sample Preparation for Proteomic Analysis: HEK-293T Sirt5-KD cells were transfected with plasmids expressing Nsp14-strep in the presence or absence of SIRT5 with 3 biological replicates for each condition.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Sirt5-KD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 stocks were propagated in Vero-E6 cells, and their sequences were verified by next-generation sequencing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For infection experiments, A549-ACE2 or Calu3 cells were seeded into 12- or 24-well plates and rested for at least 24 hours prior to infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu3</div><div>suggested: RRID:CVCL_EQ19)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Infections of HCT-8 cells with HCoV-OC43 were performed similarly.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HCT-8</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmids expressing GFP and Nsp14 proteins (from SARS-CoV-2, SARS-CoV and HCoV-OC43) with a C-terminus strep tag were a gift from Nevan Krogan (1, 2), and are also available on Addgene (pLVX-EF1alpha-SARS-CoV-2-nsp14-2xStrep-IRES-Puro, Addgene #141380).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-EF1alpha-SARS-CoV-2-nsp14-2xStrep-IRES-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mammalian expression plasmids for SIRT5 and SIRT5-H158Y with a myc-his tag in a pCDNA 3.1 vector were available in the Verdin lab (26).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDNA 3.1</div><div>suggested: RRID:Addgene_20407)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Early passage HEK293T cells were transfected with 1.5 μg of dCas9-KRAB-MeCP2 repressor plasmid (Addgene #110824) and 0.5 μg of Piggyback Transposase (gift from Maxim Greenberg), using PEI 25K transfection reagent (Polysciences Inc, Cat. 23966-1), according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>dCas9-KRAB-MeCP2</div><div>suggested: RRID:Addgene_110821)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Nsp14 is cytotoxic, and we used 0.5 μg of Nsp14-strep plasmid for a six-well plate and 4 μg for a 10-cm dish.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Nsp14-strep</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Other co-transfecting plasmids, such as pcDNA-SIRT5, were used at the same concentration except when specifically mentioned.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA-SIRT5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein purification and enzymatic assays: Nsp10 and Nsp14 proteins from the Wuhan strain of SARS-CoV-2 (NC_045512.2) were codon-optimized, ordered as Gblocks (IDT), and cloned into a pVFT1S expression vector using a HiFi DNA Assembly kit (NEB)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVFT1S</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Methyltransferase assays were performed in reaction buffer (50 mM Hepes, pH 7.0, 6 mM KCl, 2 mM DTT, 1 mM MgCl2, and 0.1 mg/ml BSA) in presence of 0.1 mM NAD+ and 10 μM SAM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SAM</div><div>suggested: (SAM, RRID:SCR_010951)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analyses were run using GraphPad Prism version 9.1.2 for macOS (GraphPad Software, USA, www.graphpad.com).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A genome index was constructed using GRCh38 genome build with Gencode v38 annotation of the transcriptome, and Genbank MT246667.1 for the sequence of SARS-CoV-2, USA/WA-1/2020 isolate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gencode</div><div>suggested: (GENCODE, RRID:SCR_014966)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Differential gene expression analysis was done with DEseq2, which was also used to generate normalized gene counts (71).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>DEseq2</div><div>suggested: (DESeq2, RRID:SCR_015687)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Over-representation of biological gene sets in the gene clusters was investigated using the R clusterProfiler package and enricher function (72).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>clusterProfiler</div><div>suggested: (clusterProfiler, RRID:SCR_016884)</div></div></td></tr></table>
  Results from OddPub: Thank you for sharing your code and 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: We did not find any issues relating to colormaps.
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  Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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  No protocol registration statement was detected.
  Results from scite Reference Check: We found no unreliable references.
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Abluftreinigung für Rösterei-Betriebe - Röstereibedarf.de

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1. rafo 06 Jan 2022
  
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  Abluftreinigung für Rösterei-Betriebe<img width="650" height="366" src="https://sp-ao.shortpixel.ai/client/to_auto,q_lossy,ret_img,w_650,h_366/https://roestereibedarf.de/wp-content/uploads/2021/07/caffekarte-985cbb94.jpg" class="scale-with-grid wp-post-image" alt="" srcset="https://sp-ao.shortpixel.ai/client/to_auto,q_lossy,ret_img,w_650/https://roestereibedarf.de/wp-content/uploads/2021/07/caffekarte-985cbb94.jpg 650w, https://sp-ao.shortpixel.ai/client/to_auto,q_lossy,ret_img,w_300/https://roestereibedarf.de/wp-content/uploads/2021/07/caffekarte-985cbb94-300x169.jpg 300w, https://sp-ao.shortpixel.ai/client/to_auto,q_lossy,ret_img,w_260/https://roestereibedarf.de/wp-content/uploads/2021/07/caffekarte-985cbb94-260x146.jpg 260w, https://sp-ao.shortpixel.ai/client/to_auto,q_lossy,ret_img,w_50/https://roestereibedarf.de/wp-content/uploads/2021/07/caffekarte-985cbb94-50x28.jpg 50w, https://sp-ao.shortpixel.ai/client/to_auto,q_lossy,ret_img,w_133/https://roestereibedarf.de/wp-content/uploads/2021/07/caffekarte-985cbb94-133x75.jpg 133w, https://sp-ao.shortpixel.ai/client/to_auto,q_lossy,ret_img,w_600/https://roestereibedarf.de/wp-content/uploads/2021/07/caffekarte-985cbb94-600x338.jpg 600w" sizes="(max-width: 650px) 100vw, 650px" />Was hilft bei Rauch, Gerüchen und gesetzlich festgelegten Emissions-Grenzwerten im Röster-Alltag? Im spannenden Kaffee-Interview mit der Firma ReiCat erfahren wir alles über deren umweltbewusste und authentische Lösungen für Kaffee-Röster!Herr Manuel Marcelli, vielen Dank für Eure Zeit. Bitte erzähle, was ist die Geschichte hinter dem Produkt bzw. der Firma ReiCat?Die Geschichte der Firma ReiCat ist sehr alt, sie kommt von der ehemaligen Firma Ipa Gastechnik GmbH. Seit mehr als 30 Jahren produzieren wir Katalysatoren. Die Firma Ipa Gastechnik GmbH hat sich auf zwei Gebiete spezialisiert: die Abluftreinigungsanlagen und die Gasbehandlung, genauer gesagt: Gasreinigung, Gasrecycling und Gasmixing – um die Qualität von Gas zu definieren. Aber unser Thema ist und bleibt die Abluftreinigungsanlage, oder wie wir das in unserer Business Unit nennen: Environmental Solution. Und in diesem Bereich ist die Geschichte von ReiCat einzigartig, weil wir in der Vergangenheit mehr oder weniger ein Monopol über unseren Namen Ipa Gastechnik GmbH hatten. Wir haben fast alle katalytischen Anlagen geliefert. Ich denke das war so vor 35 bis 15 Jahren. Dann vor 15 Jahren hat ReiCat eine Pause gemacht und sich auf die Gasbehandlung konzentriert, denn da gab es größere Projekte mit höheren Margen. Also haben wir vor 10 Jahren nur noch gelegentlich Katalysatoren geliefert. Was noch interessanter ist: seit 2015 haben wir verstanden, dass der Markt wieder wechselt und dass wir – ohne arrogant klingen zu wollen – etwas Besser als die Konkurrenz anbieten können. Wir haben gesehen, dass es viele Chancen gab, Teile vom Markt zu übernehmen. So kam es, dass wir uns Ende 2015/Anfang 2016 auf Kaffee spezialisiert haben. Am Anfang haben wir nur wenige Anlagen verkauft, mittlerweile haben wir schon richtige Standardmodelle.Für wen sind diese Katalysatoren geeignet und für welche Röstermarken eignen sie sich?Man muss dazu sagen, in der Vergangenheit haben wir nur Tailor Made Einheiten gehabt. Sehr einfach gesagt, gibt es heute drei Gruppen. Eine ist die ReiCatino® Reihe. Die ReiCatino® sind elektrisch betrieben und für Shop-Röster von ein bis 30kg. Die Kunden brauchen kein Gas, somit haben wir hier eine sehr umweltfreundliche Lösung, die die Entfernung von Geruch und Rauch gewährleistet. Eine mittlere Lösung heißt ReiCat Gourmet, diese ist gasbetrieben und für Röstereien von 30-60kg. Wir würden sie als Hybridlösung bezeichnen, denn sie kann sowohl nur Rauch und Gerüche entfernen, aber durch einen Austausch des Brenners kann sie auch gleichzeitig für die Gewährleistung bzw., Einhaltung der Emissionswerte sorgen. Diese Hybridlösung wird immer bekannter und beliebter bei uns. Die dritte Variante ist die Industrielösung: Da sprechen wir von deutlich mehr als 500kg Röstungen pro Tag. Hier müssen die Emissionswerte stimmen, um die BImschG-Genehmigung zu erhalten! Wir haben teilweise große Kunden in Italien und Deutschland mit Anlagen, die über 720kg pro Charge Rösten. Bei der industriellen Anlage hat man eben den Vorteil, dass sie auch nachgerüstet werden kann, wenn beispielsweise die Richtwerte angepasst werden müssen. Wir arbeiten hier mit verschiedenen Ebenen, die man sich wie ein katalytisches Bett vorstellen kann: Es gibt einzelne Module, die man wie Schubladen hinzufügen kann. Wir bauen die Anlagen so, dass immer eine dieser Ebenen frei bleibt, um in Zukunft Platz für Nachrüstungen zu haben. Außerdem können wir unsere Anlagen mit allen Röster-Herstellern verbinden. Für uns ist irrelevant welcher Name auf der Anlage steht. Wichtig ist die Abluftmenge und die Temperatur des Katalysators. Also auch für den HB Roaster sind die ReiCat Anlagen geeignet!Wie oft müssen die Katalysatoren angepasst werden?Das ist auf jeden Fall eine kontinuierliche Entwicklung, denn die gesetzlichen Grenzwerte werden immer heruntergesenkt. In manchen Ländern wie Italien, sind die Grenzwerte für NOx und Formaldehyd deutlich niedriger als bei uns in Deutschland. Dementsprechend müssen wir die Anlagen auch immer wieder anpassen. Wir haben unsere Anlagen weltweit, und da gibt es leider keine einheitliche Regelung. Das heißt, jedes Land, jede Region und manchmal jede Stadt hat andere Regeln. Das macht es für die Röstereien oft schwer zu verstehen, welche Regeln auf sie zutreffen. Deswegen baut ReiCat die Anlagen wie gesagt auch immer für Nachrüstungen geeignet. Wir lassen immer eine „Ebene“ frei, weil wir sagen, dass wir den Standard zum jetzigen Zeitpunkt in 2021 in Deiner Zone gewährleisten können. Aber durch die freie Ebene haben wir ein noch effizienteres und flexibleres Produkt und können jederzeit nachrüsten und anpassen. Wir wissen auch jetzt schon, dass beispielsweise in 2025 wieder Anpassungen in bestimmten Ländern gemacht werden. Und darauf bereiten wir uns vor und sind bereit, wenn es so weit ist.Wie funktioniert so ein Katalysator für Kaffeeröstereien?Es werden während des Röstprozesses viele Kohlenwasserstoffe freigesetzt. Um genau zu sein 350 Verschiedene, von denen ca. 10% aromatisch sind, dh. sie riechen. Vereinfacht gesagt werden die Kohlenwasserstoffe durch unseren Katalysator in andere Komponenten umgewandelt, welche geruchs- und rauchfrei sind. Am Ende ist es die gleiche Luft, die herauskommt, nur, dass sie vorher eben riecht und die Nachbarn stört oder eben auch schädlich sein kann (z.B.: Formaldehyd). Übrigens: Bei den ReiCatino® ist es so, dass sie gesetzlich nicht vorgeschrieben sind, aber meist durch Probleme mit den Nachbarn dann doch gerne genommen werden. Bei den größeren Varianten für Röster mit über 500kg pro Tag ist es eben gesetzlich vorgeschrieben. Man braucht für den Einbau der Anlagen auch keine spezielle Genehmigung, die Behörden wollen nur wissen, ob und dass die Werte eingehalten werden.An welcher Position im Röster wird der Katalysator eingebaut?Zwischen dem Röster und dem Zyklon-Behälter gibt es einen Vorfilter, den wir mitliefern. Dieser vermeidet, dass die Häutchen in den Katalysator gelangen. Er kann schnell und einfach gereinigt werden. Nach dem Vorfilter wird der ReiCatino® eingebaut und hinter diesem wiederum muss der Kunde einen Kamin haben. Die Luft muss immer rausgehen, das ist sehr wichtig und eine Grundvoraussetzung zum Einbau.Wieviel Platz muss ich einplanen, um eine solche Anlage einzubauen?Der ReiCatino® für die kleinen Shop- und Probenröster nimmt beispielsweise sehr wenig Platz weg. In der Regel ca. 1.5m in die Höhe und 1 Meter in die Breite. Er ist sehr kompakt, und kann sowohl stehend als auch liegend positioniert werden. Meist haben die Kunden nur sehr wenig Platz, da ist unser ReiCatino® die perfekte Lösung. Die Gourmet Variante braucht weniger als 2m Höhe und weniger als 1m in Breite und Tiefe. Bei der Industrielösung ist das so pauschal schwer zu sagen, denn da haben wir verschiedene Größen. Aber unsere größte Anlage braucht ca. 4,5m Höhe, 2m Breite und 1,5m Tiefe, also sehr überschaubar.…die Kunden, die sie kaufen sind auch stolz, dass sie diese Anlage haben!Wieviel Zeit muss ich einplanen für den Einbau?Bei ReiCatino® kann das sehr schnell gehen. Die Nachbarn beschweren sich und innerhalb von zwei Wochen kann es zu einer Entscheidung kommen. Vom Design, Zeit, Leistung und für die Größe haben wir eine sehr schöne Lösung gefunden. Und die Kunden, die sie kaufen sind auch stolz, dass sie diese Anlage haben. Der ReiCatino® ist in ein bis zwei Tagen eingebaut, wenn die Voraussetzungen da sind. Und der Einbau ist eigentlich dank eines Plug-In Systems so einfach, dass man ihn auch selbst machen kann. Man braucht eine normale Industriesteckdose und wenn man dann die Anleitung befolgt und die richtige Temperatur einstellt (Unsere Empfehlung zwischen 200 und 250 °C), hat man kaum was falsch gemacht. Wir empfehlen zwar den Selbsteinbau nicht, aber wegen Covid und der Entfernung, geben wir oftmals Unterstützung via Call oder Video-Call. Der Einbau des Gourmet dauert in der Regel 2-4 Tage, bei dem Industriellen dauern die Entscheidungen meist mindestens sechs Monate, da es hier viel mehr Aspekte gibt, die zu berücksichtigen sind. Der Einbau erfolgt dann aber in ca. einer Woche. Apropos, Covid hat uns zwar besonders bei den Industrieanlagen etwas gebremst, aber ReiCatino® hat gar nicht gelitten, sondern super performed. Sie ist die mit Abstand meistverkaufte Anlage bei uns, sehr beliebt bei den Kunden und es werden immer mehr!Wie oft fallen Wartungsarbeiten an, bzw. wie oft müssen Ersatzteile ausgetauscht werden. Und haben die Röstereien einen „Garantieanspruch“?Die erwartete Lebensdauer unserer Anlage liegt bei mehr als 30.000 Betriebsstunden. Das entspricht für ReiCatino® und Gourmet Kunden mehr als 10 Jahre, bzw. mindestens 8 Jahre. (natürlich haben wir auch große Kunden, die durchgängig rösten, dann verkürzt sich die Lebensdauer). Im Fall von ReiCatino® muss der ganze Katalysator getauscht werden. Aber auch hier haben wir eine umweltfreundliche Lösung gefunden. Wenn die Anlage ausgetauscht werden muss, kann der ReiCatino® bei uns zurückgegeben werden, wo wir die Edelmetalle recylen und ihn Umweltgerecht entsorgen. Der Kunde erhält dann noch einen Gutschein für den Kauf eines neuen Katalysators. Hier sind wir sehr nachhaltig, denn wir wollen nicht, dass er einfach im Schrott landet. Seit ich in dem Unternehmen bin, kam es außerdem noch nie zum Verkauf von Ersatzteilen! Das zeigt auch, dass die Anlagen sehr effizient sind, worauf wir stolz sind. Wir sind in allen Bereichen sehr umweltfreundlich, sozusagen eine kleine flexible Green Company im guten Wachstum.Die Anlagen tragen einen großen Teil zum Thema Umweltschutz bei, gehört das auch zur Identität der Marke?Ja, auf jeden Fall! Auch die Mitarbeiter sind begeistert von dem Produkt. Viele von meinen KollegInnen beschäftigen sich auch privat mit dem Thema Umwelt und Mülltrennung. Dieses Thema ist eine Leidenschaft, man ist häufig stolz, wenn man mit Freunden und Familie spricht und wir für unsere Zukunft und unsere Kinder etwas Besseres machen. Proaktiv und Positiv. Wir haben auch schon eine Auszeichnung von der AHK Auslandshandelskammer Italien bekommen. Durch mich haben wir auch Kontakte nach Italien (Anmerkung: Herr Manuel Marcelli kommt gebürtig aus Italien). Dort haben wir große namenhafte Kunden und ein starkes Geschäft. 2019 haben wir dort eine Auszeichnung bekommen für unsere umweltfreundlichen und energieeffizienten Lösungen.Die ReiCatino® sind ja sehr individuell…?Ja, es gibt verschiedene Varianten, auch darauf sind wir stolz. Wenn man von Katalysatoren spricht, hat man oftmals ein langweiliges Thema im Kopf, worüber man nicht unbedingt gerne spricht oder sich mit beschäftigt. Aber bei uns wird eben mehr draus gemacht. Der Kunde kann gegen einen Aufpreis beispielsweise die Farbe wählen. Anfangs gab es den ReiCatino® nur in einem langweiligen grau und davon wollen wir weg! Die Standard ReiCatino® 450 sind mittlerweile alle schwarz, man kann aber eben auch andere Farben auswählen. Wir wollen, dass der Kunde stolz ist auf seine Anlage, sie beispielsweise auch auf Instagram postet, denn auch der Kunde ist damit ja sehr umweltfreundlich.Stichwort Instagram: Was haltet ihr von Social Media & Co und welche Ideen schlummern noch in ReiCat, auf die wir uns freuen können?Wir haben verschiedene Innovationen in der Pipeline, die zeitnah herauskommen werden. Wir denken, dass die Zukunft mit noch weniger Emissionen und auch in Richtung Elektro funktionieren kann. Außerdem legen wir den Fokus nicht mehr nur noch auf die Röstluft, sondern auch auf die Reinigung der Kühlluft. Außerdem sind wir dabei im Hintergrund eine neue Landing Page aufzubauen, nur für ReiCat Coffee. Mit der Veröffentlichung der Homepage präsentieren wir dann auch zeitnah unsere neuen Produkte. Wir haben erkannt, dass Marketing ein sehr wichtiger Teil von ReiCat wird. Neben der neuen Landingpage haben wir auch eine neue Broschüre, ein Video und Fokus auf Instagram gelegt. Wir wollen Erfahrungen kreieren, unsere Anlagen sind nicht nur hochwertige und effiziente technische Anlagen, sondern haben ein zeitgemäßes Design und lassen sich gut vermarkten. Damals haben wir unseren Fokus auf Mund-zu-Mund-Propaganda und Messen gelegt. Aber vor circa drei Jahren haben wir erkannt, dass es auch um die Identifizierung der Anlagen geht, und das schafft man vor allem durch Marketing. Die Kunden sind stolz darauf, eine ReiCatino® zu haben – keine langweilige graue Kiste, sondern eine Anlage mit einer Message dahinter. Es gibt außerdem einen hohen Anteil an Frauen in der Firma, die mit viel Fachwissen und Engagement tätig sind und somit das Klischee entkräften, dass Frauen in technischen Bereichen nicht so gut sind, wie Männer. Wir legen bei ReiCat viel Wert auf Gleichberechtigung und setzen uns dafür ein!
  
  Kaffee
Visit annotations in context

Annotators

rafo

URL

roestereibedarf.de/abluftreinigung-fur-rosterei-betriebe/
www.medrxiv.org www.medrxiv.org

https://medrxiv.org/cgi/content/10.1101/2022.01.03.22268599

1
1. sciscore 06 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2022.01.03.22268599: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: NSCLC patient cohort: Peripheral blood samples from NSCLC patients were collected at Winship Cancer Institute following written informed consent approved by the Institutional Review Board at Emory University.<br>IRB: NSCLC patient cohort: Peripheral blood samples from NSCLC patients were collected at Winship Cancer Institute following written informed consent approved by the Institutional Review Board at Emory University.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MesoScale Discovery Assay: V-PLEX COVID-19 Respiratory Panel 2 Kit (K15372U panel 2) were used to measure the IgG, IgM and IgA antibody against the antigens SARS-CoV-2 Spike, receptod binding domain (RBD),</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antigens SARS-CoV-2 Spike , receptod binding domain ( RBD) ,</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following this, 50 uL per well of 1X MSD SULFO-TAG™ Anti-Human IgG Antibody was added and incubated for one hour at room temperature, shaking at a speed of 700 rpm.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were incubated with either an anti-SARS-CoV spike primary antibody directly conjugated to Alexaflour-647 (CR3022-AF647) or an anti-SARS-CoV spike primary antibody directly conjugated to biotin (CR3022-biotin) for at least 4 hours at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CR3022-AF647</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV spike</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CR3022-biotin</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viruses and cells: VeroE6 cells were obtained from ATCC (clone E6, ATCC, #CRL-1586) and cultured in complete DMEM medium consisting of 1x DMEM (VWR, #45000-304), 10% FBS, 25mM HEPES Buffer (Corning Cellgro), 2mM L-glutamine, 1mM sodium pyruvate, 1x Non-essential Amino Acids, and 1x antibiotics.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VeroE6-TMPRSS2 cells were generated and cultured as previously described18. nCoV/USA_WA1/2020 (WA/1), closely resembling the original Wuhan strain and resembles the spike used in the mRNA-1273 and Pfizer-BioNTech vaccine, was propagated from an infectious SARS-CoV-2 clone as previously described19.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6-TMPRSS2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Using VeroE6-TMPRSS cells, the B.1.1.529 variant was plaque purified directly from the nasal swab, propagated once in a 12-well plate, and expanded in a confluent T175 flask to generate a working stock.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6-TMPRSS</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Focus Reduction Neutralization Assay: FRNT-mNG assays were performed on VeroE6 cells and FRNT assays were performed on Vero-TMPRSS2 cells as previously described18,21,22.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Statistical analysis was conducted using Graphpad Prism V9 and R 4.1.2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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sciscore

URL

medrxiv.org/cgi/content/10.1101/2022.01.03.22268599
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.12.30.474561

1
1. sciscore 06 Jan 2022
  
  in SciScore
  
  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
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">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.<br>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.<br>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.<br>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.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">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.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">K18-hACE2 transgenic mice were randomly divided into the following groups (6 mice/group): vehicle, melatonin 10mg/kg (MLT10)</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">For all analyses, we imaged four fields from at least two sections per mouse and analysis was performed blinded using ImageJ.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">Sample sizes were designed to give statistical power while minimizing animal use.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">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.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">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)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-mouse CD31</div><div>suggested: (Bio-Rad Cat# MCA2388P647, RRID:AB_871974)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">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.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>1:200; caveolin-1: Cell Signaling Technology , #3267</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">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.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Collagen IV</div><div>suggested: (Abcam Cat# ab6586, RRID:AB_305584)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">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).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GFP</div><div>suggested: (Abcam Cat# ab13970, RRID:AB_300798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">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.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-FLAG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>melatonin</div><div>suggested: (Novus Cat# NB100-62745, RRID:AB_964468)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture and transfection: HEK293T cells: HEK293T (RRID:CVCL 0063) cells were obtained from Sigma-Aldrich and authenticated by the provider.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">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;</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">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.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">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.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">K18-hACE2 transgenic mice were randomly divided into the following groups (6 mice/group): vehicle, melatonin 10mg/kg (MLT10)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18-hACE2</div><div>suggested: RRID:IMSR_GPT:T037657)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">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).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAG-GFP</div><div>suggested: RRID:Addgene_11150)</div></div><div style="margin-bottom:8px"><div>pCAG-p.Cys145Ala-Mpro-HA</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCAG-Mpro-HA</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis was performed blinded using ImageJ.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Molecular dynamics simulations were computed with GROMACS 2020.5 67.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GROMACS</div><div>suggested: (GROMACS, RRID:SCR_014565)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All statistical comparisons were performed using Prism 9 (GraphPad).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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:
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
  </footer>
Visit annotations in context

Annotators

sciscore

URL

biorxiv.org/cgi/content/10.1101/2021.12.30.474561
www.medrxiv.org www.medrxiv.org

https://medrxiv.org/cgi/content/10.1101/2021.12.30.21268554

1
1. sciscore 04 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2021.12.30.21268554: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, Omicron variant RBD, full-length wild type spike and the Omciron variant RBD were cloned into pVRC vector containing an HRV 3C-cleavable C-terminal SBP-His8X tag and sequence confirmed by Genwiz.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVRC</div><div>suggested: RRID:Addgene_49746)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies with manufacturer and clone information is detailed under the STAR Methods section.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>STAR</div><div>suggested: (STAR, RRID:SCR_004463)</div></div></td></tr></table>
  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 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 29. 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.
  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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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medrxiv.org/cgi/content/10.1101/2021.12.30.21268554
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1
1. TylerRick 03 Jan 2022
  
  in Public
  
  Code that is per-component instance should go into a second <script> tag.
  
  But this seems to conflict with https://hyp.is/NO4vMmzVEeylBfOiPbtB2w/kit.svelte.dev/docs
  
  The load function is reactive, and will re-run when its parameters change, but only if they are used in the function.
  
  which seems to imply that load is not just run once for the component statically, but rather, since it can be reactive to:
  
  url, params, fetch, session and stuff
  
  may be sufficiently like a per-instance callback, that it could be used instead of onMount?
  
  guidelines important point easy to forget caveat confusing
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https://medrxiv.org/cgi/content/10.1101/2021.12.25.21268392

1
1. sciscore 03 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2021.12.25.21268392: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Ethics committee approval: The CoCo Study and the analysis conducted for this article were approved by the Internal Review Board of Hannover Medical School (institutional review board no. 8973_BO-K_2020,</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Binding is further revealed by an anti-tag peroxidase-labelled antibody and colorimetric quantification.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-tag peroxidase-labelled</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pre-incubation of the Spike-protein with serum or plasma of convalescent patients or vaccinees prevents subsequent binding to ACE2 to various degrees, depending on the amount of neutralizing antibodies present.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were washed three times with PBST and incubated with an HRP-conjugated anti-His-tag antibody (clone HIS 3D5, provided by Helmholtz Zentrum München) for 1⍰h at 37⍰°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Next, without washing, an antibody mix of anti-CD3-AF532 (UCHT1; #58-0038-42; Lot # 2288218; Invitrogen; 1:50), anti-CD4-BUV563 (RPA-T4; #741353; Lot # 9333607; BD Biosciences; 1:200), anti-CD8-SparkBlue 550 (SK1; #344760; Lot #B326454; Biolegend; 1:200), anti-CD45RA (HI100, #740298, Lot # 0295003; BD Biosciences; 1:200), anti-CCR7 (G043H7; #353230; Lot # B335328; Biolegend; 1:50), anti-CD38 PerCP-eF710 (HB7; #46-0388-42; Lot # 2044748; Invitrogen; 1:100) and Zombie NIR(tm)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-CD3-AF532</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-CD4-BUV563</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-CD8-SparkBlue</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-CD45RA</div><div>suggested: (BD Biosciences Cat# 740298, RRID:AB_2740037)</div></div><div style="margin-bottom:8px"><div>anti-CCR7</div><div>suggested: (Fluidigm Cat# 3159003, RRID:AB_2714155)</div></div><div style="margin-bottom:8px"><div>anti-CD38</div><div>suggested: (Thermo Fisher Scientific Cat# 46-0388-42, RRID:AB_1834399)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the rhabdoviral pseudotyped particles were produced in 293T cells transfected to express the desired SARS-CoV-2-S variant inoculated with VSV*DG-FLuc, a replication-deficient VSV vector that encodes for enhanced green fluorescent protein and firefly luciferase (FLuc) instead of VSV-G protein (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern, Switzerland)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The assay was performed in 96-well plates in which Vero cells were inoculated with the respective pseudotyped particles/plasma mixtures.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">READER (AESKU.GROUP, Wendelsheim, Germany) and the Gen5 2.01 Software for analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gen5</div><div>suggested: (Gen5, RRID:SCR_017317)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistics: Statistical analysis was done using GraphPad Prism 8.4 (GraphPad Software, USA) and SPSS 20.0.0 (IBM SPSS Statistics, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>SPSS</div><div>suggested: (SPSS, RRID:SCR_002865)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Ethics committee approval: The CoCo Study and the analysis conducted for this article were approved by the Internal Review Board of Hannover Medical School (institutional review board no. 8973_BO-K_2020,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CoCo</div><div>suggested: (CoCo, RRID:SCR_010947)</div></div></td></tr></table>
  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 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 pages 17 and 20. 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.
  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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
  </footer>
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https://biorxiv.org/cgi/content/10.1101/2021.12.26.474192

1
1. sciscore 03 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2021.12.26.474192: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  NIH rigor criteria are not applicable to paper type.
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HRP-conjugated secondary antibodies against the T7-tag were diluted at 1:5,000 or 1:75,00 in the blocking buffer and incubated for 1 hour at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>T7-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A validated SARS-CoV-2 antibody-negative human serum control and a validated NIBSC SARS-CoV-2 plasma control were obtained from the National Institute for Biological Standards and Control, UK) and an uninfected cells control were also used to ensure that virus neutralization by antibodies was specific.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Control, UK</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A 96-well ELISA plate (R&D system) was coated with the RBD protein or the HEK-293T cell lysate at an amount of approximately 3-5 ng per well in a coating buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, pH 9.6) overnight at 4°C, with subsequent blockage with a blocking buffer (DPBS, v/v 0.05% Tween 20, 5% milk) at room temperature for 2 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped SARS-CoV-2 neutralization assay: The 293T-hsACE2 stable cell line (Cat# C-HA101, Lot# TA060720C) and pseudotyped SARS-CoV-2 (Wuhan-Hu-1 strain, D614G, Alpha, Beta, Lambda, Delta and Omicron) particles with luciferase reporters were purchased from the Integral Molecular.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-hsACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Nb–virus mixes (220 μl total) were incubated at 37°C for 1 h, after which they were added dropwise onto confluent Vero E6 cell (ATCC® CRL-1586™, for Munich) or Vero E6-TMPRSS2-T2A-ACE2 cells (BEI cat# NR- 54970, for Delta) monolayers in the six-well plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Vero E6-TMPRSS2-T2A-ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The monomeric Nbs 2-31 and 2-45 were also cloned into a pET-22b(+) vector at the BamHI and XhoI sites for periplasmic expression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-22b(+)</div><div>suggested: RRID:Addgene_12651)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The PCR fragment was then inserted into the 2-45 pET-21b(+) vector at the same restriction sites to produce the heterodimer 2-45-(GGGGS)3-2-31.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-21b(+)</div><div>suggested: RRID:Addgene_112204)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Crystallographic analysis of psNbs with RBD: The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein (GenBank: QHD43416.1), used in the crystallographic study, was cloned into a customized pFastBac vector (69), and fused with an N-terminal gp67 signal peptide and C-terminal His6 tag (70).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFastBac</div><div>suggested: RRID:Addgene_1925)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The MS data obtained from different RBD-specific VHH isolations were analyzed by AugurLlama to identify high-affinity Nbs for each RBD (15).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>AugurLlama</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were processed by Prism 9 (GraphPad) to fit into a 4PL curve and to calculate the logIC50 (half-maximal inhibitory concentration).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Automated data collection was carried out using serialEM (46) at a nominal magnification of 64,000x with a physical pixel size of 1.329 Å/pixel (0.6645 Å/pixel at super-resolution).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>serialEM</div><div>suggested: (SerialEM, RRID:SCR_017293)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing was performed on-the-fly using CryoSPARC Live version 3.2 (47–49).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Models were manually corrected in Coot (version 9.6.0) between rounds of read-space refinement in Phenix.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Maps colored by local resolution were generated using RELION 3.1 (57).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RELION</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Iterative model building and refinement were carried out in COOT (74) and PHENIX (75), respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antigens were clustered at 70% sequence identity with a minimum length coverage of 15% using CD-HIT (78), the Bio.align pairwise sequence alignment module and CATH domain identifiers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD-HIT</div><div>suggested: (CD-HIT, RRID:SCR_007105)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody hit rate calculation: We constructed a multiple sequence alignment for each antigen cluster using MAFFT (79) and selected a representative structure with highest structure coverage and resolution.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MAFFT</div><div>suggested: (MAFFT, RRID:SCR_011811)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Conservation scores range from 0 to ln(20) = 2.99, higher is more conserved.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Conservation</div><div>suggested: (Conservation, RRID:SCR_016064)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The simulation was run starting from the RBD structure (PDB 6lzg) using Gromacs 2020 version with the CHARMM36m force field (82).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gromacs</div><div>suggested: (GROMACS, RRID:SCR_014565)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequences were aligned by MUSCLE (83) with default parameters.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MUSCLE</div><div>suggested: (MUSCLE, RRID:SCR_011812)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The phylogenetic tree was then constructed by the MEGA (84) using the maximized likelihood estimation method.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MEGA</div><div>suggested: (Mega BLAST, RRID:SCR_011920)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
  </footer>
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URL

biorxiv.org/cgi/content/10.1101/2021.12.26.474192
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https://biorxiv.org/cgi/content/10.1101/2021.12.25.474155

1
1. sciscore 03 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2021.12.25.474155: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In vitro transcription and purification: The sequences of S1hp, S1.5hp, and S2hp were cloned from a gBlock containing the 5’ 1,000 nt of SARS-CoV-2 genome into the pUC19 vector using restriction sites EcoRI and KpnI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pUC19</div><div>suggested: RRID:Addgene_50005)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the coding sequences for NCAP and its truncations with N-terminal 6xHis-SUMO tag were cloned into the pEC-28a vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pEC-28a</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The gel images were obtained using an Amersham™ Typhoon™ scanner (Cytiva) and were quantified using Quantity One (BIO-RAD, version 4.6.9) or ImageJ.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Quantity One</div><div>suggested: (Quantity One 1-D Analysis Software, RRID:SCR_014280)</div></div><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data were fitted and plotted using Prism (GraphPad, version 8).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analyses: Unpaired Student’s t-tests with two tails were performed in Excel (Microsoft).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Excel</div><div>suggested: None</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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  About SciScore
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biorxiv.org/cgi/content/10.1101/2021.12.25.474155
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https://biorxiv.org/cgi/content/10.1101/2021.12.28.474244

1
1. sciscore 03 Jan 2022
  
  in SciScore
  
  SciScore for 10.1101/2021.12.28.474244: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: All animal procedures (including surgery, anesthesia, and euthanasia, as applicable) used in the current study were submitted to the Institutional Animal Care and Use Committee of the CIPHE approved by the French authorities (CETEA DSV – APAFIS#26484-2020062213431976 v6).<br>IRB: Ethics approval was given on February 5, 2020, by the French Ethics Committee CPP-Ile-de-France VI (ID RCB: 2020-A00256-33).<br>Consent: The study was conducted with the understanding and consent of each participant or their surrogate covering the sampling, storage, and use of biological samples.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Vaccination and infection of hCD40/K18-hACE2 transgenic mice: Mice of 8 to 12 weeks of age of both sexes received two intraperitoneal injections of the CD40.CoV2 vaccine (10 µg) plus polyinosinic-polycytidylic acid (Poly-IC; Oncovir) (50 µg) or poly(IC) alone three weeks apart.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Materials availability: The CD40.CoV2 vaccine generated in this study was deposited in GenBank: anti-human CD40 12E12 antibody IgG4 H chain (GenBank ID: AJD85779.1 residues 20-467) fused to SARS_CoV_2RBD (GenBank ID: UEP92470.1 residues 17-240) followed by EPEA (C-tag) and the anti-human CD40 12E12 antibody kappa L chain (GenBank ID: AJD85780.1 residues 21-236) fused sequentially to a linker (GenBank ID: AJD85777.1 residues 699-725), nucleocapsid phosphoprotein, partial [Severe acute respiratory syndrome coronavirus 2] (GenBank ID: QWE63393.1 residues 95-230), linker residues AR, Chain A, Spike protein S1 [Severe acute respiratory syndrome coronavirus 2] (GenBank ID: 7M8J_A residues 113-237), linker residues TR, and Sequence 12 from patent US 8518410</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">[IgG] Kits, MesoScale Discovery, Rockville, MD, USA) were used on all available plasma samples to measure plasma IgG antibodies to SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoVs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>plasma IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody concentrations were quantified using a reference standard (ACE2 Calibration Reagent) and are expressed as units/mL (one unit per mL concentration of ACE2 Calibration Reagent corresponds to neutralizing activity of 1 µg/mL monoclonal antibody to SARS-CoV-2 Spike protein).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 Spike protein) .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The flow cytometry panel included a viability marker, CD3, CD4, and CD8 to determine the T-cell lineage, and IFN-γ, TNF, and IL-2 antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD4</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD8</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>TNF</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IL-2 antibodies</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IL-2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Infectious stocks were grown by inoculating Vero E6 cells and collecting supernatants upon observation of the cytopathic effect.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Heterozygous K18-hACE C57BL/6J mice (strain: 2B6.Cg-Tg (K18-ACE2)2Prlmn/J) were obtained from The Jackson Laboratory.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6J</div><div>suggested: RRID:MGI:3589388)</div></div><div style="margin-bottom:8px"><div>2B6.Cg-Tg ( K18-ACE2)2Prlmn/J</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the AIM assay, PBMCs were stimulated in vitro with various concentrations of the CD40.CoV2 vaccine or an equimolar combination of 15-mer overlapping peptide pools covering the full-length sequence of vaccine antigens (vS1 + vS2 + vRBD + vN2) referred to as vOLPmix.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>vS1 + vS2 + vRBD + vN2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Distributions were plotted using SPICE version 5.22, downloaded from http://exon.niaid.nih.gov/spice (Roederer et al., 2011)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPICE</div><div>suggested: (SPICE, RRID:SCR_016603)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Graphpad Prism software version 8 was used for nonparametric statistics and plots, as described in the figure legends.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  Results from OddPub: Thank you for sharing your data.
  Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
  Our study had several limitations. These included the absence of characterization of cross-neutralizing antibodies in vaccinated mice against the B.1.1.529 Omicron variant, which emerged during the writing of this manuscript. However, we could expect that our vaccine would elicit T-cell responses against N epitopes because of the high homology (100%) between vaccine sequences and this VOC. Due to the limited availability of hCD40/K18-hACE2 mice, we did not evaluate various VOC challenges in mice. Finally we favored the analysis of T cell responses using samples from recovered individuals instead of in vivo preclinical models. Although these responses may be dependent on the “clinical history” of patients and their HLA haplotypes, they are less biased than those that would be observed in an animal model. Our results show that the in-vitro vaccine responses are directed against all vaccine proteins, which confirmed the broad HLA coverage of the vaccine sequences. In conclusion, it is becoming urgent to develop a “pan-sarbecovirus vaccine”. The development of a new protein-based vaccine with expected improved tolerability suitable for people with specific vulnerabilities and children would extend the portfolio of current vaccines and be instrumental in controlling the circulation of the virus and the emergence of new variants. By selecting a narrow range of immunodominant epitopes, presented by a wide variety of HLA alleles and less prone to genetic variations across sarbecoviru...
  Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04842682</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Dose Escalation Trial of CD40.HIVRI.Env Vaccine Combined or …</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04262921</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">French COVID Cohort</td></tr></table>
  Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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Dec 2021
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Annotating the law | Hypothes.is

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1. radera 31 Dec 2021
  
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  Annotation viewing and export Link: https://jonudell.info/h/facet Screencast: https://jonudell.net/h/facet.mp4 Description: View annotations by user, group, URL, or tag. Export results to HTML, CSV, text, or Markdown.
  
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Balisage: Semantics and the Web

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1. mrcolbyrussell 31 Dec 2021
  
  in Public
  
  I have heard it reported in the halls of XML conferences that the browser makers actively wanted XML's strict syntax so they could reduce maintenance costs on their tag soup code
  
  The costs here tend to be overstated (and stated by people without a strong background in either browsers or languages, I've found).
  
  I remember someone mentioning to Boris Zbarsky (and expecting him to agree) that IE compatibility was a source of bloat in Gecko. Of course, he corrected them.
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https://biorxiv.org/cgi/content/10.1101/2021.12.23.473975

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1. sciscore 30 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.23.473975: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">In addition, purified PCR products were inserted into pcDNA3.1, transformed into E. coli and sequencing was performed on randomly picked colonies.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were washed and incubated with anti-FLAG-APC and anti-Histidine6-PE antibodies on ice for 20 mins, followed by washing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-FLAG-APC</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Histidine6-PE</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of soluble proteins: HEK 293 cells were transfected with mammalian expression vectors encoding the relevant protein using polyethylenimine and cells incubated for approximately 3 days to allow accumulation of the secreted protein in the medium.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry: Vero-E6 cells were cultured in DMEM with 10% (v/v) FBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">cDNA encoding a fusion protein comprising of an N-terminal CD5 secretory leader sequence followed by the Wuhan-Hu-1 CoV-2 RBD (residues 319-541), linker region and FLAG epitope tag together with a C-terminal transmembrane domain and short intracellular domain fragment of platelet-derived growth factor receptor-β was synthesised (GeneArt Gene Synthesis) and inserted into the pHypermut2 vector23 This was transfected into DT40 cells by electroporation and stable transfectants derived by growth in puromycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Wuhan-Hu-1 CoV-2 RBD</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">cDNA encoding a fusion protein comprising of an N-terminal CD5 secretory leader sequence followed by the Wuhan-Hu-1 CoV-2 RBD (residues 319-541), linker region and FLAG epitope tag together with a C-terminal transmembrane domain and short intracellular domain fragment of platelet-derived growth factor receptor-β was synthesised (GeneArt Gene Synthesis) and inserted into the pHypermut2 vector23 This was transfected into DT40 cells by electroporation and stable transfectants derived by growth in puromycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHypermut2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In addition, purified PCR products were inserted into pcDNA3.1, transformed into E. coli and sequencing was performed on randomly picked colonies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, movies were corrected for motion using MotionCor 2.1.425 and the contrast transfer function parameters were estimated using GCTF 1.1826.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GCTF</div><div>suggested: (GCTF, RRID:SCR_016500)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Initial rigid-body docking of the crystal structure (PDB ID 6M0J) was performed using UCSF Chimera 1.1528 and further model building was performed in Coot 0.9.629.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After manual rebuilding real-space refinement of the coordinates was performed using Phenix 1.19.230.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phenix</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr></table>
  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: 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.
  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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biorxiv.org/cgi/content/10.1101/2021.12.23.473975
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https://biorxiv.org/cgi/content/10.1101/2021.12.21.473733

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1. sciscore 30 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.21.473733: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: All animal work was performed under the guidelines of Yale University Institutional Animal Care and Use Committee (IACUC) with approved protocols (Chen-2018-20068; Chen-2020-20358; Wilen-2018-20198).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">M. musculus (mice), 6-12 weeks old females, of C57BL/6J and BALB/c strains, were used for immunization.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Six to eight-week-old K18-hACE2 littermate-controlled mice, mixed gender (male / female) mice were divided randomly into three groups, and administered with 20 mg/kg (of mice body weight) Clone 2, Clone 6 or placebo / control, via intraperitoneal (IP) injection.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">Replication, randomization, blinding and reagent validations: Replicate experiments have been performed for all key data shown in this study.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Contamination: All cell lines tested negative for mycoplasma.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmid construction: The cDNA sequences of the paired variable heavy and light chain region of anti-RBD antibody clones were synthesized as gBlocks (IDT) and cloned by the Gibson assembly (NEB) into human IgG1 heavy chain and light chain expression plasmids, pFUSEss-CHIg-hG1(InvivoGen, pfusess-hchg1) and pFUSE2ss-CLIg-hK (InvivoGen, pfuse2ss-hclk), respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pfusess-hchg1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFUSE2ss-CLIg-hK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, containing cDNA sequence of variable region of light chain of anti-RBD antibody clones and the regions overlapping with corresponding flanking sequences of EcoRI and BsiWI restriction sites pFUSE2ss-CLIg-hK, were ordered from IDT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IgG1 antibodies were expressed in Expi293FTM cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">) bispecific antibody is a human IgG1-like bispecific antibody, generated based on CrossMab-KiH bispecific constructs, including pFUSE2ss-knobLight-hK, pFUSE2ss-knobheavy-hG1, pFUSE2ss-HoleLight-hK, pFUSE2ss-HoleHeavy-hG1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSE2ss-knobheavy-hG1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFUSE2ss-HoleLight-hK</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFUSE2ss-HoleHeavy-hG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">BLI): Antibody binding kinetics for anti-spike mAbs were evaluated by BLI on an Octet RED96e instrument (FortéBio) at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After that, the biosensors were associated with indicated concentrations of the antibodies (from 50 nM to 0.78125 nM with 2-fold dilutions, where the kinetic buffer was served as the negative control) for 200 s, then dissociated in the kinetic buffer for 1000 s. (2) 25ng/ul of Clone13A-IgG1 antibodies were captured on a AHC biosensor (ForteBio).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Clone13A-IgG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The anti-S antibodies and HRP-conjugated goat anti-mouse IgG (Sigma, 12-349) in PBS supplemented with 0.1% saponin and 0.1% bovine serum albumin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-S</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: (Millipore Cat# 12-349, RRID:AB_390192)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">15x106 293FT cells were seeded in a 150 mm plates one day before in 20 ml D10 media.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293FT</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 293T cells were seeded in 150 mm plates, and transfected with 21 µg pHIVNLGagPol, 21 µg pCCNanoLuc2AEGFP, and 7.5 µg of a SARS-CoV-2 SΔ19 or SARS-CoV-2 SA SΔ19 plasmid utilizing 198 µl PEI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pseudovirus neutralization assays were performed on 293T-hACE2 cell line 71.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-hACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">5x105 Vero-E6 cells were plated per well of a 96-well plate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody-virus complexes were added to Vero-TMPRSS2 cell monolayers in 96-well plates and incubated at 37°C for 1 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The replication-competent SARS-CoV-2 (USA-WA1/2020) virus was produced in Vero E6 cells, and the titer was determined by plaque assay using WT VeroE6.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">M. musculus (mice), 6-12 weeks old females, of C57BL/6J and BALB/c strains, were used for immunization.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6J</div><div>suggested: RRID:IMSR_JAX:000664)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Clone 5-11 were mAbs chosen from RBD-his tag protein immunized BALB/c mice.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: RRID:IMSR_ORNL:BALB/cRl)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The K18-hACE2 mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J) were purchased from the Jackson Laboratory and bred in house using a trio breeding scheme.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18-hACE2</div><div>suggested: RRID:IMSR_GPT:T037657)</div></div><div style="margin-bottom:8px"><div>B6.Cg-Tg(K18-ACE2)2Prlmn/J</div><div>suggested: RRID:IMSR_JAX:034860)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In vivo efficacy testing of humanized Clone13A to authentic SARS-CoV-2 virus: 10-12-week-old littermate-controlled female and male K18hAce2Tg+ mice were pretreated with 20 mg/kg of either control hIgG1 (purchased from BioXCell) or clone 13A mAb (produced by the Chen lab) administered IP in 300 uL of DPBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18hAce2Tg+</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Parallelly, pFUSE2-CLIg-hK is a cloning plasmid that expresses the constant region of the human kappa light chain and contains multiple cloning sites to enable cloning of the light chain variable region.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSE2-CLIg-hK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Four plasmids were employed: pFUSE2ss-knobLight-hK, pFUSE2ss-knobheavy-hG1, pFUSE2ss-HoleLight-hK, pFUSE2ss-HoleHeavy-hG1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSE2ss-knobheavy-hG1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFUSE2ss-HoleLight-hK</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFUSE2ss-HoleHeavy-hG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pFUSE2ss-knobLight-hK is pFUSE2ss-CLIg-hK with no further editing.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSE2ss-CLIg-hK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The gBlock (pPR024), containing constant region of heavy chain with two knob mutations and the regions overlapping with corresponding flanking sequences of NsiI and NheI restriction sites in pFUSEss-CHIg-hG1 was ordered from IDT, and then cloned into NsiI and NheI restriction enzymes digested pFUSEss-CHIg-hG1 backbone by the Gibson assembly (NEB).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pPR024</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pFUSE2ss-HoleLight- hK was generated by replacing the constant region of Light chain (CL) in pFUSE2ss-CLIg-hK with CH1 region of heavy chain in pFUSEss-CHIg-hG1 vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSEss-CHIg-hG1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The gBlock (pPR023), containing cDNA sequence of constant region of light chain, CH2 and CH3 with “hole” mutations, and regions overlapping with corresponding flanking sequences of NsiI and NheI restriction sites in pFUSEss-CHIg-hG1 was ordered from IDT, and cloned into NsiI and NheI restriction enzymes digested pFUSEss-CHIg-hG1 backbone through Gibson assembly (NEB).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pPR023</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pVP21-SA and pVP28-Indian were generated based on pcDNA3.1-pSARS-CoV-2-S, which was derived by insertion of a synthetic human codon- optimized cDNA (Geneart) encoding a WA1 SARS-CoV-2 S protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVP28-Indian</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA3.1-pSARS-CoV-2-S</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For pVP28-Indian, four gBlocks, contains mutations in Indian variant regions overlapping with corresponding flanking sequences of NheI and BamHI restriction sites pcDNA3.1-pSARA-CoV-2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1-pSARA-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the HIV1-based SARS-CoV-2 spike pseudotyped virus generation, WT pcDNA3.1-pSARS-CoV-2-S, pVP21-SA-variant, and pVP28-Indian variant lacking the C-terminal 19 codons were employed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVP21-SA-variant</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A pair of forward and reverse primers were utilized to amplify fragments lacking the C- terminal 19 codons with pVP21-SAvariant and pVP28-Indian variant as template separately.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVP21-SAvariant</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The amplified fragments were gel-purified and cloned into pVP21-SAvariant backbone and pVP28-Indianvariant backbone, digested with BbvCI and BamHI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVP28-Indianvariant</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant antibody generation: The top-ranked enriched IgG clones were selected and cDNAs of relative variable region of paired heavy- and light-chain were codon-optimized and cloned separately into human IgG1 heavy chain and light chain expression vectors, containing the human IgG1 constant regions (pFuse plasmids)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFuse</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The variable region of Clone 6 heavy chain was cloned into pFUSE2ss-knobheavy-hG1vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSE2ss-knobheavy-hG1vector</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The variable region of Clone 6 light chain was cloned into pFUSE2ss-knobLight-hK vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFUSE2ss-knobLight-hK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmid expression a C-terminally truncated SARS-CoV-2 S protein (pSARS-CoV-2Δ19) was obtained from Dr Bieniasz’ lab.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pSARS-CoV-2Δ19</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 293T cells were seeded in 150 mm plates, and transfected with 21 µg pHIVNLGagPol, 21 µg pCCNanoLuc2AEGFP, and 7.5 µg of a SARS-CoV-2 SΔ19 or SARS-CoV-2 SA SΔ19 plasmid utilizing 198 µl PEI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCCNanoLuc2AEGFP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">It was inserted into a piggybac transposon (Matt Wilson of Baylor College of Medicine, along with the transposase plasmid pCMV-piggybac) that had been modified to encode a CMV-IRES-bsdr cassette; resultant plasmid was named pT-PB-SARS-CoV-2-Spike- IRES-Blasti.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-piggybac</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pT-PB-SARS-CoV-2-Spike- IRES-Blasti</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This too was inserted into piggybac transposon to make pT-PB-SARS-CoV-2-UK Spike-IRES- Blasti.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pT-PB-SARS-CoV-2-UK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">hACE2 was subsequently introduced by VSV G-mediated HIV-based transduction using pHIV-CMV-hACE2-IRES-Puro to produce HOS-3734, which cell lines maintained in selection using 10 μg/mL puromycin (Sigma-Aldrich).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHIV-CMV-hACE2-IRES-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">TZMbl cells stably expressing wild type S/UK variant S were created by co-transfecting TZMbl cells with pT-PB-SARS-CoV-2- Spike-IRES-Blasti or pT-PB-SARS-CoV- 2-UK Spike-IRES-Blasti, respectively, along with pCMV-piggybac and resistant cells selected with 10 μg/mL blasticidin (Invivogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pT-PB-SARS-CoV-2-</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pT-PB-SARS-CoV-</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The control TZMbl cell line not expressing S was generated by co-transfecting pCMV- piggybac with pT-pB-IRES-Blasti and selecting for blasticidin-resistant TZMbl cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-</div><div>suggested: RRID:Addgene_99510)</div></div><div style="margin-bottom:8px"><div>pT-pB-IRES-Blasti</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody humanization: In order to humanize the antibody, we first determine the six CDR loops from murine variable domains by using the online free program “IGBLAST” (https://www.ncbi.nlm.nih.gov/igblast/).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>https://www.ncbi.nlm.nih.gov/igblast/</div><div>suggested: (IgBLAST, RRID:SCR_002873)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The 50% inhibitory concentration (IC50) was calculated with a four-parameter logistic regression using GraphPad Prism 8.0 (GraphPad Software Inc.)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed with non-linear regression using GraphPad Prism to determine the neutralization curve and the IC50 values calculated.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Image processing and 3D reconstruction using cryoSPARC 78 produced similar results.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The final map of each body was corrected for K3 detector modulation and sharpened by a negative B-factor estimated by RELION 80, and then merged in Chimera for deposition.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RELION</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The initial models were subsequently manually rebuilt in COOT 83, followed with iterative cycles of refinement in Refmac5 84 and PHENIX 85.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>COOT</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cryo-EM structures and homology models were analyzed in Pymol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Pymol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Genomic sequencing raw data are deposited to Gene Expression Omnibus (GEO) with the accession code (GSE174635).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gene Expression Omnibus</div><div>suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)</div></div></td></tr></table>
  Results from OddPub: Thank you for sharing your code and 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 54. 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.
  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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biorxiv.org/cgi/content/10.1101/2021.12.21.473733
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https://biorxiv.org/cgi/content/10.1101/2021.12.23.474009

1
1. sciscore 30 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.23.474009: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  NIH rigor criteria are not applicable to paper type.
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting PCR product containing the S197TAG substitution and carrying the C-terminal cleavable His6-tag was treated with NdeI/HindIII restriction endonucleases and then cloned into a pRBC_14-3-3γ/His6SUMO_mBAD43-204 vector 30 pretreated with the same endonucleases.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pRBC_14-3-3γ/His6SUMO_mBAD43-204</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The C-terminal His6-tag enabled the use of the phosphoserine-incorporating E.coli system [BL21 ΔserB(DE3) cells transformed by the pKW2-EFSep plasmid (chloramphenicol resistance) and the target N plasmid] having the Release Factor 1 (responsible for translation termination at TAG codons) 51.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pKW2-EFSep</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV N-containing pGEX plasmid (ampicillin resistance) was kindly provided by Prof. Fulvio Reggiori (University of Groningen, The Netherlands)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGEX</div><div>suggested: RRID:Addgene_18100)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Untagged full-length human 14-3-3γ (Uniprot ID P61981) cloned into a pET21 vector (ampicillin resistance) 52, the surface entropy reduction mutants 53 of the C-terminally truncated human 14-3-3σ (residues 1-231), i.e. Clu1 (159KKE161 → 159AAA161) and Clu3 (75EEK77 → 75AAA77), each containing an N-terminal 3C protease cleavable His6-tag and cloned into the pET28 vector (kanamycin resistance) have been described previously 37.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET21</div><div>suggested: RRID:Addgene_90313)</div></div><div style="margin-bottom:8px"><div>pET28</div><div>suggested: RRID:Addgene_21766)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To ensure phosphorylation of SARS-CoV-2 N, its S197L mutant, or SARS-CoV N in E.coli cells, target proteins were co-expressed with the catalytically active subunit of mouse protein kinase A (PKA) encoded in a separate pACYC-PKA plasmid (chloramphenicol resistance), essentially as described earlier 26.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pACYC-PKA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Crystallization and X-ray data collection: Crystallization was performed using 14-3-3σ surface entropy reduction mutants Clu1 or Clu3 (storage buffer 20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1 mM EDTA, 2 mM DTT, 2 mM MgCl2) mixed with either pS197 or pT205 phosphopeptide at a 1:5 protein:peptide molar ratio with the final protein concentration of 11.5 mg/ml.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pS197</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pT205</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Mw distributions were calculated in Astra 8.0 software (Wyatt Technology) using dn/dc=0.185.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Astra</div><div>suggested: (ASTRA, RRID:SCR_016255)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To determine KD values, the binding curves were approximated using the quadratic equation 56 in Origin 9.0 (OriginLab Corporation, Northampton, MA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>OriginLab Corporation</div><div>suggested: (Origin, RRID:SCR_014212)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Peptide residues were manually built into difference electron density maps in Coot 59 and the overall structures were refined using Buster 2.10.4 60, which used NCS information (with pruning), TLS and all-atom individual isotropic B-factor restrained refinement.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div><div style="margin-bottom:8px"><div>Buster</div><div>suggested: (BUSTER, RRID:SCR_015653)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The refined structures were validated using the comprehensive validation algorithm in Phenix 1.10 62.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phenix</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Structural illustrations were prepared using PyMOL 2.20 (Schrodinger, Inc.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr></table>
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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https://biorxiv.org/cgi/content/10.1101/2021.12.24.474091

1
1. sciscore 30 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.24.474091: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The present study was approved by the Institutional Review Board of the National Institute of Infectious<br>Consent: All volunteers provided written informed consent prior to the enrollment.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Nucleocapsid antibody was analyzed by cobas e411 plus (Roche) with Elecsys Anti-SARS-CoV-2 (Roche), and <1 was evaluated as seronegative according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To obtain comparable titers of IgA to those of IgG, we normalized the IgA titers using a converting unit, which was calculated from the binding curves of CR3022 monoclonal antibodies prepared as human IgG1 and human IgA1 isotypes (20, 43).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human IgA1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cells were washed with the medium and stained with subsequent antibodies/reagents using the Brilliant Stain Buffer Plus (BD Biosciences) for 30 min at room temperature: FITC-conjugated anti-IgA (polyclonal rabbit F(ab’)2, Dako), BV421-conjugated anti-IgG (G18-145, BD Biosciences)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-IgA</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The virus-plasma mixtures were placed on VeroE6/TMPRSS2 cells (JCRB1819) seeded in 96-well plates and cultured at 37 °C with 5% CO2 for 5 days.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6/TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plasmid vectors were transfected into 293T cells followed by infection with 0.5 MOI of G-complemented VSVΔG/Luc 24 h after the transfection (45), and then the uninfected viruses were washed out.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, the human codon-optimized DNA sequence encoding amino acids 331-529 of the SARS-CoV-2 spike (GenBank: MN994467) with an N-terminal signal peptide sequence (MIHSVFLLMFLLTPTESYVD) and C-terminal avi-tag and histidine-tag were cloned into the pCAGGS vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_127347)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To biotinylate RBD proteins, the RBD expression vectors were co-transfected into Expi293F cells together with the secreted BirA-Flag plasmid (Addgene) in the presence of 100 μM biotin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BirA-Flag</div><div>suggested: RRID:Addgene_64395)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blood was collected in Vacutainer CPT tubes (BD Biosciences), followed by centrifugation at 1800 × g for 20 min.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD Biosciences</div><div>suggested: (BD Biosciences, RRID:SCR_013311)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: The numerical data were statistically analyzed and visualized with GraphPad Prism 9 software (GraphPad)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry data were analyzed using FlowJo software (BD Biosciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr></table>
  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: We detected the following sentences addressing limitations in the study:
  A primary limitation of our study is the lack of longitudinal analysis after the third dose of vaccines in our cohort. Therefore, we cannot determine the extents to which the numbers of cross-neutralizing Bmem subset correlate with the magnitudes of cross-neutralizing antibody responses following the third vaccine dose. Owing to the paucity of Bmem subsets, detailed phenotypic and transcriptomic characterization of the cross-neutralizing Bmem subset is hampered in this study. Finally, we focused on the neutralizing antibodies but non-neutralizing antibodies also confer the protection in vivo at least in animal models. The analysis on non-neutralizing and protective antibodies against the variants may generate the distinct outcome owing to the epitope difference.
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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Navigating the WARC file format – Common Crawl

1
1. kael 29 Dec 2021
  
  in Public
  
  WARC Format
  
  The WARC format is the raw data from the crawl, providing a direct mapping to the crawl process. Not only does the format store the HTTP response from the websites it contacts (WARC-Type: response), it also stores information about how that information was requested (WARC-Type: request) and metadata on the crawl process itself (WARC-Type: metadata).
  
  For the HTTP responses themselves, the raw response is stored. This not only includes the response itself, what you would get if you downloaded the file, but also the HTTP header information, which can be used to glean a number of interesting insights.
  
  In the example below, we can see the crawler contacted http://102jamzorlando.cbslocal.com/tag/nba/page/2/ and received a HTML page in response. We can also see the page was served from the nginx web server and that a special header has been added, X-hacker, purely for the purposes of advertising to a very specific audience of programmers who might look at the HTTP headers!
  
  WARC/1.0 WARC-Type: response WARC-Date: 2013-12-04T16:47:32Z WARC-Record-ID: Content-Length: 73873 Content-Type: application/http; msgtype=response WARC-Warcinfo-ID: WARC-Concurrent-To: WARC-IP-Address: 23.0.160.82 WARC-Target-URI: http://102jamzorlando.cbslocal.com/tag/nba/page/2/ WARC-Payload-Digest: sha1:FXV2BZKHT6SQ4RZWNMIMP7KMFUNZMZFB WARC-Block-Digest: sha1:GMYFZYSACNBEGHVP3YFQNOSTV5LPXNAU HTTP/1.0 200 OK Server: nginx Content-Type: text/html; charset=UTF-8 Vary: Accept-Encoding Vary: Cookie X-hacker: If you're reading this, you should visit automattic.com/jobs and apply to join the fun, mention this header. Content-Encoding: gzip Date: Wed, 04 Dec 2013 16:47:32 GMT Content-Length: 18953 Connection: close ...HTML Content...
  
  warc web archive
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commoncrawl.org/2014/04/navigating-the-warc-file-format/
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How to Add Chapters to Your Highlights in Readwise with Headings

1
1. elbaso 28 Dec 2021
  
  in Public
  
  Simply highlight the title of each section and add a note beginning with a period (.) followed by an h (for "heading") and then the number 1 through 3 representing the section's position in the hierarchy. For example, with a book organized into parts, chapters, and sections, you would denote all parts as .h1, all chapters as .h2, and all sections as .h3.
  
  .readwise .tagging How to tag chapters and sections
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https://biorxiv.org/cgi/content/10.1101/2021.12.21.473668

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1. sciscore 24 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.21.473668: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: Toxicology: Animal experimental procedures were reviewed and approved by the University of Illinois Institutional Animal Care and Use Committee (</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Toxicity of each peptide was assessed in 5 female and 5 male CD-1 IGS mice (8 weeks old).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Animals with sex- and age-matched littermates were randomly included in experiments.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">Animal experiments were carried out in a blinded fashion whenever feasible.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">For the number of animals needed to achieve statistically significant results, we conducted a priori power analysis.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After this, cells were stained with antibodies including CD45-EF450 (1:2000, eBioscience #48-0451-82) and CD31-APC (1:100, eBioscience #17-0311-82) for 45 minutes at 4°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD45-EF450</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD31-APC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-SARS-CoV-2 Monoclonal Antibodies: Sequences for monoclonal antibodies that had received Emergency Use Authorization from the U.S. Food and Drug Administration were pulled from the KEGG database (Accession No. REGN10933, D11938; REGN10987, D11939; VIR-7831, D12014; LY-CoV555, D11936).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>D12014; LY-CoV555, D11936</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>LY-CoV555</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 were propagated in Vero E6 cells (CRL-1586; American Type Culture Collection).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were grown to 80% confluency before transfection with pCMV3-SARS-CoV-2-spike (Sino Biological) using Lipofectamine®3000 (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture: The 2019n-CoV/USA_WA1/2019 isolate as well as the P.1 variant of SARS-CoV-2 and ACE-2 expressing A549 cell lines were obtained from BEI Resources.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>A549</div><div>suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero E6 (CRL-1586; American Type Culture Collection) Vero CCL81 (American Type Culture Collection),</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero CCL81</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Assessment of ACE2 mutants: Expi293F cells transfected with pCEP4-myc-ACE2 plasmids were collected 24 h post-transfection (600 × g, 60 s), washed with ice-cold Dulbecco’s phosphate buffered saline (PBS) containing 0.2% bovine serum albumin (BSA), and stained with 1:50 RBD-sfGFP expression medium (prepared as previously described 22 and 1:250 anti-myc Alexa 647 (clone 9B11, Cell Signaling Technology) in PBS-BSA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293F</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Hemizygous K18-hACE mice with c57BL/6J background (strain#034860: B6.Cg-Tg(K18-ACE2)2Prlmn/J) were purchased from The Jackson Laboratory.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18-hACE</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>B6.Cg-Tg(K18-ACE2)2Prlmn/J</div><div>suggested: RRID:IMSR_JAX:034860)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For in vivo studies, 10 mg/kg sACE22.v2.4-IgG1 (wt), sACE22.v2.4-IgG1 and control peptide buffer were intravenously administrated into K-18 hACE-2 mice for 30 minutes prior to SARS-CoV-2 pseudo-entry virus (106 pfu) i.p. injection for 1 dpi.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>hACE-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pharmacokinetics: 8-week-old CD-1 IGS mice (3 female and 3 male per time point) were IV administered protein solutions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD-1 IGS</div><div>suggested: RRID:MGI:5461217)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To characterize different routes side-by-side, peptides were administered to C57BL/6 mice, 3 males per time point.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were grown to 80% confluency before transfection with pCMV3-SARS-CoV-2-spike (Sino Biological) using Lipofectamine®3000 (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV3-SARS-CoV-2-spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">8his-tagged monomeric sACE2 (ACE2 a.a. 1-615; pcDNA3-sACE2(WT)-8his, Addgene No. 149268, and pcDNA3-sACE2v2.4-8his, Addgene No. 149664), and human IgG1-Fc fused dimeric sACE22 (ACE2 a.a. 1-732; pcDNA3-sACE2-WT(732)-IgG1,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3-sACE2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA3-sACE2v2.4-8his</div><div>suggested: RRID:Addgene_149664)</div></div><div style="margin-bottom:8px"><div>pcDNA3-sACE2-WT(732)-IgG1</div><div>suggested: RRID:Addgene_154104)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Addgene No. 154104, and pcDNA3-sACE2v2.4(732)-IgG1, Addgene No. 154106) are previously described.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3-sACE2v2.4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Human codon-optimized S (GenBank No. YP_009724390.1) was subcloned into pCEP4 (Invitrogen) from pUC57-2019-nCoV-S(Human) (distributed by Molecular Cloud on behalf of Haisheng Yu, Chinese Academy of Medical Sciences) with a N-terminal HA leader (MKTIIALSYIFCLVFA), myc-tag (EQKLISEEDL), and linker (GSPGGA) upstream of the mature polypeptide (a.a. V16-T1273).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCEP4</div><div>suggested: RRID:Addgene_16479)</div></div><div style="margin-bottom:8px"><div>pUC57-2019-nCoV-S(Human</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Assessment of ACE2 mutants: Expi293F cells transfected with pCEP4-myc-ACE2 plasmids were collected 24 h post-transfection (600 × g, 60 s), washed with ice-cold Dulbecco’s phosphate buffered saline (PBS) containing 0.2% bovine serum albumin (BSA), and stained with 1:50 RBD-sfGFP expression medium (prepared as previously described 22 and 1:250 anti-myc Alexa 647 (clone 9B11, Cell Signaling Technology) in PBS-BSA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCEP4-myc-ACE2</div><div>suggested: RRID:Addgene_141185)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In vitro binding assays to S variants: Expi293F cells were transfected with pCEP4-myc-S plasmids as described above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCEP4-myc-S</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">H and L chains were cloned with CD5 leader sequences into pcDNA3.1(+) and expressed in Expi293F cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Images were taken with a Zeiss microscope and analyzed by Zen software (Zeiss)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Zen</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For sACE22.v2.4 without any tags or fusion partner (corresponding to ACE2 residues 19-732, protein provided by Orthogonal Biologics, Inc.), mice were IV administered (tail vein, 0.5 mg/kg) protein twice daily for 5 consecutive days (days 0-4) and sacrificed day 7.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Orthogonal Biologics</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mutations were introduced using PyMOL (https://pymol.org/2/) and the systems were solvated using TIP3P water and 150 mM NaCl using PACKMOL 71.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein residues and glycosylation sites were parameterized using AMBER ff14SB 72 and GLYCAM06 73 force fields.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>AMBER</div><div>suggested: (AMBER, RRID:SCR_016151)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-SARS-CoV-2 Monoclonal Antibodies: Sequences for monoclonal antibodies that had received Emergency Use Authorization from the U.S. Food and Drug Administration were pulled from the KEGG database (Accession No. REGN10933, D11938; REGN10987, D11939; VIR-7831, D12014; LY-CoV555, D11936).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>KEGG</div><div>suggested: (KEGG, RRID:SCR_012773)</div></div></td></tr></table>
  Results from OddPub: Thank you for sharing your code and data.
  Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
  Another limitation is our focus on intravenous delivery, whereas intratracheal or inhalation delivery may be more readily applied in patients, especially in the outpatient setting. It would be useful in future studies to use varying doses and delivery route combinations for defined disease stages to identify the optimal combination prior to initiating human efficacy trials. Finally, we chose to fuse the ACE2 peptide to an unmodified Fc from IgG1 (isoallotype nG1m1), whereas others have considered fusions to Fc mutants or IgG4 to dampen interactions with FcγR subtypes that might contribute to inflammation24, 68. We instead reasoned that IgG1 effector functions will be necessary for optimum in vivo protection. In summary, we show for the first time that an engineered decoy ACE2 peptide demonstrating much higher affinity for the SARS-CoV-2 Spike protein is efficacious in vivo against multiple SARS-CoV-2 variants. This peptide prevented viral entry into cells and the peptide’s efficacy against a variant of concern prevented lung endothelial injury and ARDS and significantly reduced mortality. The results show the potential of this engineered peptide to treat COVID-19 patients and others with inadequate antibody titer to protect against emerging, more virulent SARS-CoV-2 variants.
  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:
  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.
  Results from scite Reference Check: We found no unreliable references.
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https://biorxiv.org/cgi/content/10.1101/2021.12.21.473774

1
1. sciscore 24 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.21.473774: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The study protocol was approved by the Human Research Ethics Committees of the Northern Sydney Local Health District, the University of New South Wales, NSW Australia (ETH00520), CALHN Human Research Ethics Committee, Adelaide, Australia<br>Consent: Written informed consent was obtained from all participants before enrolment.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Archival serum or plasma collected from 25 healthy donors prior to the pandemic with an age range of 24-73 years, and a male to female ratio of 1:2.4 was used as controls.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression of surface Fc-receptors was assessed by flow cytometry using monoclonal antibodies (mAbs) against Fcγ receptor RI (CD64)-FITC, FcγRIII (CD16)-PE, CD14-PerCP</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD64)-FITC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Endpoint titre and Isotyping of anti-SARS-CoV-2 Spike and anti-RBD antibodies in sera of patients with SARS-CoV-2 infection: Anti-SARS-CoV-2 Spike and RBD IgG antibody in sera was quantified using a modified direct ELISA [27].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>RBD IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the endpoint titre of total anti-Spike or anti-RBD antibodies, 50μL of horseradish peroxidase (HRP)-conjugated mouse anti-human detection antibody added per well (Jackson ImmunoResearch, USA) (1:5000 dilution in 5% skim milk).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Spike</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human detection</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For isotyping of the anti-Spike or anti-RBD antibodies, 50μL/well of HRP-conjugated immunoglobulin subtype or IgG subclass specific detection antibodies were added.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgG subclass specific detection antibodies</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These include 1:6000 dilution of anti-total human IgG (Jackson Immunoresearch), 1:3000 dilution of anti-human IgA (α-chain-specific) (Sigma) or 1:3000 dilution of anti-human IgM (μ-chain-specific) (Sigma) antibody subtypes and 1:6000 dilution of anti-human IgG1, IgG2, IgG3 or IgG4 (Southern Biotech, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-total human IgG</div><div>suggested: (Cell Signaling Technology Cat# 13123, RRID:AB_2617178)</div></div><div style="margin-bottom:8px"><div>anti-human IgA ( α-chain-specific ) ( Sigma )</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgM ( μ-chain-specific ) ( Sigma )</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgG1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To assess whether the uptake of the microbeads opsonised with the plasma of patients with acute disease was via Fc-receptor (antibody-dependent), and other heat labile opsonins such as complements (eg C3b), either the Fc-receptors on the THP-1 cells were pre-blocked using the universal Fc receptor blocking agent (Miltenyi, USA) [23], or the plasma heat inactivated at 56°C for 30 minutes, as described [28].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-dependent) ,</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Surface plasmon resonance: Surface plasmon resonance (SPR) was performed using a Biacore T200 (Cytiva, USA) to determine the binding characteristics of the anti-Spike polyclonal antibodies in patient plasma to SARS-CoV-2 Spike antigen.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 Spike antigen .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Friedman’s test with pairwise Dunn’s tests were used to compare the repeated measures of Spike p-score, Spike and RBD end point titre, neutralisation titre and antibody affinity across the timepoints V1, V2 and V3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>V3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To rank the variables of statistical importance that associated with Spike p-score (dependent variable), multiple linear regression analysis was performed using disease severity, age, gender, DPS, anti-Spike/RBD endpoint antibody titre, anti-Spike/RBD antibody subtypes and neutralisation titre as independent variables.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Spike/RBD</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To assess whether the uptake of the microbeads opsonised with the plasma of patients with acute disease was via Fc-receptor (antibody-dependent), and other heat labile opsonins such as complements (eg C3b), either the Fc-receptors on the THP-1 cells were pre-blocked using the universal Fc receptor blocking agent (Miltenyi, USA) [23], or the plasma heat inactivated at 56°C for 30 minutes, as described [28].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>THP-1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Retroviral SARS-CoV-2 Spike pseudovirus were generated in 293T cells by co-transfecting expression plasmids containing SARS-CoV-2 Spike and MLV gag/pol and luciferase vectors using Calphos transfection kit (Takara Bio, USA) as described [20].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, pseudovirus were incubated for one hour with heat inactivated (56 °C for 30 minutes) patient serum prior to infecting 293T-ACE2 cells (kindly provided by A/Prof Jesse Bloom) by a two hour spinoculated at 800xg in 96-well white flat bottom plates in triplicates (Sigma-Aldrich, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Biotinylated recombinant SARS-CoV-2 Spike and RBD proteins: SARS-CoV-2 Wuhan-Hu-1 (GenPept: QJE37812) RBD protein (amino acid residues 319–541) and Spike protein (amino acid residue 15-1213) were cloned into pCEP4 mammalian expression vector containing N-terminal human Ig kappa leader sequence and C-terminal Avi-tag and His-tag (Invitrogen, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCEP4</div><div>suggested: RRID:Addgene_16479)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After a two hours incubation, cells were washed once with 1mL of cold PBS containing 0.5% FBS and 0.005% of sodium azide and gentle centrifugation at 335xg for five minutes at 4°C, fixed in 400μL of 1% paraformaldehyde and kept at 4°C in the dark until acquisition of data using BD FACSCalibur™ Flow cytometer.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD FACSCalibur™</div><div>suggested: (BD FACSCalibur Flow Cytometry System, RRID:SCR_000401)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A total of 2×104 events were acquired and the proportions of cells that phagocytosed the beads (% of cells that took up the beads) and their fluorescent intensities (amounts of beads taken up per cell) were analysed using BD FlowJo version 10.5.0 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All the images were Airyscan processed with Zen Black Edition (Zeiss Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Zen</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Relative Luminescence Unit (RLU) in cell lysates was measured using CLARIOstar microplate reader (BMG Labtech, Australia), percentage neutralisation of Spike pseudovirus determined and the fifty percent inhibitory (ID50) calculated using non-linear regression model (GraphPad Prism version 9.0) [29]</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Kinetic constants, including association constant (Ka), equilibrium constant (KD) (affinity) and dissociation constant (Ka) (avidity), were calculated using BIAcore evaluation software version 4.1 [30].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BIAcore</div><div>suggested: (Biacore T100 System, RRID:SCR_019679)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: All data were analysed with Prism Software (version 9.0, GraphPad, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2021.12.21.473774
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https://biorxiv.org/cgi/content/10.1101/2021.12.19.473391

1
1. sciscore 24 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.19.473391: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: donors: Convalescent plasma, Ad26.COV2.S, and some BNT162b2 samples were obtained from the HAARVI study approved by the University of Washington Human Subjects Division Institutional Review Board (STUDY00000959).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">Authentication: None of the cell lines used were authenticated or tested for mycoplasma contamination.<br>Contamination: None of the cell lines used were authenticated or tested for mycoplasma contamination.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Infected cells were then washed an additional five times with DMEM prior to adding media supplemented with anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC) to reduce parental background.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-VSV-G</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines: Cell lines used in this study were obtained from ThermoFisher Scientific (HEK293T and Expi293F) or were kindly gifted by Florian</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK-293T cells seeded in poly-D-lysine coated 100 mm dishes at ∼75 % confluency were washed five times with Opti-MEM and transfected using 24 µg of the S glycoprotein plasmid with Lipofectamine 2000 (Life Technologies).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped VSV neutralization assay: To evaluate neutralization of D614G, Delta (B.1.617.2), and Omicron (B.1.1.529) pseudotypes by plasma of vaccinees or previously infected individuals, Vero-TMPRSS2 cells in DMEM supplemented with 10% FBS, 1% PenStrep, and 8 ug/mL puromycin were seeded at 60-70% confluency into white clear-bottom 96 well plates (Corning) and incubated at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Media was removed from the cells and 40 µL from each well (containing plasma and pseudovirus) was transferred to the 96-well plate seeded with Vero-TMPRESS2 cells and incubated at 37°C for 2 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRESS2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2-RBD-Avi construct was synthesized by GenScript into pcDNA3.1-with an N-terminal mu-phosphatase signal peptide and a C-terminal octa-histidine tag, flexible linker, and avi tag (GHHHHHHHHGGSSGLNDIFEAQKIEWHE).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1-with</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">fold-on trimerization motif, and an 8× His tag in the pCMV vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV</div><div>suggested: RRID:Addgene_16459)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plasmids encoding the SARS-CoV-2 Omicron (B.1.1.529) S variant was generated by overlap PCR mutagenesis of the wildtype plasmid, pcDNA3.1(+)-spike-D19 (80)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Prism (GraphPad) nonlinear regression with “[inhibitor] versus normalized response with a variable slope” was used to determine ID50 values from curve fits with 2-3 repeats.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
  </footer>
Visit annotations in context

Annotators

sciscore

URL

biorxiv.org/cgi/content/10.1101/2021.12.19.473391
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.12.20.473401

1
1. sciscore 23 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.20.473401: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Field Sample Permit: Experiments involving animals were conducted using protocols approved by the National Research Council Canada Animal Care Committee and in accordance with the guidelines set out in the OMAFRA Animals for Research Act, R.S.O. 1990, c. A.22. b)<br>IACUC: Experiments involving animals were conducted using protocols approved by the National Research Council Canada Animal Care Committee and in accordance with the guidelines set out in the OMAFRA Animals for Research Act, R.S.O. 1990, c. A.22. b)</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">The denatured-induced unfolding of VHHs was considered to be reversible based on their small size as shown to be the case for small proteins and numerous times for VHHs 56–59, 88, 89. d) Serum stability: Three female Syrian hamsters were injected intraperitoneally (IP) with 1 mg of 1d or VHH-72 VHH-Fc diluted in 200 µL PBS.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Reactions were stopped by adding 50 µL 1 M H2SO4 to wells, and absorbance were subsequently measured at 450 nm using a Multiskan™ FC photometer (Thermo Fisher). b) Binding to cognate anti-spike glycoprotein polyclonal antibody: The four spike glycoprotein antigens were passively adsorbed as described above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-spike glycoprotein</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After blocking with PBSC, wells were emptied, washed five times with PBST and incubated at room temperature for 1 h with 100 µL of 1 µg/mL anti-SARS-CoV-2 spike rabbit polyclonal antibody (Sino Biological, Beijing, China, Cat#40589-T62) in PBSCT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following 1 h incubation at room temperature, wells were washed 10 times with PBST and incubated with HRP-conjugated polyclonal goat anti-llama IgG heavy and light chain antibody (Bethyl Laboratories, Montgomery, TX, Cat#A160-100P) for 1 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-llama IgG heavy and light chain</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To determine the presence of antibodies that block the binding of S to ACE2 (surrogate for neutralization) in the immune sera of llamas, 400 ng of chemically biotinylated SARS-CoV-2 S was mixed with 1 × 105 Vero E6 cells in the presence of 2-fold dilutions of sera (pre immune, day 21 and day 28 sera) in a final volume of 150 µL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then plates were washed 10 times with PBST and binding of VHHs to S1-Fc was detected with rabbit anti-6xHis Tag antibody HRP Conjugate (Bethyl Laboratories, Cat#A190-114P), diluted at 10 ng/mL in PBST and added at 100 µL/well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-6xHis</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 1 h incubation at room temperature, strips were washed 10 times with PBST and the binding of VHH-Fcs to denatured S was probed by incubating strips with 1 mL of 100 ng/mL anti-human Ig Fc antibody-peroxidase conjugate (Jackson ImmunoResearch, Cat#016-030-084) at room temperature for 1 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human Ig Fc antibody-peroxidase conjugate</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 1 h incubation at room temperature, plates were washed 10 times with PBST and the ACE2-Fc binding was detected using 1 µg/mL goat anti-human IgG (Fc specific) HRP conjugate antibody (Sigma, Cat#A0170) in 100 µL PBSCT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (Sigma-Aldrich Cat# A0170, RRID:AB_257868)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immune cell infiltrate was detected using rabbit polyclonal antibodies against CD3 (1:500, Dako, Cat#A0452), and Iba-1 (ionized calcium binding adaptor protein, 1:5000, Dako, Cat#019-19741)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD3</div><div>suggested: (Agilent Cat# A0452, RRID:AB_2335677)</div></div><div style="margin-bottom:8px"><div>Iba-1 ( ionized calcium binding adaptor protein</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mouse anti-SARS-CoV-2 N monoclonal antibody (1:5000, R&D Systems, Cat#MAB10474) was used for the detection of SARS-CoV-2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Mouse anti-SARS-CoV-2 N monoclonal antibody</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 N</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Conversely, a binding response was seen during the second injection for VHHs that did not compete with ACE2. c) ACE2 competition assay by flow cytometry: Experiments were performed as described in 4.3c, except that biotinylated S/Vero E6 cells were mixed with VHHs or VHH-Fcs instead of sera.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>S/Vero E6</div><div>suggested: RRID:CVCL_JX48)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped and live virus neutralization assays: a) Pseudotyped virus neutralization assays: (i) Generation of SARS-CoV-2 spike pseudotyped lentiviral particles (LVP): HEK293T cells were plated in a 100-mm tissue culture dish and transfected the next day at about 75% confluency with a combination of a lentiviral transfer vector encoding eGFPLuc (addgene#119816), the packaging plasmid psPAX2 (addgene#12260), and a plasmid encoding the viral glycoprotein of interest SARS-CoV-2 Spike-ΔS1/S2-Δ20 expressed in pcDNA3.1+.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pellet was then resuspended in PBS buffer at 1/10 of the original supernatant volume with gentle up-and-down pipetting, aliquoted and stored at −80°C. (ii) Viral neutralization assays: HEK293T-hACE2 cell line (BEI Resources, Manassas, VA, Cat#NR-52511) were seeded in poly-L-Lysine (PLL)-coated white, clear bottom 384-wells plate (NUNC, Thermo Fisher) at a density of 9,000 cells/well in 45 µL of media (DMEM without phenol red supplemented with 5% [v/v] FBS) and incubated for 24 h at 37°C, 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-hACE2</div><div>suggested: RRID:CVCL_A7UK)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Quantitative microneutralization assay was performed on Vero E6 cells with SARS-CoV-2 strains hCOV-19/Canada/ON-VIDO-01/2020, NR-53565; hCOV- 19/England/204820464/2020, NR-54000; or hCOV-19/South Africa/KRISP-EC-K005321 /2020, NR-54008.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Virus titer was determined by plaque assay on Vero cells. b) Immunohistochemistry: Lungs were immersed in 10% neutral buffered formalin and fixed for 1 week at room temperature and then transferred into 70% ethanol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VH/VHH genes were amplified using semi-nested PCR and cloned into the phagemid vector pMED1, followed by transformation of E. coli TG1 (Lucigen, Middleton, WI, Cat#60502-02) to construct two libraries with sizes of 1 × 107 and 2 × 107 independent transformants for Green and Red, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pMED1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of VHHs and VHH-Fcs: a) Expression and validation of VHHs: Positive VHHs were cloned into a modified pET expression vector (pMRo.BAP.H6) for their production in BL21(DE3) E.coli as monomeric soluble protein 84.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pMRo.BAP.H6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the VHH-72 benchmark 29, the sequence of the VHH was synthesized as a GeneBlock (Integrated DNA Technologies, Coralville, IA) flanked by SfiI sites for cloning into pMRo.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pMRo</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Production of VHHs in mammalian cells in fusion with human IgG1 Fc (VHH-Fcs): Codon-optimized genes for bivalent VHH-Fcs were synthesized and cloned into pTT5 (GenScript; Piscataway, NJ.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTT5</div><div>suggested: RRID:Addgene_52326)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped and live virus neutralization assays: a) Pseudotyped virus neutralization assays: (i) Generation of SARS-CoV-2 spike pseudotyped lentiviral particles (LVP): HEK293T cells were plated in a 100-mm tissue culture dish and transfected the next day at about 75% confluency with a combination of a lentiviral transfer vector encoding eGFPLuc (addgene#119816), the packaging plasmid psPAX2 (addgene#12260), and a plasmid encoding the viral glycoprotein of interest SARS-CoV-2 Spike-ΔS1/S2-Δ20 expressed in pcDNA3.1+.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>pcDNA3.1+</div><div>suggested: RRID:Addgene_117272)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After a final wash, cells were resuspended in 100 µL PBSB and data were acquired on a CytoFLEX S flow cytometer (Beckman Coulter, Brea, CA) and analyzed by FlowJo software (FlowJo LLC, v10.6.2</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Proteins were buffer exchanged using Amicon® Ultra-15 Centrifugal Filter Units (Millipore-Sigma</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Millipore-Sigma</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IC50 was determined from non-linear regression [Inhibitor] vs. response, Variable slope (four parameters) using GraphPad Prism version 9 (La Jolla, CA). 4.11.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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1. chaeyeonlim 23 Dec 2021
  
  in BehSci
  
  Following the meeting of the @WHO TAG-VE today, WHO classifies B.1.1.529 as a variant of concern named Omicron. We call for increased surveillance and genetic testing & thank for sharing their work in real time. More information can be found here https://who.int/news/item/26-11-2021-classification-of-omicron-(b.1.1.529)-sars-cov-2-variant-of-concern
  
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https://biorxiv.org/cgi/content/10.1101/2021.12.18.473303

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1. sciscore 23 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.18.473303: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  NIH rigor criteria are not applicable to paper type.
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies and Virtual screening for anti-nsp5 compounds are described in the Supplementary Methods Cells and virus: HEK-293T cells (purchased from ATCC, # CRL-11268) and A549 cells stably expressing the human ACE2 receptor (A549-ACE2, kindly provided by O. Schwartz, Institut Pasteur) were grown in complete Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (FBS).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-nsp5</div><div>suggested: (Grupo de la Dra. Susana Lopez; Instituto de Biotecnologia, UNAM. Cat# NSP5 (C-6, RRID:AB_2802098)</div></div><div style="margin-bottom:8px"><div>A549-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunoblot membranes were incubated with primary antibodies directed against nsp5 [31], NS3/4A (ab21124, Abcam), FLAG-tag (F3165, Sigma) or GAPDH (MA5-15738, Invitrogen), and revealed with HRP-linked secondary antibodies (Jackson ImmunoResearch) using ECL2 substrate (Pierce).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FLAG-tag</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>GAPDH</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>MA5-15738 , Invitrogen) ,</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies and Virtual screening for anti-nsp5 compounds are described in the Supplementary Methods Cells and virus: HEK-293T cells (purchased from ATCC, # CRL-11268) and A549 cells stably expressing the human ACE2 receptor (A549-ACE2, kindly provided by O. Schwartz, Institut Pasteur) were grown in complete Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal bovine serum (FBS).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Transfected HEK-293T cells were trypsinized at 6 hpt and distributed in the 384-well plates (2×104 cells per well in 50 µL).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antiviral activity and cell viability assays: A549-ACE2 cells seeded in 384-well plates (1.5×103 cells per well) were incubated with the compound of interest at the indicated concentration in DMEM-2% FBS 2 h prior to infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549-ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These plasmids were used as templates to amplify the sequence encoding nsp5 (WT or C145A), and the resulting amplicons were subcloned into the pcDNA3.1 plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pLEX expression vectors for SARS-CoV-2, IBV and hCoV-229E nsp5 are described in [26] and were kindly provided by N. Heaton (Duke University).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLEX</div><div>suggested: RRID:Addgene_117987)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Hepatitis C Virus (HCV) NS3/4A expression vector was generated by subcloning a synthetic gene which was codon optimized for expression in human cells (Genscript) into the pCI plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI</div><div>suggested: RRID:Addgene_74230)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate the reverse-Nanoluciferase reporter plasmid Rev-Nluc-CoV, a synthetic codon-optimized nucleotide sequence encoding a permuted Nanoluciferase with the NGSVRLQSSLK linker between the N- and the C-terminal domains was cloned into the pLV-CMV-eGFP lentiviral vector (Duke vector core, Duke University) in place of the eGFP insert.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>reverse-Nanoluciferase reporter</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLV-CMV-eGFP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral protease activity assays: HEK-293T cells were seeded in 96-well white opaque plates (Greiner Bio-One, 3×104 cells per well) one day before being transfected with 5 ng of the Rev-Nluc-CoV reporter plasmid and 95 ng of the pcDNA3.1-nsp4-5-6 WT, pcDNA-3.1-nsp4-5-6 C145A, pLEX-nsp5 or an empty control pCI plasmid, using polyethyleneimine (PEI-max, #24765-1 Polysciences Inc).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1-nsp4-5-6 WT</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pcDNA-3.1-nsp4-5-6 C145A</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pLEX-nsp5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To measure NS3/4A activity, 5 ng of the Rev-Nluc-HCV reporter plasmid was co-transfected with 145 ng of the pCI-NS3/4A WT or pCI-NS3/4A S139A, using the same protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCI-NS3/4A</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCI-NS3/4A S139A</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In the 384-well plate setting, ∼107 HEK-293T cells were transfected in 50 cm2 dishes with 1 µg of the Rev-Nluc-CoV plasmid and 15 µg of the nsp4-5-6 WT or nsp4-5-6 C145A expression plasmids.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Rev-Nluc-CoV</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Luna Universal One-Step RT-qPCR Kit (New England Biolabs) was used, with SARS-CoV-2 specific primers targeting the N gene region (5’-TAATCAGACAAGGAACTGATTA-3’ and 5’-CGAAGGTGTGACTTCCATG-3’) and with the following cycling conditions: 55 °C for 10 min, 95 °C for 1 min, and 40 cycles of 95 °C for 10 sec, followed by 60 °C for 1 min, in an Applied Biosystems QuantStudio 6 thermocycler.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: (BioLegend Cat# 946101, RRID:AB_2892515)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ChEMBL protocols [56]); Removal of chemical duplicates (Open Babel [57]); Selection of dominant tautomers (pH 7.4, ChemAxon Calculator Plugins version 19.0.9, https://chemaxon.com); Selection of species populating more than 25% or, if none, species that are present at more than 75% of the most frequent one, or at least the three most frequent ones; Generation of stereoisomers and low energy ring conformers generation (CORINA classic [58] version 3.4, https://www.mn-am.com);</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ChEMBL</div><div>suggested: (ChEMBL, RRID:SCR_014042)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Molecules were kept in the sdf format for docking with FlexX [40] and converted into pdbqt files using MGLTools scripts [63]for docking with Smina [39].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MGLTools</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We used Smina (version Dec 2020 based on Autodock Vina 1.1.2, exhaustiveness set to 12, 10 best score poses saved) to perform docking targeting all three PDB structures.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Autodock</div><div>suggested: (AutoDock, RRID:SCR_012746)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">It also comprised 5RH8, which gave the best results with FlexX, allowing to diversify the screening with FlexX, while providing a basis for comparison between the two programs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlexX</div><div>suggested: (FlexX, RRID:SCR_000186)</div></div></td></tr></table>
  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: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04960202</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">EPIC-HR: Study of Oral PF-07321332/Ritonavir Compared With P…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT05011513</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Evaluation of Protease Inhibition for COVID-19 in Standard-R…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT05047783</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Masitinib in Patients With Symptomatic Mild to Moderate COVI…</td></tr></table>
  Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2021.12.18.473303
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https://biorxiv.org/cgi/content/10.1101/2021.12.16.472155

1
1. sciscore 23 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.16.472155: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Mouse studies: Ethical approval: All the experimental procedures were performed in accordance with the guide for the use of laboratory animals of the University of Sao Paulo and approved by the institutional ethics committee under the protocol number 105/2021. SARS-CoV-2: SARS-CoV-2 was isolated from a COVID-19 positive-tested patient.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">SARS-CoV-2 experimental infection and treatments: Female K18-hACE2 mice, aged 8 weeks, were infected with 2×104 PFU of SARS-CoV-2 (in 40 μL) by intranasal route.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">A total of 10 photomicrographs in 40X magnification per animal were randomly obtained using a microscope Novel (Novel L3000 LED, China) coupled to a HDI camera for images capture.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of Spike RBD of SARS-CoV-2: A codon-optimized gene encoding for SARS-CoV-2 (331 to 528 amino acids, QIS60558.1) was expressed in Expi293 cells (Thermo Fisher Scientific) with human serum albumin secretion signal sequence and fusion tags (6xHistidine tag, Halo tag, and TwinStrep tag) as described before 1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HUVEC single cell donor (Lonza, cat#C2517A) cells were transduced at room temperature with ACE2 using a BacMam viral vector at a concentration of 2e9 VG/ml (Montana Molecular #C1120G Pseudo SARS-CoV-2 D614G Green Reporter) followed by incubation at 36°C for 24 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HUVEC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-Cov-2 tested in A549-ACE2 cells: A549-ACE2 cells were plated in Corning black walled clear bottom 96 well plates 24 hours before infection for confluency.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-Cov-2 tested in Calu-3 cells: Calu-3 (ATCC, HTB-55) cells were pretreated with test compounds for 2 hours prior to continuous infection with SARS-CoV-2 (isolate USA WA1/2020) at a MOI=0.5.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu-3</div><div>suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Supernatant from the Caco-2 cells are collected on day 3 post-infection and titrated on Vero 76 cells for virus titer as before.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Caco-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Vero 76</div><div>suggested: KCLB Cat# 21587, RRID:CVCL_0603)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HCoV 229E antiviral assay: HCoV 229E, (a gift from Ralph Baric, UNC, Chapel Hill) was propagated on Huh-7 cells and titers were determined by TCID50 assay on Huh-7 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Huh-7</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The virus was propagated and titrated in Vero E6 cells in a biosafety level 3 laboratory (BSL3) at the Ribeirao Preto Medical school (Ribeirao Preto, Brazil).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Penicillin 10,000 U/mL; Streptomycin10,000 μ Vero cells in DMEM (FBS 2%) incubated at 37 °C with 5% CO2 for 48 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">K18-hACE2 mice: To evaluate the effects of vandetanib in vivo, we infected the K18-hACE2 humanized mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J)7, 8, 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>B6.Cg-Tg(K18-ACE2)2Prlmn/J)7</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">K18-hACE2 mice were obtained from The Jackson Laboratory and were breed in the Centro de Criação de Animais Especiais</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18-hACE2</div><div>suggested: RRID:IMSR_GPT:T037657)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MHV-A59 with nano-Luciferase: The MHV-A59 G plasmid was engineered to replace most of the coding sequence for orf4a and 4b with nano-luciferase (nLuc).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>The MHV-A59 G</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A sequence verified G-nLuc plasmid was used with MHV-A59 wild type A, B, C, D, E and F plasmids to recover virus expressing nLuc, using our previously described molecule clone (Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59 4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>G-nLuc</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The total septal area and total area were analyzed with the aid of the Pro Plus 7 software (Media Cybernetics, Inc., MD, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Pro Plus</div><div>suggested: (Image-Pro Plus, RRID:SCR_016879)</div></div></td></tr></table>
  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: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr style="background-color:#FF0000"><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT0427541464</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Trial number did not resolve on clinicaltrials.gov. Is the number correct?</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NA</td></tr></table>
  Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2021.12.16.472155
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https://biorxiv.org/cgi/content/10.1101/2021.12.15.472864

1
1. sciscore 21 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.15.472864: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The IgG antibodies and Fabs were purified with a CaptureSelect™ CH1-XL Matrix column (Thermo Fisher Scientific) for affinity purification and a HiLoad Superdex 200 pg column (Cytiva) for size exclusion chromatography.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequences of the antibodies against SARS-CoV-2 were included in further analysis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">11 antibody (BioLegend, 901524), PE-conjugated goat anti-human IgG polyclonal antibodies (Southern Biotech, 2040-09), and propidium iodide (Invitrogen, P1304MP) for 20 minutes on ice.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (SouthernBiotech Cat# 2040-09, RRID:AB_2795648)</div></div><div style="margin-bottom:8px"><div>2040-09</div><div>suggested: (SouthernBiotech Cat# 2040-09, RRID:AB_2795648)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus production and neutralization assays: Plasmids encoding SARS-CoV, SARS-CoV-2, or other variants of the spike protein, with the ER retrieval signal removed were co-transfected with MLV-gag/pol and MLV-CMV-Luciferase plasmids into HEK293T cells to generate pseudoviruses.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Right before the end of the incubation, HeLa-hACE2 cells were suspended with culture medium at a concentration of 2 x 105/ml.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa-hACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of SARS-CoV-2 RBD: The receptor-binding domain (RBD) (residues 333-529) of the SARS-CoV-2 spike (S) protein (GenBank: QHD43416.1), was cloned into a customized pFastBac vector (Ekiert et al., 2011), and fused with an N-terminal gp67 signal peptide and C-terminal His6 tag (Yuan et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFastBac</div><div>suggested: RRID:Addgene_1925)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">YYDRxG pattern search: Over 205,000 antibody sequences including heavy and light chains were retrieved from GenBank using Biopython program (Cock et al., 2009), supplemented with sequences reported in previous publications (Cho et al., 2021; Gaebler et al., 2021; Robbiani et al., 2020; Z. Wang, J. C. C. Lorenzi, et al., 2021; Z. Wang, F. Muecksch, et al., 2021; Z. Wang, F. Schmidt, et al., 2021), and then subjected to repertoire analysis using PyIR program (Soto et al., 2020) implemented with IgBLAST (Ye et al., 2013).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Biopython</div><div>suggested: (Biopython, RRID:SCR_007173)</div></div><div style="margin-bottom:8px"><div>PyIR</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgBLAST</div><div>suggested: (IgBLAST, RRID:SCR_002873)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequence analysis and surface conservation: Sequences of sarbecoviruses were retrieved from GenBank using Biopython program, aligned using MUSCLE program (Edgar, 2004) built in European Bioinformatics Institute (EBI) web services (Madeira et al., 2019) and scored for surface conservation in ConSurf server (Ashkenazy et al., 2016; Landau et al., 2005).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MUSCLE</div><div>suggested: (MUSCLE, RRID:SCR_011812)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The conserved surface of SARS-CoV-2 RBD was visualized using the PyMOL program (Schrödinger, LLC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Iterative model building and refinement were carried out in COOT (Emsley & Cowtan, 2004) and PHENIX (Acta Crystallographica Section D: Biological CrystallographyAdams et al., 2010), respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>COOT</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Epitope and paratope residues, as well as their interactions, were identified with the PISA program (Krissinel & Henrick, 2007) using calculated buried surface area (BSA >0 Å2) as the criterion.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PISA</div><div>suggested: (PISA, RRID:SCR_015749)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Binding curves were fitted with four-parameter non-linear regression analysis to calculate the apparent equilibrium binding constant (KDApp) in GraphPad Prism 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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Visit annotations in context

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sciscore

URL

biorxiv.org/cgi/content/10.1101/2021.12.15.472864
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.12.14.472668

1
1. sciscore 20 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.14.472668: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Field Sample Permit: Human blood collection and isolation of platelets: Platelet studies using human samples were conducted according to the principles of the Declaration of Helsinki and approved by St Thomas’s Hospital, London, UK Research Ethics Committee (Ref. 07/Q0702/24).<br>IRB: Human blood collection and isolation of platelets: Platelet studies using human samples were conducted according to the principles of the Declaration of Helsinki and approved by St Thomas’s Hospital, London, UK Research Ethics Committee (Ref. 07/Q0702/24).<br>Consent: All volunteers were screened prior to entering the study and gave written informed consent.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Of these, 25 were male and 16 were female; the average age was 77 for men and 84 for women.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing 3 times with PBS, the plate was permeabilized with 0.5% Triton (Sigma) for 10 min, blocked with 1% BSA for 1 hr at room temperature (RT) and stained with anti-GFP antibody (1:1000; Abeam #ab6556) or anti-V5 tag antibody (1:500; Invitrogen #37-7500) for 2 hr at RT, and Cell Mask (1:2000; Thermo Fisher Scientific) for 30 min at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-V5</div><div>suggested: (SICGEN Cat# AB0096, RRID:AB_2333109)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blots were then incubated (4°C overnight) with primary antibodies against Spike, tubulin or TMEM16F.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibodies against Spike, tubulin</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Membranes were incubated for 1 hr at room temperature with anti-rabbit HRP-conjugated antibody (1:5,000) or anti-mouse HRP-conjugated antibody (1:10,000).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies: Immunofluorescence analysis was performed for actin (AlexaFluor 488 Phalloidin, A12379, ThermoFisher Scientific) and GFP (ab6556, Abcam).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>actin</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>GFP</div><div>suggested: (Abcam Cat# ab6556, RRID:AB_305564)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunoblots were performed with primary antibodies against Spike (Genetex, #GTX632604; 1:1,000), tubulin (Cell Signaling, #3873S; 1:10,000) and TMEM16F (Sigma-Aldrich, #HPA038958; 1:1,000).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibodies against Spike (Genetex,</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>, tubulin (Cell Signaling,</div><div>suggested: (Cell Signaling Technology Cat# 86298, RRID:AB_2715541)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped viral particles were produced as follows: HEK293T cell (2.5 × 106) were seeded in a 100 mm-dish and co-transfected by calcium phosphate with 10 μg pLVTHM/GFP (Addgene 12247), 7.5 μg psPAX2 (Addgene 12260), and 6 μg pMD2.G (Addgene 12259) or pEC120-S-D19-V5, for a total of 13.5 μg DNA for each dish.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To assess pseudoparticle infectivity, HEK-293T cells were bulk transfected with a plasmid expressing human ACE2 and then seeded in a 96-well plate (3×103 cells per well).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For platelet aggregation on Spike-expressing cells, Vero cells were kept in culture in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% of foetal bovine serum (FBS) and transfected using FuGENE HD (Promega) transfectant reagent as suggested by the manufacturer either with a plasmid encoding the Green Fluorescent Protein (GFP; pZac-GFP) or a plasmid encoding the full-length SARS-CoV-2 Spike protein with V5 tag (pSARS-COV-2-S).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The modified DNA segment was obtained by recombinant PCR using primers Fw 5’ ACGCGTCGACTTTTGTGGCAAAGGTT, Re 5’ CCCAAGCTTGGGACGCGTCGTTACGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACCACCACCTCCACCGCAGCATGATCCGCATGAGC and cloned into the pEC117-Spike-V5 vector33.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pEC117-Spike-V5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped viral particles were produced as follows: HEK293T cell (2.5 × 106) were seeded in a 100 mm-dish and co-transfected by calcium phosphate with 10 μg pLVTHM/GFP (Addgene 12247), 7.5 μg psPAX2 (Addgene 12260), and 6 μg pMD2.G (Addgene 12259) or pEC120-S-D19-V5, for a total of 13.5 μg DNA for each dish.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVTHM/GFP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>pMD2.G</div><div>suggested: RRID:Addgene_12259)</div></div><div style="margin-bottom:8px"><div>pEC120-S-D19-V5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, PRP was incubated with VSV-G or Spike pseudoparticles (1:10) for 10 min at 37°C, followed by stimulation with collagen (30 μg/ml) for 20 min (350 rpm, 37°C).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VSV-G</div><div>suggested: RRID:Addgene_138479)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis was performed using the ImageJ software (Fiji).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Fiji</div><div>suggested: (Fiji, RRID:SCR_002285)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis was performed using ImageJ software (Fiji).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis was performed using the FlowJo software v.10 (TreeStar Inc).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PRP was incubated with VSV-G (1:10), Spike pseudoparticles (1:10), His-tag recombinant SARS-CoV-2 Spike Protein S1/S2 (S-ECD) (1 ng/ml; ThermoFisher Scientific; aa11-1208; RP-87680) or Flag-tag recombinant SARS-CoV-2 Spike RBD (1 ng/ml; Bio-Techne, 10689-CV-100) for 10 min at 37°C, followed by stimulation with thrombin (0.5 units).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ThermoFisher Scientific</div><div>suggested: None</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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Mastering Zotero

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1. radera 20 Dec 2021
  
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  press Shift + Enter when you have selected a tag
  
  lista multiple
  
  #zotero
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LibGuides: Zotero : Sharing & Organizing

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1. radera 20 Dec 2021
  
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  Colour code your tags and assign them position numbers for even more options
  
  color==concept; tag==value from a dominion
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Related items: types of relations?

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1. radera 19 Dec 2021
  
  in Public
  
  hernandez-cerdan August 13, 2019 edited August 13, 2019 There are non-linear relationships between items. And a single label (a tag, or a collection) sometimes is not enough to capture these.It can be solved by adding a lot of tags, sure, but it is much more work for the user to categorize items in such detail.Also, the problem of tags/collections is that do not scale well, when you have too many, it becomes harder to navigate. I prefer broad collections, more specific tags, and then, fine detail connections using Related, but why limiting it to first neighbors?My suggestion on adding the second neighbor pair-wise relationship would be able to capture complex relationships almost for free for the user.The user only worries about pair-wise relationships as it is now. But then, clicking in the Related tab, the first neighbors are shown (as it is now) and below (there is plenty of free space) the second neighbors are shown.
  
  ok, good answer
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https://biorxiv.org/cgi/content/10.1101/2021.12.14.472513

1
1. sciscore 19 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.14.472513: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The primary antibodies used were rabbit anti-ACE2 (1:1000, ab15348, Abcam)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-ACE2</div><div>suggested: (Abcam Cat# ab15348, RRID:AB_301861)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, rabbit anti-TACE (1:1000, 3976S, Cell Signaling Technology, MA, USA), rabbit anti-ADAM10 (1:1000, 14194S, Cell Signaling Technology), rabbit anti-Flag-tag (1:1000, PM020, MBL, MA, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-TACE</div><div>suggested: (Leinco Technologies Cat# T460, RRID:AB_2831960)</div></div><div style="margin-bottom:8px"><div>anti-ADAM10</div><div>suggested: (LSBio (LifeSpan Cat# LS-C122598-1000, RRID:AB_10800328)</div></div><div style="margin-bottom:8px"><div>anti-Flag-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, mouse anti-tubulin (1:1000, CP06, Millipore), and mouse anti-VSVM (1:1000, 23H12, Absolute antibody).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-tubulin</div><div>suggested: (LSBio (LifeSpan Cat# LS-C76623-1000, RRID:AB_10633290)</div></div><div style="margin-bottom:8px"><div>CP06</div><div>suggested: (Millipore Cat# CP06, RRID:AB_2617116)</div></div><div style="margin-bottom:8px"><div>anti-VSVM</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Secondly antibodies used were HRP-linked donkey anti-rabbit IgG antibody (NA934; GE Healthcare, Piscataway, NJ, USA) and HRP-linked donkey anti-mouse IgG antibody (NA931V; GE Healthcare)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: (GE Healthcare Cat# NA934, RRID:AB_772206)</div></div><div style="margin-bottom:8px"><div>HRP-linked donkey anti-mouse IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were then incubated with anti-SARS-CoV-2 nucleocapsid (1:1000, GTX135357, GeneTex, CA, USA) primary antibody for 16 h at 4 °C and detected with anti-rabbit-Alexa488 (1:200, A11008, Invitrogen, CA, USA) secondary antibodies for 40 min at RT.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 nucleocapsid ( 1:1000 , GTX135357</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit-Alexa488</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>A11008</div><div>suggested: (Molecular Probes Cat# A-11008, RRID:AB_143165)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A704, Calu-3, VeroE6, HEC50B, and Caco-2 cells were maintained in Eagle’s minimum essential medium (EMEM; 055-08975,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu-3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Caco-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">OUMS-23, IGROV1, and 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; 041-30081,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEC50B cells infected with pseudotype viruses were selected with 300 μg/mL hygromycin for at least 1 week.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEC50B</div><div>suggested: JCRB Cat# JCRB1145, RRID:CVCL_2929)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the DSP assay using 293FT cells, DSP8-11 expressing effector cells expressing S protein and DSP1-7 expressing target cells expressing CD26 or ACE2 alone or together with TMPRSS2 were seeded in 10 cm cell culture plates (4 × 106 cells/10 mL) one day prior to the assay (S1a,b Fig).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293FT</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After an overnight incubation at 37 °C in 5% CO2, cells were treated with protease inhibitors for 1 h and added with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 for HEC50B and HEC50B-TMPRSS2 cells, and MOI of 0.1 for VeroE6, Calu-3, and A704 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEC50B-TMPRSS2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>A704</div><div>suggested: NCI-DTP Cat# A704, RRID:CVCL_1065)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">OVTOKO (JCRB1048), OVISE (JCRB1043), HEC50B (JCRB1145), VeroE6-TMPRSS2 (JCRB1819)[67], and OUMS-23 (JCRB1022) cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6-TMPRSS2 (JCRB1819)</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VeroE6-TMPRSS2 (JCRB1819) cells were cultured in DMEM containing 10% FBS and 1 mg/mL G418.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6-TMPRSS2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To establish stable cell lines expressing the S protein of SARS-CoV, SARS-CoV-2, or MERS-CoV, recombinant pseudotype lentiviruses were produced in 293T cells with psPAX2 packaging plasmid, vesicular stomatitis virus (VSV)-G-expressing plasmid and lentiviral transfer plasmid expressing S protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VSV)-G-expressing</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">recombinant pseudotype lentivirus expressing TMPRSS2 was produced using 293T cells with psPAX2 packaging plasmid and VSV-G-expressing plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>VSV-G-expressing</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IGROV1 cells (SCC203) were purchased from Merck (Darmstadt, Germany) and Caco-2 cells (RCB0988) were obtained from the RIKEN BioResource Research Center (Tsukuba, Japan).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RIKEN BioResource</div><div>suggested: (RIKEN BioResource Center, RRID:SCR_003250)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Statistical analyses were performed in Microsoft Excel 2016 (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Microsoft Excel</div><div>suggested: (Microsoft Excel, RRID:SCR_016137)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(Microsoft, Redmond, WA, USA) and GraphPad Prism 8 (GraphPad Software, San Diego, CA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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:
  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.
  Results from scite Reference Check: We found no unreliable references.
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  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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Meta Theme Color and Trickery

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1. kael 18 Dec 2021
  
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  If you want to define a theme color for light and dark mode, your best bet is to define both colors and use the first meta tag as a fallback for browsers that don’t support the media feature.
  
  <meta name="theme-color" content="#319197" media="(prefers-color-scheme: light)"> <meta name="theme-color" content="#872e4e" media="(prefers-color-scheme: dark)">
  
  js css pwa
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Share like a native app with the Web Share API

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1. kael 16 Dec 2021
  
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  If your site has multiple URLs for the same content, share the page's canonical URL instead of the current URL. Instead of sharing document.location.href, you would check for a canonical URL <meta> tag in the page's <head> and share that. This will provide a better experience to the user. Not only does it avoid redirects, but it also ensures that a shared URL serves the correct user experience for a particular client. For example, if a friend shares a mobile URL and you look at it on a desktop computer, you should see a desktop version:
  
  let url = document.location.href; const canonicalElement = document.querySelector('link[rel=canonical]'); if (canonicalElement !== null) { url = canonicalElement.href; } navigator.share({url});
  
  web share js
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Expialidocio.us

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1. kael 16 Dec 2021
  
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  Expialidocio.us is a tool for visualizing your del.icio.us posting activity. It displays a graph of your posting activity over time. You can select a timespan from this graph, and it will show you a tag cloud weighted by just those dates. Expialidocio.us was inspired by a posting by Jon Udell. Coming full circle, Udell has posted since posted about this application. Since then, I’ve published the sources.
  
  Expialidocio.us del.icio.us viz
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ged/ruby-pg: A PostgreSQL client library for Ruby

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1. TylerRick 15 Dec 2021
  
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  We tag and release gems according to the Semantic Versioning principle. As a result of this policy, you can (and should) specify a dependency on this gem using the Pessimistic Version Constraint with two digits of precision.
  
  you can (and should)...
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The VarA-CsrA regulatory pathway influences cell shape in Vibrio cholerae

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1. EMBOpress 14 Dec 2021
  
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  Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
  
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  Reply to the reviewers
  
  Description of the planned revisions
  
  From Review Commons: Please find below our point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees
  
  **************
  
  Reviewer #1 (Evidence, reproducibility and clarity (Required)):
  
  Comment #1: In this manuscript, the authors follow up on an interesting finding that varA null mutants of V. cholerae form spherical cells in stationary phase. The authors determine that this cell rounding is due to weakening of the cell wall via less production of tetrapeptide cross links. Mutation of the regulator csrA and the enzyme aspA lead to a model in which a varA mutant cell lacks aspartate leading to low cross-linked cell wall that is unable to hold the typical curved V. cholerae shape. The data are robust, and the manuscript is clearly written.
  
  Authors’ reply #1: We very much appreciate the reviewer’s accurate summary and the appraisal of the robust data and the clearly written manuscript.
  
  *
  
  Comment #2: I think the finding is quite interesting, even though it is not clear to me if this observed cell morphology has a biological function or if it is an artifact of completly removing VarA. However, this manuscript builds the foundation to further test this question.
  
  Authors’ reply #2: We agree with the reviewer. However, it is worth mentioning that this two-component system (TCS) has been first described in 1998 yet the input signal (or repression of the signal under certain conditions) still remains elusive. Maybe, this isn’t too surprising, given that studies on V. cholerae are strongly biased towards its pathogenic lifestyle, while the varA/varS system is highly conserved among Gram-negative bacteria including non-pathogenic environmental V. cholerae strains. These strains can live under very diverse conditions of slow or fast growth, including long starvation periods. Unfortunately, we still lack significant insight into this part of the V. choleraebiology. We therefore believe that the current study is very important, as the elucidation of the molecular mechanism of the observed shape transition within the varA mutant will foster fresh hypotheses on the role that the system plays in V. cholerae and what signals might be sensed.
  
  We would also like to remind the editor and reviewer(s) that a plethora of studies have been published based on varA and varS (and csrA) deletion mutants of V. cholerae with various readouts ranging from transcriptomics to quorum sensing defects, impairment of virulence, etc. Thus, the argument that the complete removal of varA might cause an artifact seems equally valid for previous work by others and, maybe, even the vast majority of studies in which TCS are investigated for which the sensed signal has yet to be identified.
  
  In conclusion, we propose to address this valid point of critique in the revised manuscript by clearly stating the caveat of the gene deletion(s). However, as the reviewer correctly stated, “this manuscript builds the foundation to further test this question.”
  
  Comment #3: The data all support the conclusions, but I do think the authors could have really confirmed their model by connecting mutations in csrA and aspA to restoration of high cross-linked cell well similar to the WT strain as done in Fig. 2. As it stands, this is still somewhat hypothetical and has not been directly demonstrated, although I do think their model is correct and these experiments will be conformation of it.
  
  Authors’ reply #3: We thank the reviewer for their comment and the assumption that our model might be correct. It is very unlikely that the csrA suppressor mutant(s) or the ∆varA∆aspA mutant maintain the low level of cross-links and the high level of dipeptides that we observed for the ∆varA mutant. Indeed, it would be unclear how the cells could restore the Vibrio shape that we visualized in the phase contrast image under such conditions. However, as this point seems very important to the reviewer (see also comment #7 below), we will perform the suggested cell wall analysis of these mutants and include the new data in the revised manuscript.
  
  Comment #4: I also have a few other suggestions to improve the manuscript, but in sum I think it is a well-done research study that will be interesting to research in V. cholerae and other gamma proteobacteria.
  
  Authors’ reply #4: Once again, we thank the reviewer for their kind words.
  
  Major comments:
  
  Comment #5: 1. The enrichment for suppressors is very creative and connected the varA impact on cell morphology to misregulation of csrA as 10/10 mutants were ultimately linked to this gene. However, insertion in aspA should also suppress this phenotype, and I am curious why this gene was not identified in the transposon suppressor screen.
  
  Authors’ reply #5: This is a very relevant and important comment. The reason why we did not isolate ∆varA-aspA::Tn mutants is most likely due to a growth defect that we observed for the double ∆varA∆aspA mutant compared to the ∆varA-csrA suppressor mutant(s). In the figure on the right, respective growth curves are shown [∆varA∆aspA in orange and the ∆varA-csrA suppressor mutant ∆varA-Tn A in gray]. Any ∆varA-aspA::Tn mutant is therefore expected to be outcompeted by the ∆varA-csrA suppressor mutants during the enrichment process. We will include this information and the corresponding data (e.g., final growth curves after 3 biologically independent experiments) in the revised manuscript.
  
  [figure not shown in online form]
  
  Comment #6: 2. The authors should complement at least one of their varA/csrA mutants with csrA.
  
  Authors’ reply #6: Agreed. We are in the process of performing the suggested experiment and will include the results in the revised manuscript.
  
  Comment #7: 3. The changes in cell wall structure are not directly connected to the genetic identification of csrA and aspA. Yes, I agree their model makes sense, but to really nail it down they should analyze the cell wall composition in the varA/csrA and varA/aspA double mutants and show it returns to WT levels of crosslinking.
  
  Authors’ reply #7: As mentioned above, we will perform those cell wall analyses (see also authors’ reply #3 above), as requested.
  
  Comment #8: 4. Does deletion of aspA in the WT or varA mutant impact the growth rate?
  
  Authors’ reply #8: This is indeed the case in the ∆varA background but not in the WT background (as shown under authors reply #5). These data will be included in the revised manuscript.
  
  *
  
  Minor comments
  
  Comment #9: 5. Are the round cells able to divide? The data in Fig. S2 would suggest they can based on the increase in CFUs from hour 6 to hour 8, but the authors never comment on this point and it might be worth addressing in the discussion.
  
  Authors’ reply #9: We never observed diving round cells. Indeed, the increase of the optical density and CFUs from 6h to 8h is most likely based on those bacteria that have not yet changed their cellular morphology and therefore keep dividing (see below the imaging/quantification for this timeframe taken from Fig. 2). What we observed though is that upon dilution into fresh medium, the round cells start to elongate and then divide resulting in newborn Vibrio shaped cells. We will include these new data in the revised manuscript.
  
  [figure not shown in online form]
  
  *
  
  Comment #10: 6. Fig. S2-Why does the OD600 increase from 8 to 24 hours but the CFUs decrease in the varA mutant?
  
  Authors’ reply #10: This is an interesting observation that might reflect the presence of dead but not yet lysed cells in these cultures. Indeed, while it looks as if the OD600 values are still increasing for the ∆varA mutant at 24h, we cannot exclude at this point that the OD600 values increased during the 16h-time interval and went again down at 24h (e.g., like shifting the WT peak to later time points/the right of the X-axis). Notably, the purpose of this figure was mostly to i) indicate the slower growth of the ∆varA mutant while ii) emphasizing that late during growth (e.g., 24h here) the strain can still reach similar OD600 values as well as CFUs/ml as the WT strain. We will change Fig. S2 to better emphasize these two points in the revised manuscript.
  
  Comment #11: 7. Lines 309-A little bit more detail here would help the reader. Are the authors examining whole cell lysates or lysates from specific cellular components? I am actually very surprised this worked as there are so many proteins in crude cell lysates.
  
  Authors’ reply #11: Indeed, these are whole cell lysates, which were prepared as described in the methods section (lines 482 onwards; “SDS-PAGE, Western blotting and Coomassie blue staining”). We fully agree that there are many proteins in the crude cell lysates and realized that we might not have explained well enough that only the gel region containing the overproduced band in the ∆varA strain and the same location in the WT sample were analyzed by mass spectrometry (even though we were referring to the “gel pieces” in line 498 onwards). Please accept our sincerest apologies for this neglect. During the revision, we will ensure that this information is explicitly stated and that these details are included in the main text and the methods section.
  
  Comment #12: 8. Lines 320-321-I don't think there is evidence that CsrA enhances aspA RNA translation, merely that CsrA enhances AspA protein production. It is likely through increasing translation, but this cannot be concluded without direct evidence.
  
  Authors’ reply #12: We fully agree and thank the reviewer for this important comment. Indeed, we meanwhile know that the aspA mRNA levels also increase in the ∆varA mutant strain (which might or might not be linked to enhanced translation). We will add these transcript level data to the revised manuscript and discuss all possibility that could explain the AspA overproduction.
  
  Comment #13: 9. Line 348-350-I do not understand the logic of this sentence stating that the "..until now, the signal that abrogates VarA phosphorylation..." as this manuscript does not contribute to our understanding of the VarS signal.
  
  *Authors’ reply #13: We apologize that this sentence or the logic behind it wasn’t clear. As this is a combined result and discussion section, the aim of the sentence was to put the observed shape transition of the bacteria into a broader context, which required us to mentioned that the input signal is still unknown. We will make sure that this becomes more obvious in the revised manuscript by rephrasing this sentence.
  
  Comment #14: 10. I am curious if the total volume of the round versus curved cells is constant at 20 hours. This should be easy to determine using ImageJ and worth reporting.
  
  Authors’ reply #14: We are not entirely sure how this question is relevant for the study (e.g., for this report on the observed shape transition phenotype it doesn’t matter if the cells maintain the same volume or not). However, given the importance for the reviewer, we will perform these volume measurements on our images and add a sentence to the revised manuscript on the analysis’ outcome (plus include the data as a supplementary panel).
  
  *
  
  Reviewer #1 (Significance (Required)):
  
  Comment #15: Understanding changes to cell morphology and their biological implications is a growing area of microbiology. This study makes a new contribution to this area by demonstrating a round, spherical form of V. cholerae that is driven by alterations to the cell that decrease cell-wall cross linking.
  
  Authors’ reply #15: Once again, we thank the reviewer for this summary and for placing our study into context. We agree that cell morphological changes and the underlying molecular mechanism(s) are an exciting and growing area of microbiology.
  
  Reviewer #2 (Evidence, reproducibility and clarity (Required)):
  
  Comment #16: In this manuscript, Rocha et al studied the effect of the VarA response regulator on cell shape of Vibrio cholerae. VarA is part of a two-component system that also includes the histidine kinase VarS. It has previously been shown that VarA activates the expression of three redundantly acting regulatory RNAs called CsrB, CsrC, and CsrD. All three Csr RNAs share the same regulatory principle, which is to sequester the activity of the RNA-binding protein CsrA. CsrA in turn can bind hundreds of mRNA species in the cell, which in the majority of cases results in reduced translation of these mRNAs (in addition to various other modes of action that have been reported). Here, the authors discovered that deletion of the varA gene results in an abnormal, spherical cell shape in stationary phase grown V. cholerae cells. Biochemical analysis revealed an unusual peptidoglycan composition in varA-deficient cells, showing increased levels of dipeptides, a reduction of tetrapeptides, and an overall decrease in peptide-cross linkage. Interestingly, the varA phenotype was complemented by the addition of conditioned medium from wild-type cells, which are likely to provide peptidoglycan building blocks in trans. The authors further discover that varA-deficiency results in AspA over-production, which could be linked to the activity of CsrA. The authors speculate that high AspA levels deplete the cell of aspartate, which is required to produce peptidoglycan precursors. The manuscript is interesting, well-written, and the rationale of the experiments is easy to follow.
  
  Authors’ reply #16: We thank the reviewer for this excellent summary and the kind words on the quality of the manuscript.
  
  Comment #17: However, I have two major points of criticism, which reduce my enthusiasm for this work. First, the molecular pathways that links varA-deficiency to increased AspA levels is incomplete: please clarify how CsrA activates AspA levels and if this phenotype is linked to direct binding of CsrA to the aspA mRNA and if so how is activation is achieved at the molecular level.
  
  Authors’ reply #17: We thank the reviewer for the comment. However, we never had the intention to decipher the entire pathway and it is indeed possible that intermediate regulators might be involved. Notably, the first part of the signaling pathway (VarA -> CsrB,C,D -> CsrA) seemed well established in the literature and we truly believe that our work supports this part of the pathway (given the numerous csrA suppressor mutants that we obtained in the varA-minus background). For the link between CsrA and AspA, we indeed do not provide direct evidence. Nonetheless, we discuss recent work in Salmonella by mentioning “Interestingly, previous studies identified the aspA mRNA amongst hundreds of direct CsrA targets in Salmonella using the CLIP-seq technique to identify protein-RNA interactions (32)”. Notably, this finding by Holmqvist et al. (2016, EMBO J.) has been reproduced for E. coli by Potts et al. (2017, Nat. Commun.; see Supplementary Data file 1, CsrA CLIP-seq data; with three highly significant peaks corresponding to aspA mRNA binding), an information that we will add to the revised manuscript. Of course, neither Salmonella nor E. coli belongs to the same genus as V. cholerae (though, of course, all are gamma-Proteobacteria). Thus, to accommodate the reviewer’s comment, we will revise the manuscript to include the caveat that direct CsrA binding of the aspA mRNA has been shown in both Salmonella and E. coli but that it is still feasible that intermediate regulatory proteins might be involved in the case of V. cholerae. We will also revise the model to show such potential intermediate steps between CsrA and AspA.
  
  Comment #18: Second, I am not convinced about the biological relevance of the findings. The authors speculate in the discussion section that the VarA-pathway could modulate cell shape under physiological conditions, however, I am not sure such conditions exist given that VarA activity is not only controlled by VarS, but rather integrates information from multiple histidine kinases. I have a several additional comments, which I listed below.
  
  Authors’ reply #18: We regret the referee’s personal opinion that our findings might not be of biological relevance.
  
  However, we respectfully disagree with the notion that such physiological conditions would never occur just because several histidine kinases can feed into VarA signaling. Indeed, as discussed above under authors’ reply #2, the (VarS/)VarA-CsrA pathway is highly conserved in Vibrio species and other proteobacteria. Yet, for V. choleraemost studies have focused on virulence-inducing conditions, while the species’ environmental lifestyle has been vastly neglected in the past. Indeed, even our own work on several V. cholerae’s phenotypes (natural competence for transformation [Meibom*, Blokesch* et al., 2005, Science]; T6SS production in pandemic strains [Borgeaud et al., 2015, Science]; pilus-mediated aggregation [Adams et al., 2019, Nat. Microbiol]; etc.) has remained unknown for decades, given the chitin dependency for their induction – a substrate not commonly studied in lab settings. Interestingly, several of these findings have also initially been considered biologically irrelevant, “artifacts”, or even non-reproducible by reviewers in the past, while nowadays all these phenotypes have been extensively reproduced by many different research groups and are well accepted in the field as biologically highly relevant. Thus, we truly believe that one should be open to new phenotypes and, as reviewer #1 rightfully acknowledged, consider that “this manuscript builds the foundation to further test this question.”
  
  It should also be noted that the (VarS/)VarA-CsrA system has been studied for >15 years based on deletion strains, as we did in this study, and the readouts of these studies have been well accepted in the field without provision of the physiological conditions that would mimic the situation of these knock-out strains.
  
  Collectively, we truly believe that there are still many understudied physiological conditions for V. cholerae; however, finding the right conditions could take years and is therefore beyond the scope of the current study.
  
  Major points:
  
  Comment #19: - Figs. 1 and S1: I think it is interesting that the varS mutant strain does not share the cell shape phenotype with the varA mutant. As pointed out by the authors, this result indicates that varA activity is controlled by another histidine kinase. While I believe it might be beyond the scope of this manuscript to determine which other histidine kinases signal towards VarA, I think it would be useful to measure and compare CsrB/C/D levels in WT, DvarA, and DvarS cells.
  
  Authors’ reply #19: Thanks for this comment. We fully agree that finding the secondary histidine kinase is beyond the scope of this study. In the revised manuscript, we will, however, include the CsrB/C/D levels of the WT, ∆varA, and ∆varS strains, as suggested by the reviewer.
  
  Comment #20: - Figs. 1, S1, and 4C: The regulatory logic implied by these results suggest that deletion of varA results in reduced CsrB/C/D levels, which in turn leads to higher activity of CsrA in the cell. Thus, it would be useful to test if A) over-production of CsrB, CsrC, or CsrC can rescue the phenotype of an varA mutant and if B) combined deletion of csrB/C/D will phenocopy the mutation of varA.
  
  Authors’ reply #20: These are also very good suggestions. Notably, this has been done in the past for the V. cholerae system (Lenz et al., 2005, Mol. Microbiol.). Indeed, after receiving the reviewer’s comment, we immediately asked these authors to kindly share their csrB,C,D overproduction plasmids as well as the triple knock-out strain with us (as all of these constructs have been extensively verified in their published work). Unfortunately, we are not entirely sure whether it will be possible to receive these constructs any time soon, as we were told that such shipment might take >1 year (though, upon further discussion, this timeframe was lowered to ~3 months). If we manage to receive these published constructs in a reasonable timeframe, we will certainly perform the suggested experiments.
  
  Comment #21: - Figs. 4B & 5C: I was somewhat surprised by these results. Given that AspA overproduction is suggested to cause cell shape abnormalities in the varA mutant, I would have expected additional transposon insertion in aspA. The fact that mutations only occurred in csrA could indicate that additional (CsrA-controlled) could be involved in the phenotype.
  
  Authors’ reply #21: See authors’ reply #5 above, where we explain that the ∆varA∆aspA strain has a slight growth disadvantage. For this reason, any ∆varA-aspA::Tn transposon mutant would likely be outcompeted by the csrAsuppressor mutants in our genetic screen.
  
  Minor points:
  
  Comment #22: - Throughout study: italicize gene names
  
  Authors’ reply #22: Gene names have been italicized in the initial manuscript; however, strain names - such as strain ∆varA – haven’t been italicized, in accordance with several of our previous publications. However, for the revision, we will italicize all strain names to accommodate the reviewer’s request.
  
  Comment #23: - Figs. 1C and S3C: please quantify the results of these western blots and indicate how many replicates were performed.
  
  Authors’ reply #23: We apologize for this oversight – indeed, all Western Blot were performed three independent times, as is good scientific practice. We will add this information into the methods section of the revised manuscript.
  
  Concerning the quantification: the primary claim of these figures is that the HapR protein is still produced in the ∆varA mutant in the different pandemic strain backgrounds, while the luxO-mutated strains have a significant defect in HapR production (as we have previously reported; Stutzmann and Blokesch, 2016, mSphere). These data are qualitatively very clear in the Western Blots and can be considered as “black or white” results.
  
  However, for the revision we will quantify the bands’ intensities of the performed Western blots and provide these quantitative data, as requested by the reviewer.
  
  Comment #24: - Fig. 5A and B: in order to properly quantify the levels of AspA in the cell (and link them to CsrA activity in the transposon mutants), I think it would be better to add a tag to the chromosomal aspA gene and perform quantitative Western blot analysis.
  
  Authors’ reply #24: We respectfully disagree. Firstly, this is not a subtle difference that we observe in these cell lysates/the corresponding stained gel bands but a rather strong difference when WT is compared to the mutants (see, for instance, a copy of panel 5B below as a kind reminder). Together with the genetic experiments that follow afterwards, the link seems very solid to us. Secondly, adding a tag could change the proteins abundance (change of the protein’s production/degradation dynamics) and/or activity, which could cause more confusion than needed (and a loss of the spherical cell shape if the enzyme loses its activity through the tagging).
  
  However, as mentioned above under authors’ reply#12, we meanwhile observed that the aspA mRNA levels also increase in the ∆varA mutant. Thus, we will provide qRT-PCR data in the revised manuscript (and discuss all options on how the increase of the transcript and subsequently the protein might be caused, as mentioned above under authors’ reply #12), which we truly believe will fulfill the reviewer’s request for quantification.
  
  [figure not shown in online form]
  
  Reviewer #2 (Significance (Required)):
  
  Comment #25: I think this manuscript starts with an interesting observation, which is that varA mutant cells of V. cholerae display an aberrant cell shape. The manuscript also provides several important findings explaining the molecular basis of this phenotype.
  
  Authors’ reply #25: Once again, we thank the reviewer for the kind words.
  
  Comment #26: However, as pointed out in my report, I think the manuscript is yet incomplete in connecting this information to identify the underlying regulatory mechanism.
  
  Authors’ reply #26: As mentioned above, the focus of this study was never on the elucidation of the entire regulatory pathway. Instead, we aimed at deciphering the molecular mechanism behind an observed phenotype - that is, the cell wall modification in the varA-deficient strain that leads to the bacterium’s spherical shape, which can be restored to the WT Vibrio shape by peptidoglycan precursor cross-feeding from neighboring cells – followed by the identification of several regulators and enzymes that trigger these phenotypes. Overall, we consider this a very complete study. However, as mentioned above, we will certainly discuss in the revised manuscript that the step between CsrA and AspA could be indirect in V. cholerae, in contrast to what was experimentally shown for Salmonella and E. coli.
  
  **Referee Cross-commenting**
  
  Comment #27: As pointed out in my review, I think this manuscript is well written and easy to follow. However, I agree with reviewer #1 that the underlying phenotype is most likely an artifact, which limits the biological relevance of this study. In addition, I am missing the molecular mechanism that connects CsrA with AspA production in V. cholerae.
  
  Authors’ reply #27: See authors’ reply #18 above. We disagree that there is any strong indication that the observed phenotype is an artifact. Given that it is state-of-the-art to study TCS by deleting their genes, our study isn’t any more prone to being an artifact than any other study on TCSs.
  
  We truly believe that it is also important to not take reviewer #1’s comment out of context by stating “I agree with reviewer #1 that the underlying phenotype is most likely an artifact”. Indeed, he/she provided a rather encouraging statement in which he/she mentions the possibility of an artifact but also clearly states that this study is interesting and builds the foundation to further investigate the newly observed phenotype(s): “I think the finding is quite interesting, even though it is not clear to me if this observed cell morphology has a biological function or if it is an artifact of completly removing VarA. However, this manuscript builds the foundation to further test this question.”
  
  Moreover, whether there is a direct (as in Salmonella and E. coli) or indirect connection between CsrA and AspA production is not a key aspect of the current study, as discussed above.
  
  *
  
  PeerReviewed
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  Referee #2
  
  Evidence, reproducibility and clarity
  
  In this manuscript, Rocha et al studied the effect of the VarA response regulator on cell shape of Vibrio cholerae. VarA is part of a two-component system that also includes the histidine kinase VarS. It has previously been shown that VarA activates the expression of three redundantly acting regulatory RNAs called CsrB, CsrC, and CsrD. All three Csr RNAs share the same regulatory principle, which is to sequester the activity of the RNA-binding protein CsrA. CsrA in turn can bind hundreds of mRNA species in the cell, which in the majority of cases results in reduced translation of these mRNAs (in addition to various other modes of action that have been reported). Here, the authors discovered that deletion of the varA gene results in an abnormal, spherical cell shape in stationary phase grown V. cholerae cells. Biochemical analysis revealed an unusual peptidoglycan composition in varA-deficient cells, showing increased levels of dipeptides, a reduction of tetrapeptides, and an overall decrease in peptide-cross linkage. Interestingly, the varA phenotype was complemented by the addition of conditioned medium from wild-type cells, which are likely to provide peptidoglycan building blocks in trans. The authors further discover that varA-deficiency results in AspA over-production, which could be linked to the activity of CsrA. The authors speculate that high AspA levels deplete the cell of aspartate, which is required to produce peptidoglycan precursors. The manuscript is interesting, well-written, and the rationale of the experiments is easy to follow. However, I have two major points of criticism, which reduce my enthusiasm for this work. First, the molecular pathways that links varA-deficiency to increased AspA levels is incomplete: please clarify how CsrA activates AspA levels and if this phenotype is linked to direct binding of CsrA to the aspA mRNA and if so how is activation is achieved at the molecular level. Second, I am not convinced about the biological relevance of the findings. The authors speculate in the discussion section that the VarA-pathway could modulate cell shape under physiological conditions, however, I am not sure such conditions exist given that VarA activity is not only controlled by VarS, but rather integrates information from multiple histidine kinases. I have a several additional comments, which I listed below.
  
  Major points:
  
  Figs. 1 and S1: I think it is interesting that the varS mutant strain does not share the cell shape phenotype with the varA mutant. As pointed out by the authors, this result indicates that varA activity is controlled by another histidine kinase. While I believe it might be beyond the scope of this manuscript to determine which other histidine kinases signal towards VarA, I think it would be useful to measure and compare CsrB/C/D levels in WT, DvarA, and DvarS cells.
  
  Figs. 1, S1, and 4C: The regulatory logic implied by these results suggest that deletion of varA results in reduced CsrB/C/D levels, which in turn leads to higher activity of CsrA in the cell. Thus, it would be useful to test if A) over-production of CsrB, CsrC, or CsrC can rescue the phenotype of an varA mutant and if B) combined deletion of csrB/C/D will phenocopy the mutation of varA.
  
  Figs. 4B & 5C: I was somewhat surprised by these results. Given that AspA overproduction is suggested to cause cell shape abnormalities in the varA mutant, I would have expected additional transposon insertion in aspA. The fact that mutations only occurred in csrA could indicate that additional (CsrA-controlled) could be involved in the phenotype.
  
  Minor points:
  
  Throughout study: italicize gene name
  
  Figs. 1C and S3C: please quantify the results of these western blots and indicate how many replicates were performed.
  
  Fig. 5A and B: in order to properly quantify the levels of AspA in the cell (and link them to CsrA activity in the transposon mutants), I think it would be better to add a tag to the chromosomal aspA gene and perform quantitative Western blot analysis.
  
  Significance
  
  I think this manuscript starts with an interesting observation, which is that varA mutant cells of V. cholerae display an aberrant cell shape. The manuscript also provides several important findings explaining the molecular basis of this phenotype. However, as pointed out in my report, I think the manuscript is yet incomplete in connecting this information to identify the underlying regulatory mechanism.
  
  Referee Cross-commenting
  
  As pointed out in my review, I think this manuscript is well written and easy to follow. However, I agree with reviewer #1 that the underlying phenotype is most likely an artifact, which limits the biological relevance of this study. In addition, I am missing the molecular mechanism that connects CsrA with AspA production in V. cholerae.
  
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https://biorxiv.org/cgi/content/10.1101/2021.12.07.471597

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1. sciscore 14 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.07.471597: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  NIH rigor criteria are not applicable to paper type.
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were incubated on ice for 15 minutes with anti- HA antibody (Invitrogen HA Tag Mouse anti-Tag, DyLight® 650 conjugate, Clone: 2-2.2.14; 1:100 dilution) and Streptavidin-PE (Thermo Fisher scientific; catalog number S866; 1:100 dilution).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti- HA</div><div>suggested: (Claes Ãrvel lab; Karolinska Institute Cat# anti-MuV rabbit 144, RRID:AB_2747378)</div></div><div style="margin-bottom:8px"><div>anti-Tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Competitive indirect enzyme linked immunoassay (competitive ELISA): Nunc™ MaxiSorp 96-well Immuno-Plates (Thermo Fisher Scientific, Illkirch, France) were coated with 200 μL/well of AffinityPure goat anti-mouse IgG+IgM (H+L) antibody (Jackson Immuno Research Laboratories Inc., Pennsylvania, USA) at 10 μg/mL in 50 mM potassium phosphate buffer, and incubated overnight (16h) at 22 ± 2 °C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG+IgM</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293 Freestyle™ were transiently transfected at a density of 2.5 106 cells/mL in 100mL Freestyle medium (Thermo-Fisher) by addition of 150 μg plasmid and 1.8 mL of linear polyethylenimine (PEI, 0.5 mg/ml) (Polysciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then, the culture medium of each plate containing the VERO E6 cells is removed and 500 μL of each VHH/virus mixture is added to each well in duplicate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VERO E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Gap repair transformations were made in plasmid pNT VHH72 between restriction sites NheI and NotI with 1 μg of digested vector and a molar ratio of 12:1 (library/digested vector).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pNT</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Genes coding for the various RBD domains were cloned in the pCAGGS RBD-SARS-CoV-2 plasmid, which was a kind gift from Florian Krammer lab.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS RBD-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Reads were demultiplexed and each sample was processed separately using the Galaxy platform (https://usegalaxy.org/) using the functions described in Blankenberg et al 45.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Galaxy</div><div>suggested: (Galaxy, RRID:SCR_006281)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After seven days at 37°C, 120 rpm, 8% CO2, supernatant was purified using HiTrap Protein A for VHH-Fc constructs or HisTrap Excel for RBD constructs, following the manufacturer’s instructions (GE Healthcare).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HisTrap Excel</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Affinity measurement by BioLayer Interferometry: Binding kinetics were determined using an Octet RED96 instrument (ForteBio).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BioLayer</div><div>suggested: (Harvard Medical School Center for Macromolecular Interactions Core Facility, RRID:SCR_018270)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The initial structure of VHH72 was built by homology modelling using the MODELLER software 46 and the coordinates of VHH72 in PDB structure 6WAQ as template 18.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MODELLER</div><div>suggested: (MODELLER, RRID:SCR_008395)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis of the trajectories was achieved using in-house scripts written in the macrolanguage of CHARMM v42b153.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHARMM</div><div>suggested: (CHARMM, RRID:SCR_014892)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Figures were produced with PyMol [PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.] and Gnuplot 5.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div><div style="margin-bottom:8px"><div>Gnuplot</div><div>suggested: (Gnuplot, RRID:SCR_008619)</div></div></td></tr></table>
  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: We detected the following sentences addressing limitations in the study:
  As a result, a second generation of antibodies might be required to overcome these limitations and could be obtained by reshaping the initial sequences by conferring them the necessary properties, such as affinity and selectivity, to have optimal therapeutic efficacy for treatment in humans. From this perspective, many teams have proposed a wide range of methods to generate candidates with the expected properties, mostly increased affinity.29-32 Affinity maturation aims at improving biological activity by adjusting the kinetic parameters of the binding to the target, which in turn may confer greater therapeutic efficacy.25,33 However, the magnitude of this effect depends largely on the epitope recognized by the antibody and the initial affinity along with the format of the antibody and its valence.25 In the context of the current COVID-19 pandemic, several studies have described affinity maturation of VHH or conventional antibodies to enhance their binding to SARS-CoV-2 antigens, by CDR-swapping approaches 34, saturation mutagenesis in CDRs 35,36 or light-chain shuffling36. In recent years, Deep Mutational Scanning (DMS) approaches have emerged as a powerful tool for understanding protein/protein interactions. Deep Mutational Scanning (DMS) explores in a selected protein all possible unique substitutions, i.e. all unique mutations for each position. DMS defines mutational landscape of the protein and helps to understand the interaction modalities as recently shown for the RBD...
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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collignon.github.io collignon.github.io

Integrating hypothesis highlights with obsidian.md

1
1. radera 13 Dec 2021
  
  in Public
  
  On top of biredictional links, it also has the #tag functionality, which creates a further network layer
  
  enlaces crean redes "fisicas"; tags crean redes "logicas"
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collignon.github.io/2021/02/hypothesis-with-obsidian/
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Beethoven y los Upanishads - Filosofía - Revista Esfinge

1
1. radera 12 Dec 2021
  
  in Public
  
  Según leemos en el artículo «Beethoven and Indian Philosophy»[1], en un texto escrito, y en realidad copiado por Beethoven, y que es mencionado, incluido y comentado en el libro Beethoven’s Letters with explanatory notes by Dr. A.C. Kalischer (trans. J. S. Shedlock), 1926, se muestran dos textos de filosofía de la India que, aunque no especifica de dónde son, es casi evidente que son de los Upanishads y de un himno védico respectivamente, no identificados en esta obra
  
  "siguiendo la pista": referencia externa link
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theoryofmusic.wordpress.com theoryofmusic.wordpress.com

Beethoven and the Upanishads – Theory of Music

1
1. radera 12 Dec 2021
  
  in Public
  
  Beethoven’s Letters with explanatory notes by Dr. A.C. Kalischer, J.M. Dent & Sons, Ltd., London & Toronto, 1926, pp.393-394
  
  "fuente original (secundaria)"; now, come back to esfinge
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theoryofmusic.wordpress.com/tag/beethoven-and-the-upanishads/
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Aristóteles – UNA HISTORIA CURIOSA

1
1. radera 12 Dec 2021
  
  in Public
  
  Alejandro tenía el hábito de inclinar ligeramente la cabeza sobre el hombro derecho, de baja estatura con cutis blanco, cabello ondulado de color castaño claro y ojos heterócromos, el izquierdo marrón y el derecho gris, que no se sabe si eran de nacimiento o por un traumatismo craneal.
  
  citation needed
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https://biorxiv.org/cgi/content/10.1101/2021.12.05.471263

1
1. sciscore 11 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.05.471263: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  NIH rigor criteria are not applicable to paper type.
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The absence of the polyhistidine tag in the purified protein was verified by western blot using antihistidine antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antihistidine</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 96-hr culturing (27°C, orbital shaking at 125 rpm) and centrifugation, the high-titre baculovirus supernatant produced was collected and used to infect Sf9 cells at a density of 3 × 106 Sf9 cells/ml by 1:8 dilution of the supernatant virus stock.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Sf9</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The [S:A222V + S:D614G]-1-up, [S:A222V + S:D614G]-2-up, [S:A222V + S:D614G]-3-down, S:D614G-1-up and S:D614G-2-up cryo-EM density maps were deposited in the EM Data Bank with codes EMD-13916, EMD-13917, EMD-13918, EMD-13919 and EMD-13920, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>S:A222V + S:D614G]-1-up, [S:A222V + S:D614G]-2-up</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmid pSPIKE (a generous gift from Cesar Santiago, CNB-CSIC) was designed to include the region encoding the SARS-CoV-2 protein S ectodomain (residues 15-1213) with substitutions to proline at residues 986 and 987 and of 668RRAR671 furin cleavage site to alanine, with a N-terminal gp67 signal peptide for secretion, and C-terminal foldon trimerization motif, a thrombin protease recognition site and 9x His and Myc tags, into the insect expression plasmid pFastBac.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pSPIKE</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pFastBac</div><div>suggested: RRID:Addgene_1925)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmid pACE2TEV was prepared from that previously used to express the N-terminal peptidase domain of human ACE2 (Lan et al. 2020), by including a cleavage site for TEV protease, to allow removing the C-terminal 6×His tag in an additional purification step.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pACE2TEV</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plots, curve fittings and numerical calculations were performed with the program GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">2D classification was performed in cryoSPARC (Punjani et al. 2017) and 310,162 and 165,304 particles were selected.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After several rounds of refinement in Refmac and model building in Coot, acceptable refinement metrics were obtained.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Point mutations at position 222 (A-->V) of the S1-NTD domain (residues 13-305) and 614 (D-->G) of the S1/S2 furin cleavage site were introduced by using the mutagenesis tool of PyMol 2.0 (glycan-free) or an in-house topology editing tool (glycosylated; see gitlab.com/KomBioMol/gromologist).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr></table>
  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: We did not find any issues relating to colormaps.
  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.
  Thank you for including a protocol registration statement.
  Results from scite Reference Check: We found no unreliable references.
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  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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Drp1 SUMO/deSUMOylation by Senp5 isoforms influences ER tubulation and mitochondrial dynamics to regulate brain development

1
1. scibot 11 Dec 2021
  
  in Public
  
  RRID:AB_1095181
  
  DOI: 10.1016/j.isci.2021.103484
  
  Resource: (MBL International Cat# M180-3, RRID:AB_10951811)
  
  Curator: @Naa003
  
  SciCrunch record: RRID:AB_10951811
  
  Curator comments: anti-HA-tag antibody MBL International Cat# M180-3
  
  What is this?
  
  RRID:AB_10951811 RRIDCUR:Incorrect RRIDCUR:Unresolved
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sciencedirect.com/science/article/pii/S2589004221014553
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PHP: PHP tags - Manual

1
1. lariloran 10 Dec 2021
  
  in Public
  
  If a file contains only PHP code, it is preferable to omit the PHP closing tag at the end of the file;
  
  but if you are embedding php with html then enclose php code with opening and closing tag;
  
  The type of a variable is not usually set by the programmer; rather, it is decided at runtime by PHP depending on the context in which that variable is used;
  
  #tips
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https://medrxiv.org/cgi/content/10.1101/2021.11.29.21267000

1
1. sciscore 10 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.11.29.21267000: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: All methods and procedures were performed following the relevant guidelines and regulations approved by the Ethics Committee of the Universidad de Santiago of Chile.<br>Consent: Informed consent was obtained from all participants and/or their legal guardians.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The Taq DNA Polymerase (pET-28a_6H-TAQ_E602D) was obtained from Dr. Robert Tjian</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-28a_6H-TAQ_E602D</div><div>suggested: RRID:Addgene_166944)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In addition, NdeI and BamHI were used to clone it in a pET-19 vector with N-terminal 10x His-tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-19</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The analysis was performed using GraphPad Prism 8 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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https://medrxiv.org/cgi/content/10.1101/2021.12.06.21267328

1
1. sciscore 10 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.06.21267328: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: This study was approved by Thomas Jefferson University Institutional Review Board as minimal risk, and each participant provided written consent.<br>Consent: This study was approved by Thomas Jefferson University Institutional Review Board as minimal risk, and each participant provided written consent.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Cohort Selection: This is a prospective cohort of pregnant patients consented for collection of samples at delivery, including maternal blood on admission and cord blood at delivery as part of an ongoing delivery cohort biorepository.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The detection antibody, SULFO-TAG either with anti-human IgG (or anti-human IgM antibody (was diluted to 2 μg/ml in Diluent 100 (MSD) and added to the wells and incubated at RT for 1h on a plate shaker.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (RevMAb Biosciences Cat# 31-1019-MK, RRID:AB_2783627)</div></div><div style="margin-bottom:8px"><div>anti-human IgM</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Additional serological outcomes included: correlation between epitope levels, relation between antibody epitopes and latency to delivery Statistical Analysis: Statistical analysis conducted using SPSS v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPSS</div><div>suggested: (SPSS, RRID:SCR_002865)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Figures were generated using the stats, ggplot2, and corrplot packages in R.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ggplot2</div><div>suggested: (ggplot2, RRID:SCR_014601)</div></div></td></tr></table>
  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: We detected the following sentences addressing limitations in the study:
  The limited positive serology reported (65-70% of PCR+ mothers) demonstrates the limitations of examining isolated antibody epitopes, and specifically of RBD only epitope. SARS CoV-2 Immunity: While prior studies have focused on SARS-CoV-2 spike protein, we evaluated a range of epitopes. Our results demonstrated high sensitivity of our platform with detection of spike (full length)- IgM and IgG in ∼90% of documented PCR infection. We found that, as in non-pregnant patients, high levels of N-IgG and IgM titers in those with a history of COVID-1923. Studies in non-pregnant individuals identified IgM peaks in N and S epitopes in second week of infection24. We found that maternal nucleocapsid antibodies demonstrated the highest specificity for documented COVID-19 infection and were highly expressed in both maternal and cordblood samples. Nucleocapsid proteins of many coronaviruses are highly immunogenic and highly expressed during acute infection25. While the focus of vaccine and monoclonal antibody therapeutics have been on spike antigen, these results, consistent with other studies in non-pregnant adults, highlights the potential import of N-antibodies in SARS-CoV-2 immunity and target for therapy. Finally, similar to the reports cited above18,19,22, we found a high degree of correlation and a linear fit with a slope of 1.0, between maternal and cordblood IgG with cordblood IgG concentrations, indicating highly efficient transfer of CoV-2 IgG antibodies. Finally, in examining s...
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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https://biorxiv.org/cgi/content/10.1101/2021.12.05.471310

1
1. sciscore 10 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.05.471310: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells and pseudotype assay: 293T, Huh-7 (human liver cells), and BHKs were maintained under standard cell culture conditions in DMEM with L-glutamine, antibiotics, and 10</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Huh-7</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Westernblot: Viral pseudotypes were concentrated and 293T producer cells were lysed in 1% SDS and clarified as described previously1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Spike sequences from HCoV-229E (AB691763.1), MERS-CoV (JX869059.2), and SARS-CoV-1 (AY278741) were codon-optimized, appended with a carboxy-terminal FLAG tag sequence separated by a flexible poly-glycine linker and cloned into pcDNA3.1+ as previously described1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1+</div><div>suggested: RRID:Addgene_117272)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Amino acid sequences for the receptor binding domain of the spike glycoprotein were aligned using ClustalW multiple sequence alignment with default parameters.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ClustalW</div><div>suggested: (ClustalW, RRID:SCR_017277)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A maximum likelihood phylogenetic tree was inferred with PhyML v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PhyML</div><div>suggested: (PhyML, RRID:SCR_014629)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The final tree was then visualized as a cladogram with FigTree v1.4.4 (https://github.com/rambaut/figtree).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FigTree</div><div>suggested: (FigTree, RRID:SCR_008515)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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Birds Aren’t Real, or Are They? Inside a Gen Z Conspiracy Theory.

1
1. chrisaldrich 09 Dec 2021
  
  in Public
  
  Except that the creator of Birds Aren’t Real and the movement’s followers are in on a joke: They know that birds are, in fact, real and that their theory is made up.
  
  Linking to a New York Times tag archive would not be considered evidence by any self-respecting conspiracy theorist.
  
  New York Times post-fact conspiracy theories birds aren't real
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nytimes.com/2021/12/09/technology/birds-arent-real-gen-z-misinformation.html
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https://biorxiv.org/cgi/content/10.1101/2021.12.04.471219

1
1. sciscore 09 Dec 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.12.04.471219: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  NIH rigor criteria are not applicable to paper type.
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Soluble detergent extracts were incubated with Glutathione resins for 2 hr at 4°C prior to washing three times with PBS supplemented with NaCl 200 mM and 0.1% Triton and processed for western blot analysis with GFP antibody (Novus NB600-313).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GFP</div><div>suggested: (Novus Cat# NB600-313, RRID:AB_10002194)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK 293 cells were transiently transfected with the GFP tagged constructs using the phosphate calcium method.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293</div><div>suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">4.6 Immunofluorescence: HeLa cells were transfected using the Genejuice transfection reagent (Novagen) according to the manufacturer protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa</div><div>suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The PDZ domains were cloned into pETG-41A plasmid vectors as an N-terminal fusion to a histidine tag and a maltose-binding protein (His-MBP-tag).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pETG-41A</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Invitrogen), PDZ domains of ZO-1, LNX2 and PARD3 were subcloned into pDEST™17 vector and MPP5-PDZ into pDEST™15 vector, allowing the production of recombinant proteins as a fusion to an N-terminal histidine and GST tag, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pDEST™17</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pDEST™15</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pCMV ALFA E vector was constructed as follows: DNA sequence encoding the ALFA tag (MSRLEEELRRRLTE) followed by a linker (GGGGS) fused to the sequence corresponding to the alpha-variant of SARS-CoV2 E cDNA (GenBank: BCM16077.1) synthetized by Eurofins.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV ALFA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The ALFA-linker-E sequence was subsequently cloned into a pCMV backbone vector using ClaI and XhoI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV</div><div>suggested: RRID:Addgene_16459)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequences corresponding to the PDZ domains were cloned into the pCMV GFP vector using EcoRI and XhoI sites.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV GFP</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Models were rebuilt using COOT (Emsley et al., 2010), and refinement was done with phenix.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>COOT</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">refine of the PHENIX suite (Adams et al., 2010).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All structural figures were generated with the PyMOL Molecular Graphics System, Version (Schrödinger).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr></table>
  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 24. 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.
  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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2021.12.04.471219
blog.readwise.io blog.readwise.io

How to Tag Your Highlights While You Read with Inline Tagging in Readwise

1
1. ChandlberB 08 Dec 2021
  
  in Public
  
  To use inline tagging, simply add the note .probability in addition to the highlight. When it's imported into Readwise, the passage will be tagged accordingly.
  
  .ReadWise
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update.lib.berkeley.edu update.lib.berkeley.edu

Zotero

1
1. radera 07 Dec 2021
  
  in Public
  
  Do you … ? … save random URLs in a Word or Google Doc? … save article PDFs on your desktop and as email attachments? … have a pile of article printouts sitting on your desk? … write down citations on sticky notes and post them to your monitor? … stay up late the night before a paper is due reconstructing your citations? If you answered yes to any of the above … the answer is YES, you need Zotero
  
  zotero
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update.lib.berkeley.edu/tag/zotero/
www.titanwolf.org www.titanwolf.org

Question Seeking citation parser

1
1. radera 07 Dec 2021
  
  in Public
  
  We recently faced a similar problem and ended up writing our own parser based on ParsCit but using Wapiti instead of CRF++ for the conditional random fields model. Like Mike mentions above, the problem with ML-based parsers is getting good tagged training data; for this we wrote a visual editor that lets you tag the results (and save them as training data). This approach works pretty well for parsing bibliographies. If anyone is interested, we've made both parser and editor available here at anystyle.io.
  
  anystyle.io
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titanwolf.org/Network/q/6a0a1bac-a417-4943-a810-72dd51c0e08d/y
forum.obsidian.md forum.obsidian.md

Using YAML FrontMatter as data - Resolved help - Obsidian Forum

1
1. radera 06 Dec 2021
  
  in Public
  
  Chuckle123VIP12d@SamAdams, I added tags to the frontmatter, and use them to denote project details. I use Templater for templates, and it gives me the ability to dynamically populate the Path tag. I use Dataview to display the status of my projects on my Workbench. This may not be the intended purpose, but the ability to add tags to the frontmatter allows me to use it as I see fit. — Aliases: Tags: Project Path: <% tp.file.folder(true) %> Status: _Open Priority: 1 — ```dataview Table replace(Path, “Projects/”, “”) as Project, Status, Priority From #Project Where Status = “Open” Sort Priority, Project ``` ce
  
  answer
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forum.obsidian.md/t/using-yaml-frontmatter-as-data/12773
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Enhanced transcriptional heterogeneity mediated by NF-κB super-enhancers

1
1. EMBOpress 06 Dec 2021
  
  in Review Commons
  
  Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
  
  Learn more at Review Commons
  
  Reply to the reviewers
  
  Reviewer 1
  
  Reviewer #1 (Evidence, reproducibility and clarity (Required)):
  
  *The manuscript by Wibisana et al. describes an impressive set of experiments that analyse the NFkB response at the single-cell level, using a variety of cutting-edge techniques (live cell imaging, single-cell RNA-seq, single-molecule RNA FISH, and single-cell ATAC-seq) in chicken DT40 B-cells.
  
  In the fist half of the paper, the authors perform a detailed characterization of the cell-to-cell variation arising from a homogeneous stimulation with various doses of anti-IgM. They observe that the NFKB TF RelA forms clear nuclear 'foci' upon stimulation in DT40 cells: this was anecdotally shown in a different cell-type by the same authors in ref 7, but (to my knowledge) has never been systematically studied. This allows them to quantitatively analyse the foci formed in response to stimulation, and they show that this is dose-dependent, heterogeneous and biomodal, and exhibits properties of cooperativity. In parallel, the authors analyse the resulting stimulus-driven changes in gene expression, first using single-cell RNA-seq, and then, elegantly, using RNA FISH, which allows them to directly compare the number of RelA foci to gene expression in individual cells. Like the RelA foci, they find that cell-to-cell gene expression is heterogeneous and bimodal (this has been described before). Interestingly, though, they are able to show that individual stimulus-responsive genes exhibit distinct patterns of cell-to-cell hetereogeneity: they can categorize 4 clusters of responding genes according to different patterns of cell-to-cell variation at distinct stimulus doses, and moreover they show that while the heterogeneity of NFKBIA arises due to bimodal expression levels, that of CD83 is simply due to broad variation between cells. Although focused on NFkB, there is a lot of information here with some important (and non-intuitive) implications that could apply to many other stimulus-driven or developmental responses that exhibit heterogeneous patterns of gene expression. A more in-depth analysis of the single-cell datasets would certainly be very worthwhile and fruitful.
  
  In the second half of the paper, the authors attempt to use their single-cell data, alongside ATAC-seq genomic analyses, to draw inferences about how or whether the model genes NFKBIA and CD83 are regulated by super-enhancers (SEs). Both of these genes are associated with SEs that gain accessibility upon stimulation (recapitulating the authors' findings in ref 8 in a different cell-type), and the CD83 promoter exhibits co-accessibility with two regions within an adjacent SE. The authors also show that both genes are sensitive to treatment with 1.6-HD, a compound that disrupts liquid-like condensates (a characteristic that has been reported for SEs), and CD83 is sensitive to an inhibitor of Brd4 (which has been associated to SE function). However, while these findings could be considered to be suggestive of regulation by SEs, they are clearly not definitive (nor do the authors claim so).
  
  Finally, the authors show (figure 4a-c) that while the level of stimulus-driven gene upregulation correlates with co-accessibility with both SEs and typical enhancers (TEs), the cell-to-cell heterogeneity of gene expression correlates only with co-accessibility with SEs. This would agree with a model in which SE-regulated gene regulation may generally impart heterogeneous or switch-like gene expression. *
  
  **Specific comments**
  
  • The experiments are adequately presented, and the authors indicate that not only the sequencing data but also the analysis code is available. Nevertheless, the methods section is rather terse, and could benefit from more detail to understand the various analyses, particularly concerning the analyses of SEs in figures 3 and S7, where it is often difficult to understand how peaks or genes are categorized.
  
  Response: We thank the Reviewer for pointing this out and we agree that the Methods section was not described in detail, particularly in how the SEs were analyzed and categorized. Therefore, we have added more details on how SEs were categorized in the Methods section as follows:
  
  “ Peak calling and enhancer identification from ATAC-seq data were performed using Homer v4.10.4 (http://homer.ucsd.edu/homer/) using the bam files generated from the Cell Ranger pipeline. Tag directories were created for the bam file from each condition using the “makeTagDirectory” program with the “--sspe -single -tbp 1” option. Peak calling was performed using the “findPeaks” program with the “-style super -typical -minDist 5000 -L 0 -fdr 0.0001” option. This procedure stitches peaks within 5 kb and ranks regions by their total normalized number reads and classifies TE and SE by a slope threshold of 1. Peak annotation was subsequently performed using the “annotatePeaks.pl” program with the GRCg6a.96 annotation file. The consequent peak files were merged between each stimulation condition for the SE and TE peaks separately using the “mergeBed” program of bedtools. Peak annotation was performed for the second time for the merged peaks to create the final SE and TE peaks. ATAC fold-change was then calculated between both conditions for the merged peaks separately for SE and TE. Genes associated with both SE and TE were assigned only to the SE.”
  
  Similarly, we have added more details for other analyses in the Method section and the main sentences.
  
  The imaging, scRNA-seq and RNA-FISH experiments are well-presented, although the supplementary figures 4 and 5 include key results that would merit inclusion within the main figures. *
  
  Response: We thank the Reviewer for this comment. We have included supplementary figures 4b and 5d in the main figures (new Fig. 2g) since both of these figures represent the raw data revealing the differences between smFISH counts and RNA-seq derived gene expression.
  
  It is strking that although all the conclusions about SEs are drawn almost exclusively from analysis of ATAC-seq data, no raw ATAC-seq data is directly shown in any figure (even in the browser snapshots of figure 4d & e). It would be important to show the actual ATAC data from which the inferences of figures 3 and 4 are drawn, especially so that it is possible to visualize the implication of a particular 'ATAC fold-change' or of 'ATAC-gained enhancers'. Response: We have added a browser snapshot of the ATAC-seq data, presenting the super-enhancer region assigned to both CD83 and NFKBIA* (new Fig. 3c).
  
  Reviewer #1 (Significance (Required)):
  
  • This manuscript can be considered as a follow-up of the authors' previous paper (Michida 2020, ref 8), here focusing on cell-to-cell heterogeneity rather than on the overall magnitude of the stimulus-induced response. Overall, the experiments are well-performed and bring new data to an interesting angle of gene regulation. However, the analyses presented do not seem to fully exploit the data, and the authors do not manage to present any strong conclusions, particularly relating to the possible involvement of super enhancers.
  
  Response: To strengthen our conclusions about the possible involvement of super-enhancers in regulating heterogeneity, we performed additional analyses on the properties of the SE including the number of transcription factors, NF-κB and PU.1 binding motifs and the length of the enhancers, according to a previous report (Michida et al., 2020, Cell Rep). This was also conducted to confirm whether the ATAC-seq-based SE identification method presents results consistent with those provided by H3K27Ac-ChIP-based methods utilized in the previous study (Michida et al., 2020, Cell Rep). SEs revealed longer genomic length (new Supplementary Fig. 8a) and this length was positively correlated with the ATAC signal (new Supplementary Fig. 8b). Furthermore, gained and lost SE revealed a correlation with enhanced gene expression upregulation and downregulation, respectively, compared to TE (new Fig. 3g). We also demonstrated that SE-regulated genes have a higher Fano factor change, which is consistent with the state of an SE whether it is gained or lost (new Fig. 5a, 5b). For binding motif analysis, we observed a slightly higher PU.1 motif density at SEs (new Supplementary Fig. 11), corresponding to the results of the previous study (Michida et al., 2020, Cell Rep). Interestingly, only the density of NF-κB and not PU.1 was correlated with ATAC signal change in SE (new Fig. 4a), suggesting that those SEs were controlled by nuclear translocation of NF-κB.
  
  As a mechanism to produce gene expression heterogeneity in phenotypically identical cells, we observed that co-accessibility, which has been reported to be concordant with genomic contacts is correlated to Fano factor change, indicating that gene expression heterogeneity possibly stems from cis-regulatory interactions. NF-κB activation has been reported to increase the heterogeneity in some genes and is attributed to the accumulation of Ser5p RNAPII (Wong et al., 2018, Cell Rep). Additionally, Ser5p RNAPII has been reported to accumulate at enhancer regions (Koch et al., 2011, Nat Struct Mol Biol), and that the accumulation of RNAPII is suggested to assist in gene expression activation through enhancer-promoter contact (Thomas et al., 2021, Mol Cell). Our results support these conclusions since co-accessibility or putative cis-regulatory interactions correlate to Fano factor changes. SE can form phase-separated transcription hubs containing multiple enhancers and/or promoters, which may enable the higher diffusion rate of active enhancers; therefore, it may induce a higher possibility of genomic DNA interactions (Gu et al., 2018, Science). In contrast, the enrichment of TATA motif has also been proposed to generate transcriptional heterogeneity (Faure et al., 2017, Cell Syst). Therefore, we examined this possibility with our data. However, we observed a higher occurrence of TATA box in genes associated with lost SE (new Supplementary Fig. 18) which might have caused gene expression heterogeneity in unstimulated cells. This heterogeneity might be due to the differences in Pol II loading intervals (Tunnacliffe & Chubb, 2020, Trends Genet) however the noise associated with gained SE is possibly generated by the fluctuation of high-order biomolecular assembly. Therefore, we believe that the source of heterogeneity in these conditions were different.
  
  Additionally, we performed Hill function analysis to reveal the threshold behavior of gene expression in our analysis since previously gained SEs were associated with threshold gene expression (Michida et al., 2020, Cell Rep). In this study, we presented that threshold behavior in gained SE is related to motif density of NF-κB (Fig. 4d), however, threshold behavior does not seem to be related to heterogeneous gene expression.
  
  Following these results, we concluded that NF-κB activated SE has two closely related but distinct functions for gene control: (1) enhanced heterogeneity and fold-changes and (2) switch-like expression. These are controlled by different mechanisms stemming from chromatin status: (1) frequency of cis-regulatory genomic interactions possibly mediated by phase separation and (2) cooperative binding of NF-κB to DNA. These differences were well represented by expression profiles of CD83 (higher heterogeneity and weak bimodal expression) and NFKBIA (lower heterogeneity and strong bimodal expression).
  
  For instance, the existence of multiple gene clusters that exhibit distinct patterns of heterogeneity implies that switch-like gene activation occurs on a per-gene basis, rather than corresponding to an all-or-nothing activation of individual cells. This would be an exciting finding, and the authors have the data to test this. Likewise, the division of heterogeneous gene expression into bimodal (like NFKBIA) or unimodal (like CD83) distributions could be a nice paradigm if systematically applied to the other 1335 differentially-expressed genes identified by the authors. * Response: We appreciate this comment. Following your comment, we analyzed the relationship between heterogeneity and bimodality (switch-like expression or high Hill coefficient) for the remaining genes. We observed that SE having a high Hill coefficient contained a higher number of NF-κB motif in SE (new Fig. 4), indicating that cooperative binding of NF-κB to DNA shaped non-linear gene expression profiles as we indicated in a previous paper (Michida et al., 2020, Cell Rep). Additionally, as described in the earlier section, we observed that heterogeneity arises from cis-regulatory genomic interaction. We compared these gene groups and observed that these properties were not completely shared (new Supplementary Fig. 15), indicating that bimodality and heterogeneity originated from different mechanisms. We assume that those differences are mediated through a combination of chromatin accessibility and the biophysical properties of NF-κB.
  
  In contrast, although the authors try to use their data to investigate gene regulation by SEs, these inferences are all somewhat indirect, and the authors themselves do not manage to draw any definitive conclusions. Response: We appreciate this comment. We performed the additional computational analysis and carefully interpreted the data. Additionally, we have now concluded that SEs have two major biological functions: (1) gene expression heterogeneity, which is mediated via cis-regulatory interactions (Fig. 5) and (2) bimodal gene expression, which is mediated by NF-κB binding (new Fig. 4). The latter finding has also been reported in a mouse primary B cell, albeit the mechanism causing heterogeneity was a novel conclusion of this study.
  
  I feel that the authors are under-selling their data here. As-is, the data represents more of a resource than a study with a clear message, but I believe that with more in-depth analysis the authors could make a much more significant advance, particularly concerning the cell-to-cell heterogeneity of gene expression. I would be very enthusiastic to review the same data again with a more detailed analysis, which I believe would enormously improve the manuscript. Response: We appreciate this comment. As described in this report and the revised manuscript, we performed a considerably detailed computational analysis and gained several novel insights to answer the question regarding the functional roles of SE. We are grateful to learn that gene expression patterns may be estimated from ATAC-seq profiles and that they may even be controlled. We hope that this Reviewer would observe the scientific value of our study and provide us with your valuable feedback on our revised manuscript.
  
  Reviewer #2
  
  Reviewer #2 (Evidence, reproducibility and clarity (Required)):
  
  Imaging and single cell sequencing analyses of super-enhancer activation mediated by NF-κB in B cells" by Wibisana et al. examined the relationship between super-enhancers, NF-κB nuclear aggregation, and target gene regulation. The authors have generated a large amount of data from fluorescent microscopy, scRNA-seq, scATAC-seq, smRNA FISH. While this is an impressive dataset in terms of diverse technically advanced methods employed, it is not clear what to take as a main conceptual advance. What could be the functional implications of observed cell-cell variability in B cell transcriptional responses to environmental stimuli? In addition to this general point, the following are specific comments that could improve the manuscript.
  
  In Figure 2, smRNA FISH foci of CD83 and NFKBIA are quantified as # of spots per cell (Supplementary figure 5). But it is difficult to see in Figure 2 the colocalization of any mRNA spots with RelA foci. Ideally, it will be convincing to show by DNA FISH that these target loci are indeed located within NF-κB occupied super-enhancer puncta. Even with the current RNA FISH data, some colocalization analysis could have been performed. * Response: In Figure 2, we were unable to perform accurate colocalization analysis with the current smFISH data as the probes used by us map to exons. Moreover, we have also previously performed DNA-FISH; nevertheless, it was difficult to assess co-localization between the DNA and RelA proteins secondary to the degradation of RelA-GFP proteins. Therefore, we decided to perform intronic smRNA-FISH, which can be used to pinpoint the site of active transcription (Levesque and Raj, 2013, Nat Methods). The results, along with the quantification results, are presented in the new Fig. 2f.
  
  Supplementary Figure 5a shows lower correlations of # GFP-RelA foci to CD83 transcripts in comparison to NFKBIA. Even though the foci and smRNA FISH spots are derived from high resolution imaging data, we should remember that any snapshot measurements have limited information content for gene regulatory relationships. Live cell studies (for example, from the groups of Suzanne Gaudet, Kathryn Miller-Jensen, and Myong-Hee Sung) have shown that time-integrated measures (e.g. maximum fold change and area under the curve of RelA signaling time course in single cells) are better correlates to transcriptional output of target genes (Lee REC et al 2014 Mol Cell; Wong VC et al 2019 Biophysical J; Sung MH et al. 2014 Science Signal; Martin EW et al. 2020 Science Signal). *
  
  Response: We thank the Reviewer for this valuable comment. One of the reasons for a lower correlation between GFP-RelA foci and CD83 transcripts compared to NFKBIA may be the difference in expression timing of CD83 and NFKBIA and the timing of nuclear localization of GFP-RelA. RelA localizes in the nucleus 10−30 mins after cell stimulation, and NFKBIA is an early responsive gene, however, CD83 is expressed later (new Supplementary Fig. 17). Therefore, this time difference possibly affects correlation accuracy. Although we agree that high-throughput time-course measurement of RelA-GFP combined with smFISH measurements, such as that reported in Wong VC et al., 2019, will be ideal, it is technically difficult since DT40 are suspension cells and the smFISH protocol requires multiple washing and centrifugation steps. Thus, with this experimental setup, we were unable to perform the time-course analysis.
  
  Nonetheless, we measured the time-course foci formation at the same single-cells (new Supplementary Fig. 1b) and observed that it effectively represents Figure 1a, which is a snapshot of the population dynamics of RelA foci across time. Additionally, the observed dynamics, which revealed a steep initial increase and slight decrease with time, effectively recapitulates the previous reports (Lee et al., 2014, Mol Cell; Wong et al., 2019, Biophys J).
  
  In our analysis, we performed imaging analysis to demonstrate that NF-κB foci formation is switch-like, and this formation might be involved in the formation of phase-separated condensates enhancing DNA to DNA contact. The number of foci may depend upon the intracellular concentration of NF-κB, and fold change in the RelA signal may be correlated with gene expression as previously reported (Lee et al., 2014, Mol Cell; Wong et al., 2019, Biophys J). However, there is another report presenting that promote/enhancer proximity is not related to gene expression (Alexander et al. 2019, eLife). Although we were unable to perform this analysis owing to the limitations stated above, we tried to find the relationship between RelA foci and gene expression by performing biochemical perturbations (Fig 1e-f, Fig 5h) and presented that these foci are related to gene expression.
  
  The analyses have been performed using DT40 cells. In the Methods section, no description was provided about what type of B cells DT40 is, even though few outside of the field may not know that the cells were immortalized from chicken. This is an important consideration, because some nuclear bodies and genome organization features are different between host species and they also depend on whether the cells are primary or transformed. Because the authors do not discuss this point, it seems possible that the findings about NF-κB aggregates and super-enhancers may not necessarily hold true for primary B cells. *
  
  Response: We thank the Reviewer for pointing out these issues. We have added the following description on DT40 cells in the Methods section describing that DT40 cells are chicken bursal lymphoma cells.
  
  DT40 B lymphocytes have been widely used as a B cell model for studying B cell receptor signaling (Mori et al., 2002, J. Exp. Med.; Patterson et al., 2002, Cell; Saeki et al., 2003, EMBO J.) due to its high gene targeting efficiency. We also previously confirmed that anti-IgM stimulation induces the NF-κB signaling pathway in mouse primary splenic B cells and DT40 and that the signaling molecules and dynamics in these cells are well conserved (Shinohara et al., 2014, Science; Shinohara et al., 2016, Sci. Rep.; Inoue et al., 2016, NPJ Syst. Biol. Appl.). However, we understand the Reviewer’s concerns. Therefore, we have provided the track view of primary B cell ATAC-seq data to demonstrate that the chromatin accessibility changes upon anti-IgM stimulation in CD83 and NFKBIA were similarly observed in primary B cell data (new Supplementary Fig. 9b) and that the upregulation and association with SE of CD83 and NFKBIA were also observed in primary B cell (new Supplementary Fig. 9a).
  
  Similarly, the GFP-RelA expressing DT40 cell generation should be described with more detail (beyond "provided by ..."). N-terminal or C-terminal fusion? Did the fusion construct contain an artificial promoter (e.g. CMV) or an upstream fragment of the genomic Rela locus (chicken or human)? Methods of transfection and cloning of stable lines? These choices affect the interpretation of the data, so they must be fully described and justified. *
  
  Response: We thank you for pointing this out. We have added the following details on the RelA-GFP construct in the Methods section:
  
  Mouse RelA-eGFP with eGFP on the C terminal was cloned into a pGAP vector containing Ecogpt resistance gene targeting endogenous GAPDH locus. This construct was further electroporated into wild-type cells and selected using Ecogpt to produce RelA-GFP-expressing DT40 cells.
  
  DT40 cells were cultured in 39 degrees. Michael White and colleagues have shown that high temperatures can alter NF-kappaB dynamics and function (https://www.pnas.org/content/115/22/E5243). Did the authors try lower temperatures to ascertain that the NF-kB aggregates and other major findings are still observed in 37 degrees? *
  
  Response: We performed the experiments at 39 degrees to mimic the natural body temperature of chicken since DT40 cells were derived from chicken bursal lymphoma (Saribasak and Arikawa, 2006, Subcell Biochem). Previously, we cultured DT40 cells at 37 degrees and observed that the cell growth was inhibited, and thus, we believed that it was not ideal to perform experiments of DT40 cells at 37 degrees.
  
  Reviewer #2 (Significance (Required)):
  
  It is not clear what to take as a main conceptual advance.
  
  Response: Considering the original manuscript, we agree with the Reviewer on the lack of strong emphasis on the conclusions of our study. Therefore, in this revised manuscript, we have focused on the comprehensive mechanism of heterogeneity and switch-like activation in gene expression control. As we described in the comments to Reviewer #1, we performed an additional in-depth computational analysis on SE and TE. Consequently, we demonstrated that enhanced heterogeneity and expression fold-changes mediated by SE are defined by the number of cis-regulatory genomic interactions in open chromatin regions (Figure 5), however, switch-like expression (bimodal patterns) is determined by the number of NF-κB binding in SE (new Figure 4). The latter finding has also been reported in a mouse primary B cell in our previous study (Michida et al. 2020, Cell Rep.). However, the mechanism causing heterogeneity is a novel conclusion obtained in this study. We also concluded that these similar, albeit quantitatively and slightly different characteristics in gene control can be achieved through a combination of chromatin accessibility of host cells and biophysical properties of NF-κB molecule, which is involved in phase separation.
  
  What could be the functional implications of observed cell-cell variability in B cell transcriptional responses to environmental stimuli?
  
  Response: We performed gene ontology analysis to reveal how the heterogeneously expressed genes (cluster 4) (Fig. 2d) presented enrichment for immune-related functions (Supplementary Fig. 5b). This result supports a previous study, which stated that variability in gene expression is related to function (Osorio et al., 2019, Cells).
  
  This discussion is incorporated in the manuscript as follows:
  
  “We observed that genes with an increased heterogeneity upon increasing stimulation dose are enriched with cell-type-specific immune regulatory genes (Supplementary Fig. 5b), supporting a previous report where heterogeneity in gene expression is tied to biological functions and may be used by cells as a bet-hedging or a response distribution mechanism (Osorio et al., 2019, Cells), where cells exhibit heterogeneity to enable response to changing environment and also allowing dose-dependent fractional activation respectively. This was observed in CD83, a B cell activation marker, demonstrating the involvement of heterogeneity in B cell development.”
  
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12 must know Zotero tips and techniques | The Ideophone

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1. radera 05 Dec 2021
  
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  Tag incomplete items to find them back and fix them later
  
  truco
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reactjsexample.com reactjsexample.com

Apps - React.js Examples

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Gumroad

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1. bbarker 03 Dec 2021
  
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  Has can be thought of as a type-indexed heterogeneous map, which is type safe,but requires access to compile-time type tag information. ZLayer can be thoughtof as a more powerful version of Java and Scala constructors, which can buildmultiple services in terms of their dependencies.
  
  scala zio zlayer
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Recommendation on Open Educational Resources (OER)

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1. cogdog 02 Dec 2021
  
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  automatic OER processing
  
  I am unsure of what "automatic OER Processing" might mean/ Can anyone help?
  
  The closes I came was in the section of a International Journal of OER paper by Stephen Downes: A Look at the Future of Open Educational Resources where in the Artificial Intelligence section he illustrates an example of AI creating OER (?)
  
  What is relevant to open education is that the services offered by these programs will be available as basic resources to help build courses, learning modules, or interactive instruction. For example, Figure 3 illustrates a simple case. It takes the URL of an image, loads it, and connects an online artificial intelligence gateway offered by Microsoft as part of its Azure cloud services using an API key generated from an Azure account.
  
  The Azure AI service automatically generates a description of the image, which is used as an alt tag, so the image can be accessible; the alt tag can be read by a screen reader for those who aren’t able to actually see the image. In this case, the image recognition technology automatically created the text “a large waterfall over a rocky cliff,” along with a more complete set of analytical data about the image.
  
  Yes this is interesting and is a useful tool for content creation, but to me seems a far leap to creating educational content.
  
  ai
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Volume 1 Number 3

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Far East (Singapore): Defence of Singapore: account written by son of Admiral Beamish

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Correspondence, memoranda, draft articles of association and notes on foundation of Alfred Herbert (Australasia) Pty Ltd

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https://biorxiv.org/cgi/content/10.1101/2021.11.25.470011

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1. sciscore 01 Dec 2021
  
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  SciScore for 10.1101/2021.11.25.470011: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The study and corresponding experiments were approved by the local ethics committee (S64089) and all patients gave their written informed consent.<br>Consent: The study and corresponding experiments were approved by the local ethics committee (S64089) and all patients gave their written informed consent.<br>Euthanasia Agents: At day 4 post-infection, animals were euthanized by intraperitoneal injection of 500 μL Dolethal (200 mg/mL sodium pentobarbital, Vétoquinol SA).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Female Syrian hamsters (Mesocricetus auratus) were purchased from Janvier Laboratories and kept per two in individually ventilated isolator cages (IsoCage N Bio-containment System, Tecniplast) at 21°C, 55% humidity and 12:12 day/night cycles.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">No randomization methods were used and confounders were not controlled, though all caretakers and technicians were blinded to group allocation in the animal facility and to sample numbers for analysis (qPCR, titration, and histology).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">No randomization methods were used and confounders were not controlled, though all caretakers and technicians were blinded to group allocation in the animal facility and to sample numbers for analysis (qPCR, titration, and histology).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">Group size was calculated based on the independent t-test with an effect size of 2.0 and a power of 80% (effect size = delta mean/SD = 1 log10 decrease in viral RNA/0.5 log10), resulting in 5-6 animals/group.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">His-tag labeled SARS-CoV-2 RBD (The Native Antigen Company) was biotinylated with the EZ-Link Sulfo-NHS-LC-Biotin kit (Thermofisher Scientific) according to the manufacturer’s protocol, corresponding to 1-3 biotin groups per antibody molecule.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Sulfo-NHS-LC-Biotin</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After the 60 minutes incubation, B cells were washed with FACS buffer and stained with PerCP-cy5.5 anti-human CD19 antibody (Biolegend, 363016)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human CD19</div><div>suggested: (BioLegend Cat# 363016, RRID:AB_2564207)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">FITC anti-human CD3 antibody (Biolegend, 300306) and PE streptavidin (Biolegend, 405203) for 25 minutes on ice.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human CD3</div><div>suggested: (BioLegend Cat# 300306, RRID:AB_314042)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">As a positive and negative control, an anti-SARS-CoV-2 RBD mAb (40150-D004, Sino Biological) and anti-SARS-CoV-2 nucleocapsid antibody (MBS2563841, MyBioSource) were used, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 nucleocapsid antibody ( MBS2563841</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">First, mouse anti-human IgG (Fc) antibody (Human Antibody Capture Kit, Cytiva) was immobilized on a CM5 chip according to manufacturer instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Monoclonal antibody neutralization assays: a. Production of S-pseudotyped virus and serum neutralization test (SARS2, SARS1, MERS, 229E): VSV S-pseudotypes were generated as described previously56.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two hours later, the medium was replaced by medium containing anti-VSV-G antibody (I1-hybridoma, ATCC CRL-2700) to neutralize residual VSV-G input.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-VSV-G</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, serial dilutions of antibodies were mixed separately with live SARS-CoV-2 Wuhan, alpha, beta and gamma virus strains, incubated at 37 °C for 1h, and added to the monolayer of Vero E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>1h</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody protein treatments (anti-SARS-CoV-2 mAbs or human IgG1 isotype control Trastuzumab/Herceptin® (Roche)) were initiated 24 hours post infection by intraperitoneal injection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human IgG1</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK-293T cells (SARS-CoV, SARS-CoV-2 and MERS-S) or BHK-21J cells (229E) were transfected with the respective S protein expression plasmids, and one day later infected (MOI = 2) with GFP-encoding VSVΔG backbone virus (purchased from Kerafast).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>BHK-21J</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To quantify nAbs, serial dilutions of serum samples were incubated for 1 hour at 37 °C with an equal volume of S pseudotyped VSV particles and inoculated on Vero E6 cells (SARS-CoV and SARS-CoV-2) or Huh-7 cells (229E and MERS-S) for 18 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Huh-7</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Shortly, 104 VeroE6 cells/well were seeded in 96-well plates one day prior to the titration and inoculated with 10–fold serial dilutions of virus solutions and cultured for 3 days at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Infectious virus was isolated by serial passaging on Huh7 and Vero E6 cells58; passage 6 virus was used for the study described here.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Huh7</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Intramuscular pDNA electroporation: 3B8 was delivered in vivo, encoded in the CMV-driven pcDNA3.4 vectors, as an equimolar mixture of the 3B8 heavy and light chain plasmids (jointly referred to as ‘p3B8’).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.4</div><div>suggested: RRID:Addgene_131198)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis was performed using Graphpad Prism 9.0 (Graphpad Software). b.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad</div><div>suggested: (GraphPad, RRID:SCR_000306)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Epitope binning graphs were made in Microsoft Excel and clustering was done with ClustVis web tool (</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Microsoft Excel</div><div>suggested: (Microsoft Excel, RRID:SCR_016137)</div></div><div style="margin-bottom:8px"><div>ClustVis</div><div>suggested: (ClustVis, RRID:SCR_017133)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization IC50 values were determined by normalizing the serum neutralization dilution curve to a virus (100%) and cell control (0%) and fitting in Graphpad Prism (GraphPad Software, Inc.). b.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Graphpad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Both strains were subjected to sequencing on a MinION platform (Oxford pore) directly from the nasopharyngeal swabs</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MinION</div><div>suggested: (MinION, RRID:SCR_017985)</div></div></td></tr></table>
  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: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT03831503</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Active, not recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Study of INO-A002 in Healthy Dengue Virus-naive Adults</td></tr></table>
  Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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Florida Amendment 4, Voting Rights Restoration for Felons Initiative (2018) - Ballotpedia

1
1. juliannedo 01 Dec 2021
  
  in Public
  
  Counties could add additional early voting days from October 22 through October 26 and/or November 4. ↑ Counties could add additional early voting days from October 22 through October 26 and/or November 4. ↑ Tampa Bay Times, "Confusion clouds restoration of Florida felons’ voting rights," accessed December 17, 2018 ↑ My News 13, "Amendment 4 Could Be Delayed 60 Days, DeSantis Says," accessed December 17, 2018 ↑ Palm Beach Post, "EXCLUSIVE: DeSantis to act quickly on water, Supreme Court, Broward sheriff," accessed December 17, 2018 ↑ https://www.tallahassee.com/story/news/2018/12/14/ron-desantis-wants-lawmakers-have-look-amendment-4/2314818002/ Tallahassee Democrat, "Critics angry after Ron DeSantis asks Florida lawmakers to review Amendment 4 implementation," accessed December 17, 2018] ↑ Law 360, "BREAKING: 11th Circ. Sides With Fla. In Felon Voting Rights Dispute," accessed September 11, 2020 ↑ Document Cloud, "Jones v Florida," accessed September 11, 2020 ↑ Jump up to: 9.0 9.1 9.2 Florida Division of Elections, "Voting Restoration Amendment 14-01," accessed April 20, 2017 ↑ The Brennan Center, "Voting rights restoration efforts in Florida," accessed July 17, 2018 ↑ Jump up to: 11.0 11.1 Florida Commission on Offender Review, "Executive Clemency Timeline: 1991-2015," accessed December 7, 2017 ↑ Jump up to: 12.0 12.1 Florida Commission on Offender Review, "Rules of Executive Clemency," accessed December 7, 2017 ↑ Miami Herald, "Florida’s lifetime ban on voting by felons is unconstitutional, federal judge rules," February 1, 2018 ↑ CBS 12, "Court backs state in felons' rights fight," April 25, 2018 ↑ Jump up to: 15.0 15.1 15.2 15.3 15.4 15.5 Florida Department of State, "Campaign Finance Database," accessed December 11, 2018 Cite error: Invalid <ref> tag; name "fin" defined multiple times with different content ↑ Jump up to: 16.0 16.1 16.2 Florida Secretary of State, "Initiative #14-01 Petition," accessed April 20, 2017 ↑ Jump up to: 17.0 17.1 17.2 17.3 17.4 17.5 Note: This text is quoted verbatim from the original source. Any inconsistencies are attributable to the original source. Cite error: Invalid <ref> tag; name "quotedisclaimer" defined multiple times with different content Cite error: Invalid <ref> tag; name "quotedisclaimer" defined multiple times with different content Cite error: Invalid <ref> tag; name "quotedisclaimer" defined multiple times with different content Cite error: Invalid <ref> tag; name "quotedisclaimer" defined multiple times with different content ↑ Jump up to: 18.0 18.1 Floridians for a Fair Democracy, "Homepage," accessed November 30, 2017 ↑ St. Peters Blog, "Supreme Court OKs gambling control, felon voting rights amendments," April 20, 2017 ↑ Jump up to: 20.0 20.1 Bernie Sanders Tweet, September 26, 2018 ↑ USA Today, "Ex-felons in Florida need their voting rights back," February 11, 2018 ↑ Jump up to: 22.0 22.1 22.2 22.3 22.4 22.5 Tampa Bay Times, "Where they stand: Candidates for governor on vote for felons," January 30, 2018 ↑ Florida Politics, "‘Julián Castro 2020’? Former HUD head addresses Democrats in Miami," accessed October 1, 2018 ↑ Twitter, "Second Chances Florida September 18, 2018 Tweet," accessed September 20, 2018 ↑ Democratic Progressive Caucus of Florida, "2018 Ballot Amendments Recommendations," accessed October 14, 2018 ↑ Florida Rights Restoration Coalition, "Homepage," accessed January 6, 2017 ↑ Politico, "ACLU to storm 2018 midterms," January 6, 2018 ↑ Our Revolution, "Voting Rights Restoration for Felons Initiative: Voting Restoration Amendment," accessed September 2, 2017 ↑ Florida Politics, "Committee backing voting restoration amendment raises $1.1M in November," December 13, 2017 ↑ Wear TV, "Amendment would restore felons' right to vote in Florida," accessed July 25, 2018 ↑ Jump up to: 31.0 31.1 31.2 31.3 31.4 31.5 League of Women Voters of Florida, "Amendments," accessed September 13, 2018 ↑ News-Press, "We support restoration of an ex-felon's voting rights," September 13, 2018 ↑ Florida TaxWatch, "2018 Florida Voter Guide," accessed October 5, 2018 ↑ Jump up to: 34.0 34.1 34.2 Second Chances Florida, "Freedom Partners Chamber of Commerce Endorses Amendment 4," September 13, 2018 ↑ Libertarian Party of Florida, "LPF Voting Recommendations for the 2018 FL Ballot," accessed October 19, 2018 ↑ Second Chances Florida, "FOR IMMEDIATE RELEASE: Florida Conference of Catholic Bishops Announces Support for Amendment 4," October 18, 2018 ↑ TBYR, "2018 Florida Constitutional Amendments Recommendations," accessed November 1, 2018 ↑ News 965, "SINGER JOHN LEGEND WILL RALLY IN ORLANDO TO RESTORE FELON’S VOTING RIGHTS," accessed October 1, 2018 ↑ WLRN, "ACLU Of Florida Explains Its Stand On Constitutional Amendments Four, Six And 11," accessed September 26, 2018 ↑ Tallahassee Democrat, "Amendment 4 will save taxpayer money and give felons a second chance | Opinion," accessed September 20, 2018 ↑ Florida Department of State, "Floridians For A Sensible Voting Rights Policy," accessed November 30, 2017 ↑ Floridians For A Sensible Voting Rights Policy, "Homepage," accessed December 21, 2017 ↑ Floridians For A Sensible Voting Rights Policy, "About," accessed December 21, 2017 ↑ Florida Politics, "Tampa attorney argues against proposed voter restoration amendment," November 3, 2017 ↑ Florida Family Action, "2018 Ballot Amendment Voter Guide," accessed October 19, 2018 ↑ Human Rights Defense Center, "FLORIDA AMENDMENT 4 – HRDC FACT SHEET," accessed November 14, 2018 ↑ Jump up to: 47.0 47.1 47.2 Ballotpedia staff, "Email correspondence with Woment Against Registry representative," accessed November 14, 2018 ↑ Tampa Bay Times, "Column: Reject effort to restore voting rights for most felons," August 31, 2017 ↑ Tallahassee Democrat, "The case against Amendment 4 on felon voting rights | Opinion," accessed September 20, 2018 ↑ Florida Today, "Why you should vote to restore felons' voting rights | Our view," February 5, 2018 ↑ New York Times, "Florida’s 1.5 Million Missing Voters," January 2, 2018 ↑ Washington Post, "Floridians should scrap these retrograde, racist voting laws," January 27, 2018 ↑ Tampa Bay Times, "Times recommends: Yes on Amendment 4," accessed October 8, 2018 ↑ Sun Sentinel, "Five good — seven bad — amendments for Florida’s Constitution | Editorial," accessed October 8, 2018 ↑ Naples News, "Editorial: Our final recommendations on amendments," accessed October 10, 2018 ↑ Tallahassee Democrat, "Florida's constitutional amendments: Vote 'yes' on 4 and 11, 'no' on rest | Our opinion," accessed October 12, 2018 ↑ Herald Tribune, "Editorial: In support of Amendments 3, 4," accessed October 3, 2018 ↑ Your Observer, "Florida voters will decide dozens of ballot questions. Here are six for consideration," accessed October 13, 2018 ↑ Treasure Coast Palm, "How to vote on 12 constitutional amendments on Nov. 6 ballot | Our view," accessed October 13, 2018 ↑ Jacksonville, "Editorial: Sorting out confusing amendments for the voters," accessed October 15, 2018 ↑ My Palm Beach Post, "Editorial: Time to restore voting rights to 1.5 million Floridians," accessed October 15, 2018 ↑ Daily Commercial, "Our Opinion: Our recommendations on the amendments," accessed October 23, 2018 ↑ The Independent Florida Alligator, "The Alligator's endorsements for Constitutional amendments and referenda," accessed October 31, 2018 ↑ The Orlando Sentinel, "Editorial: Florida's Election 2018: Our endorsements for governor, U.S. Senate, U.S. House and the amendments," accessed October 31, 2018 ↑ Miami Herald, "Learn how 12 Florida amendments affect your life, and your wallet, before you vote," accessed November 4, 2018 ↑ News-Press, "Editorial: Proposed amendments too much of a gamble; vote 'no' on 11 of them," accessed October 8, 2018 ↑ National Conference of State Legislatures, "Felon Voting Rights," November 28, 2017 ↑ FairVote, "Average Margin of Victory," accessed August 7, 2017 ↑ Usenix.org, "Computing the Margin of Victory in IRV Elections," accessed August 7, 2017 ↑ Florida Constitution Revision Commission, "Amendments, Election of 11-5-68," accessed December 7, 2017 ↑ Florida Constitution Revision Commission, "Constitution of 1885," accessed December 7, 2017 ↑ Florida Constitution Revision Commission, "Constitution of 1868," accessed December 7, 2017 ↑ Florida Secretary of State, "Tabulation of Official Votes Cast in the General Election (1968)," accessed December 7, 2017 ↑ Florida Constitution Revision Commission, "Constitution of 1838," accessed December 7, 2017 ↑ Florida Commission on Offender Review, "Clemency," accessed January 30, 2018 ↑ Jump up to: 76.0 76.1 76.2 United States Court of Appeals for the 11th Circuit, "Johnson v. Bush," April 12, 2005 ↑ United States District Court for the Southern District of Florida, "Johnson v. Bush," September 21, 2000 ↑ Jump up to: 78.0 78.1 78.2 78.3 United States District Court for the Northern District of Florida, "Hand v. Scott," February 1, 2018 ↑ NPR, "Voting Rights Process For Florida Felons Unconstitutional, Judge Says," February 2, 2018 ↑ Tampa Bay Times, "Judge strikes down Florida’s system for denying felons’ voting rights," February 2, 2018 ↑ CBS News, "Ruling on voting rights for felons in Florida could impact upcoming elections," February 2, 2018 ↑ News 4 JAX, "State says it should control rights restoration," February 12, 2018 ↑ Florida Politics, "State, voting rights group disagree on how to handle clemency process," February 12, 2018 ↑ Orlando Sentinel, "Florida ordered to redo how it restores felons' voting rights," March 26, 2018 ↑ Florida Politics, "Rick Scott, Cabinet appeal voting rights ruling," April 6, 2018 ↑ Florida Politics, "State requests more time in felons’ rights battle," April 25, 2018 ↑ CBS 12, "Court backs state in felons' rights fight," April 25, 2018 ↑ Florida Commission on Offender Review, "Rules of Executive Clemency (2007)," accessed January 30, 2018 ↑ Florida Politics, "Charlie Crist applauds Terry McAuliffe for beating his record on restoring voting rights," April 20, 2018 ↑ Jump up to: 90.0 90.1 The Sentencing Project, "6 Million Lost Voters: State-Level Estimates of Felony Disenfranchisement, 2016," October 6, 2016 ↑ Florida Politics, "Felon voter restoration advocates make their case to Supreme Court," November 23, 2016 ↑ Tampa Bay Times, "Voting rights ballot initiative clears Supreme Court legal hurdle," April 20, 2017 ↑ Florida Supreme Court, "Advisory Opinion," April 20, 2017 ↑ Tampa Bay Times, "Petition drive for 2016 would make it easier for ex-felons to regain voting rights," December 21, 2014 ↑ Florida Politics, "Voting restoration amendment has 900,000 signatures," November 29, 2017 ↑ Florida Department of State, "Campaign Finance Database," accessed February 13, 2018 ↑ Florida Secretary of State, "FAQ - Voting," accessed October 17, 2019 ↑ Jump up to: 98.0 98.1 Florida Division of Elections, "National Voter Registration Act (NVRA)," accessed October 6, 2019 ↑ Jump up to: 99.0 99.1 Florida Division of Elections, "Register to Vote or Update your Information," accessed October 6, 2019 ↑ Florida Division of Elections, "Election Day Voting," accessed September 29, 2019 ↑ Jump up to: 101.0 101.1 Florida Division of Elections, "Florida History: Voter ID at the Polls," accessed September 29, 2019 ↑ National Conference of State Legislatures, "Voter Identification Requirements|Voter ID Laws," June 5, 2017 ↑ The Washington Post, "Do I need an ID to vote? A look at the laws in all 50 states," October 27, 2014 ↑ United States Census Bureau, "QuickFacts - Florida," accessed May 9, 2018 ↑ Florida Demographics, "Florida Cities by Population," accessed May 9, 2018 Only the first few references on this page are shown above. Click to show more.
  
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ballotpedia.org/Florida_Amendment_4,_Voting_Rights_Restoration_for_Felons_Initiative_(2018)
Nov 2021
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.11.17.468943

1
1. sciscore 30 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.11.17.468943: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">1.5 Cytotoxicity assays: Vero cell lines (ATCC® CCL-81™) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">1.1 Cloning, protein overexpression and purification of SARS-CoV-2 PLpro: A fragment of SARS-CoV-2 ORF pp1a/ab encoding the PLpro domain and corresponding to amino acids 746-1060 of non-structural protein 3 (YP_009742610.1) was cloned into pETM11(EMBL), which encodes N-terminal hexa-his tag followed by a tobacco etch virus (TEV) protease cleavage site.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pETM11</div><div>suggested: RRID:Addgene_108943)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Final rounds of manual refinement with either Refmac48 or Phenix49 together with manual model building applying COOT resulted in the final refined structures.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>COOT</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Microsoft Excel and GraphPad Prism (version 8.3.1) were used for analyzing the results and preparation of corresponding figures.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Microsoft Excel</div><div>suggested: (Microsoft Excel, RRID:SCR_016137)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture supernatant was harvest 42 h post-infection and viral RNA was purified using MagMAX™</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MagMAX™</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">EC50-values were calculated by fitting the data using GraphPad Prism version 8.00 (GraphPad Software, La Jolla California USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 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:
  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.
  Results from scite Reference Check: We found no unreliable references.
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  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2021.11.17.468943
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https://biorxiv.org/cgi/content/10.1101/2021.11.23.469714

1
1. sciscore 30 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.11.23.469714: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">As a control, a same number of cells were stained with BV421 anti-hCD45 antibody (Biolegend, #368522) and the top 3% of the BV421-positive cells were sorted.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-hCD45</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Then the cells were washed two times and resuspended in FACS buffer containing the secondary antibodies at a 1:1000 dilution: AF647-labeled donkey anti-goat IgG (Invitrogen, #A32849) or AF488-labeled goat anti-rabbit IgG (Invitrogen, #A32731).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>AF647-labeled donkey anti-goat IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: (Thermo Fisher Scientific Cat# A32731, RRID:AB_2633280)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HRP-conjugated goat anti-human IgG Fc secondary antibody was used to detect the bound ACE2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To compare GFP expressions in ACE2- and LRRC15-positive cells, pseudovirus-infected cells were stained for surface ACE2 and LRRC15 as described above with following secondary antibodies: AF405-labeled donkey anti-goat IgG (Invitrogen, #A48259) and PE-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch, #711-116-152)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>AF405-labeled donkey anti-goat IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>PE-labeled donkey anti-rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were subsequently incubated with recombinant anti-LRRC15 antibody (Abcam, #ab150376) and SARS-CoV-2 spike antibody [1A9] (GeneTex, #GTX632604) at 1:100, followed by incubation with 1:500-diluted Alexa Fluor 555 conjugated goat anti-rabbit IgG antibody (Abcam, #ab150078) and 1:200-diluted Alexa Fluor 488 conjugated goat anti-mouse IgG antibody (Abcam, #ab150117) for 60 min at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-LRRC15</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: (Abcam Cat# ab150117, RRID:AB_2688012)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For A375 cells, 5 µg/mL blasticidin (Gibco, #A1113903) and 1 µg/mL puromycin (Gibco, #A1113803) were added as appropriate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A375</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A375-dCas or HeLa-dCas cells were generated by transducing with pLenti-dCas9-VP64-Blast (Addgene, #61425).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa-dCas</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">7.8 x 107 A375-dCas cells were transduced with the CRISPRa library at ~0.3 MOI to make 2.4 x 107 transduced cells, which is sufficient for the integration of each sgRNA into ~500 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A375-dCas</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 8 x 106 HEK293T cells were plated in 10-cm tissue culture dishes and transfected using Lipofectamine2000 (Invitrogen) with plasmids encoding different CoV spike proteins or VSV-G protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For 1:1 ratio co-culture, 1 x 104 HeLa-ACE2 cells and 1 x 104 HeLa or HeLa-sgLRRC15 cells were plated per well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HeLa-sgLRRC15</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Microscopic analysis: 8 x 103 HeLa-ACE2 cells and 3.2 x 104 HeLa or HeLa-sgLRRC15 cells (1:4 ratio) were co-plated per well in 8-well chamber slides (Nunc).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HeLa-ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A375-dCas or HeLa-dCas cells were generated by transducing with pLenti-dCas9-VP64-Blast (Addgene, #61425).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLenti-dCas9-VP64-Blast</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Stable ACE2 expressing HeLa cells (HeLa-ACE2) were generated by transducing HeLa-dCas cells with pLENTI_hACE2_HygR (Addgene, #155296) followed selection with hygromycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLENTI_hACE2_HygR</div><div>suggested: RRID:Addgene_155296)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For ectopic expression of LRRC15, a lentiviral vector pCDH-MSCV-T2A-Puro (System Biosciences, #CD522A-1) was modified to enable zeocin selection instead of puromycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDH-MSCV-T2A-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A codon-optimized LRRC15 ORF was cloned into pCDH-MSCV-T2A-Zeo with a C-terminal 3xFLAG tag and used to transduce HeLa-ACE2 followed by zeocin selection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDH-MSCV-T2A-Zeo</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sgRNAs were cloned into pXPR_502 (Addgene, #96923) with assistance from the Genome Engineering and iPSC Center (GEiC) at Washington University in Saint Louis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pXPR_502</div><div>suggested: RRID:Addgene_96923)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression vectors for SARS-CoV-2 Wuhan-Hu-1 (Addgene, #149539), SARS-CoV-2 B.1.167.2 (Addgene, #172320), SARS-CoV-2 B.1.1.7 (Addgene, #170451), SARS-CoV-2 B.1.351 (Addgene, #170449), SARS-CoV-2 P.1 (Addgene, #170450), SARS-CoV-1 (Addgene, #170447), MERS-CoV (Addgene, #170448) and VSV-G (Addgene, #12259) were used.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VSV-G</div><div>suggested: RRID:Addgene_138479)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Generation of genetically modified cell lines: Individual sgRNAs (sgLRRC15 #1: GACATGCAGGCACTGCACTG; sgLRRC15 #2: AGTGTCAGCCCGGGACATGC; sgACE2: GTTACATATCTGTCCTCTCC) targeting the candidate genes were cloned into linearized pXPR_502 (Addgene, #96923) for CRISPR activation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GACATGCAGGCACTGCACTG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 30 min incubation at 4°C, the cells were washed two times, fixed with 4% formaldehyde for 15 min and washed and resuspended in FACS buffer before analyzing by flow cytometry using FACSCelesta (BD Biosciences) or Cytek</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Cytek</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data was analyzed with Flowjo software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Flowjo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">GraphPad Prism 9 software was used to perform nonlinear regression curve-fitting analyses of binding data to estimate dissociation constants (KD).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To compare LRRC15 expression in lung cell lines infected with SARS-CoV-2 vs mock controls [61], we accessed the raw count data from GSE147507 and performed differential expression analysis using DESeq2 as above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>DESeq2</div><div>suggested: (DESeq, RRID:SCR_000154)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Statistical significance was determined using GraphPad Prism 9 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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Visit annotations in context

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sciscore

URL

biorxiv.org/cgi/content/10.1101/2021.11.23.469714
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.11.24.469842

1
1. sciscore 30 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.11.24.469842: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bound phages were detected by HRP-conjugated anti-M13 antibody (Sino Biological, China)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-M13</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bound VHHs were detected by HRP-conjugated anti-Myc-tag antibody and TMB substrate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Myc-tag</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After incubation samples were added to a monolayer of Vero E6 cells and incubated in a 5% CO2 incubator at 37 °C for 96-120 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cloning, expression and purification of recombinant SARS-CoV-2 RBD protein: The RBD nucleotide sequence of SARS-CoV-2 Wuhan-Hu-1 isolate (Genbank accession number MN908947, from 319 to 545 aa) was synthesized (Evrogen, Russia) and cloned into the pCEP4 mammalian expression vector (Thermo Fisher Scientific, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCEP4</div><div>suggested: RRID:Addgene_16479)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VHHs coding sequences were PCR amplified and cloned into a pHEN1 phagmid vector (33)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHEN1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In the second round PCR nanobodies sequences were assembled together and amplified using pHEN1-F and pHEN1-R primers.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHEN1-F</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pHEN1-R</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VHH-coding sequences were sequenced with Lac-prom (5’-CTTTATGCTTCCGGCTCGTATG-3’) and pIII-R (5’ CTTTCCAGACGTTAGTAAATG 3’) primers according to the protocol of the BigDyeTerminator 3.1 Cycle Sequencing kit for the Genetic Analyzer 3500 Applied Biosystems (Waltham, MA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pIII-R</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">EC50 values were calculated using four-parameter logistic regression using GraphPad Prism 9 (GraphPad Software Inc, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequences were aligned with MUSCLE (v3.8.31) (35).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MUSCLE</div><div>suggested: (MUSCLE, RRID:SCR_011812)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.1/3.0 aLRT) (36).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PhyML</div><div>suggested: (PhyML, RRID:SCR_014629)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Graphical representation and edition of the phylogenetic tree were performed with MEGA X (34).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MEGA</div><div>suggested: (Mega BLAST, RRID:SCR_011920)</div></div></td></tr></table>
  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:
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2021.11.24.469842
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https://biorxiv.org/cgi/content/10.1101/2021.11.23.469747

1
1. sciscore 30 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.11.23.469747: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Target proteins were detected using the following antibodies: mouse anti-Strep tag II (Millipore Sigma: 71590); rabbit anti-Caspase 3 (Cell signaling: 9662); rabbit anti-Cleaved-Caspase 3 (Cell signaling: 9664) and mouse anti-GAPDH antibody (Cell signaling: 2118).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Strep tag II</div><div>suggested: (Abnova Cat# PAB16601, RRID:AB_10677207)</div></div><div style="margin-bottom:8px"><div>anti-Caspase 3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Cleaved-Caspase 3</div><div>suggested: (Affinity Biosciences Cat# BF0711, RRID:AB_2846190)</div></div><div style="margin-bottom:8px"><div>anti-GAPDH</div><div>suggested: (Cell Signaling Technology Cat# 2118, RRID:AB_561053)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A549 and 293T cell lines were maintained in the high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Corning Cat#: 10-017-CV) with 10% fetal bovine serum (FBS, Gibco Cat#: 100-438-026) and 100 U/mL Penicillin-Streptomycin (Gibco Cat#:</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Calu-3 cells were maintained in Eagle’s Minimum Essential medium (EMEM) (Quality Biological Cat#: 112-018-101) with 10% FBS and 100 U/mL Penicillin-Streptomycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu-3</div><div>suggested: BCRJ Cat# 0264, RRID:CVCL_0609)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Measurement of Mammalian Cell-specific Activities: 2 x 104 293T or 1 x 104 A549 and Calu-3 cells/well were seeded into a 96-well plate and cultured at 37°C/5% CO2 overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>A549</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the mammalian ORF3a study, a lentiviral constitutive expression vector pLVX-EF1alpha-IRES-Puro (Takara) that carries the ORF insert (provided by Dr. Nevan J.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-EF1alpha-IRES-Puro</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Fission Yeast Plasmid Transformation and Inducible SARS-COV-2 Gene Expression: The SARS-COV-2 gene-carrying pYZ1N plasmids were transformed into a wild type fission yeast SP223 strain by electroporation (5, 57).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pYZ1N</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Calu-3 cells were maintained in Eagle’s Minimum Essential medium (EMEM) (Quality Biological Cat#: 112-018-101) with 10% FBS and 100 U/mL Penicillin-Streptomycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Quality Biological</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical Analysis: Pair-wise t-test or one-way ANOVA was calculated using software Prism 9 (GraphPad, San Diego, CA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 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 pages 24 and 17. 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.
  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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
  </footer>
Visit annotations in context

Annotators

sciscore

URL

biorxiv.org/cgi/content/10.1101/2021.11.23.469747
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.11.24.469776

1
1. sciscore 30 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.11.24.469776: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: All animal experiments were performed according to Institute of Laboratory Animal Resources guidelines and the protocol was approved by the National Cancer Institute Animal Care and Use Committee.<br>Euthanasia Agents: All mice were anesthetized via isoflurane inhalation (3 - 5 % isoflurane, oxygen flow rate of 1.5 L/min) prior and during BLI using the XGI-8 Gas Anesthesia System.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">6–8-week-old male and female mice were used for all the experiments.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were washed three times and incubated with the goat anti-human-IgG Fc secondary antibody conjugated with alkaline phosphatase (AP, Southern Biotech) at a 1:1000 dilution in blocking buffer for 1 h at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human-IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody-dependent cellular phagocytosis was determined by flow cytometry, gating on THP-1 cells that were triple-positive for GFP, efluor450 and efluor670 cellular dyes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GFP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Samples were washed, sonicated, and incubated with goat anti-guinea pig C3 antibody conjugated with biotin (Immunology Consultants Laboratory) at RT for 1 h followed by incubation with streptavidin R-Phycoerythrin (PE,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-guinea pig C3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Before and after injection, serum samples were collected at 0 min, 10min, 1 h, 6 h, 24 h and 48 h and the ACE2-Fc serum concentration was estimated by indirect ELISA in which SARS-CoV-2 RBDwt (200 ng/well) were used as capturing molecule and the goat-anti-human IgG conjugated with AP (1:1000 dilution) were used as secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgG</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IgG1 Fc tag and 8xHis tag) (45), plasmids encoding the respective genes were transfected to 293F cells with the same protocol as described above.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293F</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To determine viral titers, hACE2-expressing 293T cells (gift from Dr. Allison Malloy, USUHS) were infected with serial PsV dilutions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody-dependent cellular phagocytosis was determined by flow cytometry, gating on THP-1 cells that were triple-positive for GFP, efluor450 and efluor670 cellular dyes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>THP-1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Dilutions from infected cell homogenates were applied on Vero E6 monolayer. 24 hour post infection, infected Vero E6 cells were washed with PBS, lysed with Passive lysis buffer and transferred into a 96-well solid white plate (Costar Inc) and nanoluciferase activity was measured using Tristar multiwell Luminometer (Berthold Technology) for 2.5 seconds by adding 20 µl of Nano-Glo® substrate in nanoluc assay buffer (Promega Inc).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">96-well Nunc Maxisorp plates (Sigma) were coated with SARS-CoV-2 RBDwt (residue 319-541) (50ng), RBDB.1.351 (50 ng), S-2P (75 ng), SB.1.1.7 (75 ng), SB.1.351 (75ng), SP.1 (75 ng), SB.1.526 (75 ng) and SARS-CoV RBD (50ng) per well in Tris-buffered saline (TBS) at 4 °C overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SB.1.1.7</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For in vivo PsV-based inhibition assays, 6-8-week-old K18-hACE2 mice were intranasally (i.n) treated with Synagis (control IgG, 25 µg), M27 or M81 (5 or 25 µg) one hour before challenge by SARS-CoV-2 PsVD614G or PsVB.1.617.2 (i.n., ∼108 RLU)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18-hACE2</div><div>suggested: RRID:IMSR_GPT:T037657)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For PK studies, C57BL/6J mice were intravenously (i.v.) injected with 100 μg (5 mg/kg) of two engineered ACE2-Fc M81 or M86.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6J</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate SARS-CoV-2 RBDwt (residue 319-541 or residue 319-537, for crystallization), RBDB.1.1.7 (residue 329-527, N501Y) and RBDB.1.351 (residue 329-527, K417N/E384K/N501Y), the respective codon optimized DNA segments fused with an N-terminal secretion peptide and a C-terminal 6xHis tag were cloned into the pACP-tag (m)-2 vector using either EcoRI/NotI for RBDwt (319–541), RBD B.1.1.7 and RBDB.1.351 or BamHI/XhoI for RBDwt (319–537) as restriction enzymes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pACP-tag</div><div>suggested: RRID:Addgene_101126)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Monomeric ACE2wt and engineered ACE2LFMYQY2HA plasmids encoding ACE2 (residue 1-615) with C-terminal HRV-3C-cleavable 8xHis tag (45) were transfected to FreeStyle 293F cells and the resulting protein was purified over Ni-NTA columns (Cytiva).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2LFMYQY2HA</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">GraphPad Prism was used to display the mean and SEM for all groups and used to calculate the area under the curve (AUC) within the concentration range of 0.05-2.5 nM using 5% binding as baseline (Fig. 2D & S2)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Iterative cycles of model building and refinement were done in Coot (79) and Phenix (80).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Structural analysis and Fig. generation were performed in PyMOL (81)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All samples were acquired on an LSRII cytometer (BD Biosciences) and data analysis performed using FlowJo v10 (Tree Star)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bioluminescence Imaging (BLI) of SARS-CoV-2 infection: All standard operating procedures and protocols for IVIS imaging of SARS-CoV-2 infected animals under ABSL-3 conditions were approved by IACUC, IBSCYU and YARC.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>YARC</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Images were acquired and analyzed with Living Image v4.7.3 in vivo software package (Perkin Elmer Inc).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Living Image</div><div>suggested: (Living Image software, RRID:SCR_014247)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data were processed and plotted using GraphPad Prism 8 v8.4.3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  Results from OddPub: Thank you for sharing your data.
  Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
  Thus far, the in vivo protective potential of an ACE2-Fc therapeutic has been tested only once in a Syrian hamster model (28) which has several limitations due to its inability to fully recapitulate SARS-CoV-2 pathogenesis and severity. To better test our lead ACE2-Fc variant we utilized a well characterized K18-hACE2 mouse model (67). Due to the constitutive high endogenous human ACE2 expression, this model is highly susceptible to SARS-CoV-2 infection and the disease progression partially recapitulates the severe pathological features of SARS-CoV-2 infection in humans. The model has also been used extensively for evaluating contributions from direct neutralization and Fc-effector activities mediated by nAbs (68) and a non-neutralizing Ab (69). However, a high basal level of hACE2 on target cells in this model, particularly in the brain, poses a significant obstacle for soluble ACE2-based antivirals such as our engineered ACE2-Fc to surmount and achieve protection. Despite these limitations, we detected a strong benefit to the administration of ACE2740 LFMYQY2HA–Fc GASDALIE variant both prophylactically and therapeutically in K18-hACE2 mice. In both settings, ACE2-Fc treatments were associated with markedly improved in vivo efficacy, e.g., a reduction in virus-induced body weight loss, pro-inflammatory cytokine responses and mortality, particularly in the therapeutic context. Given the human Fc-mouse FcγR mismatch may compromise Fc-effector functionality of ACE-Fcs in K18-hA...
  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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 56, 51 and 60. 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.
  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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  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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Microsoft Word - Differences in Vaccine and SARS-CoV-2 Replication Derived mRNA- Implications for Cell Biology and Future Disease_11-24-21.Submit-0SF.docx

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1. IngoF 29 Nov 2021
  
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  Jiang et al.
  
  I would be cautious with the interpretation of Jiang et al. paper since the expressed spike proteins contains his-tag and the authors IMO didn't do a proper control so one can't exclude that the effect(s) seen it due to the his-tag.
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The Next Chapter of Readwise: Our Own Reading App

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1. akamatsm 26 Nov 2021
  
  in Public
  
  Whenever you tag or add an annotation to a resurfaced highlight, you can see that update reflected in the margin of the original document (and vice versa).
  
  Yes! Will this include margin notes taken with digital ink that is then recognized by OCR? (e.g. like myscript/kobo is doing). That would be truly transformative. Much more personal and flexible than typing some stuff into the margins.
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Blog Paper: Advanced Note Taking Technology: Luddite, Productive: 9781926892108: Amazon.com: Books

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1. chrisaldrich 26 Nov 2021
  
  in Public
  
  https://www.amazon.com/Blog-Paper-Advanced-Taking-Technology/dp/1926892100/
  
  Doing some research for my Paper Website / Blog.
  
  Similar to some of the pre-printed commonplace books of old particularly with respect to the tag and tag index sections.
  
  I sort of like that it is done in a way that makes it useful for general life even if one isn't going to use it as a "blog".
  
  How can I design mine to be easily photographed and transferred to an actual blog, particularly with Micropub in mind?
  
  Don't forget space for the blog title and tagline. What else might one put on the front page(s) for identity? Name, photo, address, lost/found info, website URL (naturally)...
  
  Anything else I might want to put in the back besides an category index or a tag index? (Should it have both?)
  
  paper websites blogging commonplace books journals stationery
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compendiumofawesome.wordpress.com compendiumofawesome.wordpress.com

tmesis – A Compendium of Awesome Things

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1. fncll 24 Nov 2021
  
  in Public
  
  Apparently, there was a poem written not too long ago by an Australian author and poet named John O’Grady[iii] entitled Tumba Bloody Rumba. I won’t include the poem here in its entirety, partly because its frequent use of the word bloody may offend some. Suffice it to say, the poem makes ample use of colourful tmesis with words such as “Tumba-bloody-rumba” and “kanga-bloody-roos.” The result, thanks in no small part to the almost hypnotic power of the word, is that tumbarumba has now become a synonym for tmesis in the English language.
  
  tumbarumba = tmesis in Australia
  
  #tmesis
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CNIC (Computerised National Identity Card)

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1. dmq 21 Nov 2021
  
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  CNIC (Computerised National Identity Card)
  
  dmq public Annotations
  
  thẻ thẻ mới thẻ khác dmq-tag mqd-tag vn vietnam Vietnam's
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https://biorxiv.org/cgi/content/10.1101/2021.11.02.466951

1
1. sciscore 19 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.11.02.466951: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  NIH rigor criteria are not applicable to paper type.
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The blots were then probed with SARS-CoV-2 spike antibody (NR-52947, BEI Resources, NIAID, NIH) in blocking buffer for 12 hr at 4°C, followed by secondary Goat Anti-Rabbit IgG antibody (ab6721, Abcam, RRID:AB_955447) incubation for 2hr.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Rabbit IgG</div><div>detected: (Abcam Cat# ab6721, RRID:AB_955447)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Actin was labelled using antibody against beta-actin [AC-15] (HRP) (ab49900, Abcam, RRID: AB_867494).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody against beta-actin</div><div>detected: (Abcam Cat# ab49900, RRID:AB_867494)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The HEK293 cells were transfected with 100 ng/well of pGL3-Fluc plasmid using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol at around 75-90% confluency in a 96 well plate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells and virus: The following cell lines were used in this study, namely HEK 293T cells (CRL-1573, ATCC, RRID: CVCL_0045)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293T</div><div>detected: (NIH-ARP Cat# 103-306, RRID:CVCL_0045)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, HEK 293T cells stably expressing human ACE2 (NR-52511, BEI Resources, NIAID, NIH, RRID:</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, Vero-E6 cells (CRL-1586, ATCC, RRID: CVCL_0574).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-E6</div><div>detected: (IZSLER Cat# BS CL 87, RRID:CVCL_0574)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">NR-52282, BEI Resources, NIAID, NIH) was propagated and quantified by plaque assay in Vero-E6 cells as described before (Case et al., 2020)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cytotoxicity assay: HEK-ACE2 cells were seeded in 0.1 mg/mL poly-L-lysine (P9155-5MG, Sigma-Aldrich) coated 96-well plate to reach 70-80% confluency after 24 Hrs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Virus infection: HEK ACE2 cells were seeded in poly-L-lysine coated 24-well plate to reach 80% confluency at the time of infection.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Nsp1 expression and purification: The gene construct encoding the Nsp1 from SARS-CoV-2 in pCDNA 5-3X-Flag-Nsp1 was amplified and sub-cloned into pET28a with N-terminal His-tag (Schubert et al., 2020; Thoms et al., 2020) using appropriate primers (Supplementary table 1).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDNA 5-3X-Flag-Nsp1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pET28a</div><div>suggested: RRID:Addgene_114156)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">the cultures were then transferred at 16°C at 120 rpm, and the expression of pET28a-His-Nsp1 and pET28a-His-Nsp1Δ40 were induced by adding 1 mM of Isopropyl β-d-1-thiogalactopyranoside (IPTG) and allowed to grow for 18 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET28a-His-Nsp1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pET28a-His-Nsp1Δ40</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The HEK293 cells were transfected with 100 ng/well of pGL3-Fluc plasmid using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol at around 75-90% confluency in a 96 well plate.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGL3-Fluc</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plasmid expressing the Nsp1 protein (pcDNA 3.1-Nsp1) was co-transfected at 100 ng/well concentration.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA 3.1-Nsp1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 48 hr incubation, cells were fixed with 4% paraformaldehyde, and crystal violet (C6158, Merck) staining was done to visualize the plaques. Plasmids: pLVX-EF1alpha-SARS-CoV-2-nsp1-2xStrep-IRES-Puro expressing SARS CoV-2 NSP1 was a kind gift from Prof. Nevan Krogan (Gordon et al., 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pLVX-EF1alpha-SARS-CoV-2-nsp1-2xStrep-IRES-Puro</div><div>suggested: RRID:Addgene_141367)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Other plasmids used in this study include Plasmids pRL-TK (mammalian vector for weak constitutive expression of wild-type Renilla luciferase), pGL4 (mammalian vector expressing firefly luciferase), pIFN-β Luc (IFN beta promoter-driven firefly luciferase reporter).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pRL-TK</div><div>suggested: RRID:Addgene_11313)</div></div><div style="margin-bottom:8px"><div>pGL4</div><div>suggested: RRID:Addgene_48744)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plasmid pMTB242 pcDNA5 FRT-TO-3xFLAG-3C-Nsp1_SARS2 was a kind gift from Prof. Ronald Beckmann.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pMTB242</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Finally, the library was screened against the 18S rRNA interacting interface of Nsp1-C-ter using the Surflex-dock program, which is available in SYBYL v2.1 (Jain, 2003).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Surflex-dock</div><div>suggested: (Surflex-Dock, RRID:SCR_000196)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data was analysed by using ThermControl software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ThermControl</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The two subsequent 100 ns runs from MD simulations were further subjected to perform the MM-PBSA by using the python script (mmpbsa.py) to calculate the binding energy of the two drugs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>python</div><div>suggested: (IPython, RRID:SCR_001658)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Relative intensity of bands was quantified using Fiji/imageJ.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Fiji/imageJ</div><div>suggested: None</div></div></td></tr></table>
  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:
  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.
  Results from scite Reference Check: We found no unreliable references.
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  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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https://biorxiv.org/cgi/content/10.1101/2021.10.29.466470

1
1. sciscore 19 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.29.466470: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies: Monoclonal antibodies MAb362 isotypes IgG1 and IgA1 has been described before(32).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgA1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">REGN10987, S309 and CR3022 antibodies heavy and light variable region sequences(33, 34, 58) were synthesized and cloned into pcDNA3.1 vector (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA) in-frame with human IgG heavy or light chain Fc fragment.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CR3022</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">. 2G12 monoclonal antibody was expressed in ExpiCHO-S™ cells through co-transfection of plasmids encoding light and IgG heavy chains(59), using the ExpiFectamine™ CHO transfection kit (Gibco™, Thermo Fisher Scientific, Waltham, MA, USA) according to manufacturer instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>2G12</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Monoclonal antibodies 4A8 and 1A9 were purchased from BioVision (Milpitas, CA, USA) and GeneTex (Irvine, CA, USA), respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>1A9</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-6x-His-tag polyclonal antibody, and both HRP-conjugated anti-mouse IgG Fc and anti-human IgG Fc were purchased from Invitrogen™ (Waltham, MA, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-6x-His-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We used a rabbit anti-6X-His antibody (Invitrogen™, Waltham, MA, USA) to detect histidine-tagged proteins or mouse 1A9 antibody (GeneTex, Irvine, CA, USA) for specific detection of SARS-CoV-2 SΔTM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-6X-His</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Membranes were washed three times with PBS-T and then incubated with secondary HRP-conjugated anti-rabbit IgG (Abcam, Cambridge, UK) or anti-mouse IgG (Invitrogen™, Waltham, MA, USA) antibodies diluted in 0.5% (w/v) skim milk/PBS-T and incubated for one hour at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">As secondary antibodies, HRP-conjugated anti-human kappa antibody (SouthernBiotech, Birmingham, AL, USA) diluted 1:4000 in PBS was used in wells treated with MAb362, CR3022 and S309 antibodies, while HRP-conjugated anti-human IgG Fc (Invitrogen™, Waltham, MA, USA) diluted 1:10,000 in PBS was used in wells treated with REGN10987, 4A8 and 2G12 antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human kappa</div><div>suggested: (Abgent Cat# AT4000a, RRID:AB_1554597)</div></div><div style="margin-bottom:8px"><div>S309</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">S1). smFRET imaging: Labeled SΔTM spikes (100-200 nM) were incubated in the absence or presence of unlabeled ACE2 or the indicated antibody at a monomer:ACE2 or monomer:antibody ratio of 1:3 for 90 minutes at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two-tailed nonparametric Spearman test with 95% confidence was performed to evaluate the correlation level between the occupancy of SΔTM in the open conformation due to allosteric antibody binding and ACE2 binding (Figs. 4 and 5).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2 binding (Figs. 4</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">glycoprotein ectodomain (SΔTM) (residues Q14–K1211) with SGAG substitution at the furin cleavage site (R682 to R685), and proline substitutions at K986 and V987, was synthesized by GenScript® (Piscataway, NJ, USA) and inserted into pcDNA3.1(−).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SΔTM hetero-trimers for smFRET experiments were expressed by co-transfection with both the untagged SΔTM (D614 or D614G) construct and the corresponding 161/345A4-tagged SΔTM plasmid at a 2:1 molar ratio.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SΔTM</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SΔTM concentration was also estimated by densitometric analysis of protein bands on immunoblots with the monoclonal antibody 1A9 as described below, and using ImageJ software v1.52q (NIH, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All smFRET data were processed and analyzed using the SPARTAN software (www.scottcblanchardlab.com/software) in Matlab (Mathworks, Natick, MA, USA)(65).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Matlab</div><div>suggested: (MATLAB, RRID:SCR_001622)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">smFRET trajectories were idealized to a 3-state hidden Markov model and the transition rates were optimized using the maximum point likelihood algorithm(66), implemented in SPARTAN.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPARTAN</div><div>suggested: (SPARTAN, RRID:SCR_014901)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Dissociation constants (KD) were determined using GraphPad Prism version 9.2.0 (GraphPad Software, San Diego, CA, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Structural analysis: Protein structures from RCSB PDB were visualized and analyzed using PyMOL™ software version 2.0.7 (The PyMOL Molecular Graphic System,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL™</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr></table>
  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 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 21. 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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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Visit annotations in context

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sciscore

URL

biorxiv.org/cgi/content/10.1101/2021.10.29.466470
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.11.15.468737

1
1. sciscore 19 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.11.15.468737: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 nsp12, nsp10 and nsp7 gene were cloned into a modified pET-21b vector with the C-terminus possessing a 6×His-tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-21b</div><div>suggested: RRID:Addgene_132607)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The nsp8 gene was cloned into the modified pET-32a vector with the N-terminus possessing a trx-His6-tag and PreScission Protease site.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-32a</div><div>suggested: RRID:Addgene_120288)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The nsp7-pET-21b plasmid was transformed into E. coli Rosetta-gami2 (DE3) and the transformed cells were cultured at 37 °C in LB with a final concentration of 100 μg/ml ampicillin and 25 μg/ml chloramphenicol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>nsp7-pET-21b</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The nsp14 gene (nsp14-ExoN (residues 1–289)) was cloned into the modified pET-28b vector with the N-terminus possessing a MBP-tag and PreScission Protease site.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-28b</div><div>suggested: RRID:Addgene_47327)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The PCR product was digested with Xho1 and BamH1 and ligated into pET-15b vector, and transformed into E-coli DH5α to obtain clones.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET-15b</div><div>suggested: RRID:Addgene_108953)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
  </footer>
Visit annotations in context

Annotators

sciscore

URL

biorxiv.org/cgi/content/10.1101/2021.11.15.468737
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.11.08.467773

1
1. sciscore 19 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.11.08.467773: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Field Sample Permit: Mouse studies: Mouse studies were conducted at Murigenics (Vallejo, CA) under IACUC approved protocols.<br>IACUC: Mouse studies: Mouse studies were conducted at Murigenics (Vallejo, CA) under IACUC approved protocols.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Female Balb/c mice (Envigo), 6–8 weeks old were used for all studies.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Membranes were blocked for 1h in 5% skim milk in TBST and then probed with an anti-S2 mouse monoclonal antibody (GeneTex) at a 1:1000 dilution for 2h-overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-S2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Membranes were washed (0.05% Tween 20 in 1X TBS) and then probed with a Rabbit anti-Mouse HRP antibody (Bethyl labs) for 1h before washing and detection with a SuperSignal West Femto Maximum Sensitivity Substrate (Pierce).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Mouse HRP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A parallel blot using a mouse anti-actin antibody (Thermo Fisher) was used to ensure equivalent protein amounts per well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-actin</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Extracellular staining was performed in FACS buffer (PBS + 2% FBS + 2mM EDTA) with the following antibodies: CD4 (GK1.5, Biolegend), CD8 (53-6.7, BD).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD4</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD8</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Intracellular staining was then performed in permeabilization buffer with the following antibodies: IFNγ (XMG21.2, Invitrogen), TNFα (MP6-XT22, eBiosciences), IL2 (JES6-5H4, eBiosciences), IL4 (11B11, Biolegend), IL10 (JES5-16E3, Biolegend).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IL2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IL4</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IL10</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>JES5-16E3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The intensity of the light being emitted is inversely proportional to the amount of anti-SARS-CoV-2 neutralizing Spike antibodies bound to the VSVΔG – Spike ΔCT particles.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 neutralizing Spike</div><div>suggested: (Creative Diagnostics Cat# CABT-CS064, RRID:AB_2891088)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plates were incubated with anti-nucleocapsid protein primary antibody cocktail (clones HM1056 and HM1057) (EastCoast Bio, North Berwick, ME) for 60 minutes at 37°C (Battelle Memorial Institute, Patent Number 63/041,551 Pending, 2020).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-nucleocapsid protein</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plates were washed and the secondary antibody (goat anti-mouse IgG Horse Radish Peroxidase (HRP) conjugate; Fitzgerald, North Acton, MA) was added to the wells, and the plates were incubated for 60 minutes at 37°C1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Wells were washed and incubated with 25 μL of 1 μg/mL SULFO-TAG labeled anti-species antibody (MSD), diluted in DPBS + 1% BSA (Sigma-Aldrich, St. Louis, MO), for 1 hour at room temperature on an orbital shaker.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MSD</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Western analysis: HEK293F cells seeded at 5e5 cells/mL were infected with an MOI of 1 IU/cell.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293F</div><div>suggested: RRID:CVCL_6642)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The VSVΔG virus was transduced in HEK293T cells previously transfected with the spike glycoprotein of the SARS-CoV-2 coronavirus (Wuhan strain) for which the last 19 amino acids of the cytoplasmic tail were removed (ΔCT).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After the incubation of the serum/plasma-pseudotyped virus complex, the serum/plasma-pseudotyped virus complex was transferred to the plate containing Vero E6 cells (ATCC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Female Balb/c mice (Envigo), 6–8 weeks old were used for all studies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Balb/c</div><div>suggested: RRID:IMSR_ORNL:BALB/cRl)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequence of a E1 (578-3404 bp)/E3 deleted virus (2,125-31,825 bp) was assembled into pUC19 from VR-594-derived and synthetic (SGI-DNA) fragments.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pUC19</div><div>suggested: RRID:Addgene_50005)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pA68-E4d-Spike plasmids were linearized, purified using a Nucleospin kit (Machery-Nagel) and transfected into 2 mL of 293F cells (0.5 mL/mL) using TransIT-Lenti (Mirus bio).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pA68-E4d-Spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Spike sequences were PCR amplified and cloned into PacI/BstBI sites of a pUC02-VEE vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pUC02-VEE</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vector generation: The ChAd68 nucleotide sequence was based on the wild-type sequence obtained by MiSeq (Ilumina sequencing) of virus obtained from the ATCC (VR-594).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MiSeq</div><div>suggested: (A5-miseq, RRID:SCR_012148)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Analysis of flow cytometry data was performed using FlowJo software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data processing was performed using the R programming language and graphed using GraphPad Prism.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT03639714</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Active, not recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Study of a Personalized Neoantigen Cancer Vaccine</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT03953235</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Study of a Personalized Cancer Vaccine Targeting Shared Ne…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04776317</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Chimpanzee Adenovirus and Self-Amplifying mRNA Prime-Boost P…</td></tr></table>
  Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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biorxiv.org/cgi/content/10.1101/2021.11.08.467773
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https://biorxiv.org/cgi/content/10.1101/2021.10.25.465714

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1. sciscore 19 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.25.465714: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: The KYODOKEN Institutional Animal Care and Use Committee approved the protocols for these studies (approval number 20200312) and monitored health conditions.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">half-siblings—a 19-month-old male named “Puta” and a 19-month-old female named “Christy”—were immunized.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Blotted membranes were incubated overnight at 4°C with the C9 antibody or the C-terminally 6×His-tagged homodimer of nanobodies—the dilution ratios of P158, P334, and P543 were 1:5000, 1:1000, and 1:2500, respectively—in Tris-buffered saline (TBS, pH 7.4) containing 0.005% Tween 20 (TBST) and 5% skim milk.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C9</div><div>suggested: (LSBio (LifeSpan Cat# LS-C9-1000, RRID:AB_1276501)</div></div><div style="margin-bottom:8px"><div>P334</div><div>suggested: (Leinco Technologies Cat# P334, RRID:AB_2831621)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In the case of nanobody-based blotting, after 3 washes with TBST, the membranes were incubated with 1:5000-diluted anti-His-tag antibody (MBL) in TBST containing 5% skim milk at room temperature for 1 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His-tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The membranes were soaked with 1:5000-diluted HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (GE Healthcare) in TBST containing 5% skim milk for 30 min at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-6×His-tagged antibody-positive fractions were gathered and concentrated via VIVAspin 6 size exclusion columns (30,000-MWCO) to reach a volume under 0.5 ml.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-6×His-tagged</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 3 washes with PBST, HRP-conjugated anti-alpaca VHH antibody (Jackson) at a dilution of 1:5000 was reacted at room temperature for 30 min.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-alpaca VHH</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing, the cells were incubated with an anti-His antibody (Abcam) on ice for 30 min and then Alexa 647-conjugated anti-rabbit IgG (Dako).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing with PBST, an appropriately diluted anti-His-tagged antibody and anti-C9-tagged antibody in blocking buffer were added and reacted at room temperature for 1 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His-tagged</div><div>suggested: (StressMarq Biosciences Cat# SPC-167, RRID:AB_2703750)</div></div><div style="margin-bottom:8px"><div>anti-C9-tagged</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell culture and transfection: HEK and K562 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM: Invitrogen) supplemented with 10% foetal bovine serum (FBS) and antibiotics (1% penicillin and streptomycin).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K562</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped virus production: HIV-1-based SARS-CoV-2 spike pseudotyped virus was prepared as follows: LentiX-HEK293T cells were transfected using a polyethyleneimine transfection reagent (Cytiva) with plasmids encoding the C-terminally C9-tagged full-length SARS-CoV-2 spike variants (original, alpha, beta, and delta) and HIV-1 transfer vector encoding a luciferase reporter, according to the manufacturer’s protocol.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>LentiX-HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Microscopy analyses for cell staining: HEK cells were transiently transfected with plasmids encoding the C-terminally C9-tagged full-length SARS-CoV-2 spike variants using Lipofectamine 3000 (Thermo) according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The expression vector of the SARS-CoV-2 S2 domain (residues 744-1213) was constructed by removing an N-terminal part of the extracellular domain of the SARS-CoV-2 spike (residues 31-743) and subcloned into the pcDNA3.1(+) vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1 ( + )</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The synthesized genes were subcloned in the pMES4 vector to express N-terminal PelB signal peptide-conjugated and C-terminal 6×His-tagged nanobodies into the bacterial periplasm.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pMES4</div><div>suggested: RRID:Addgene_98223)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The lentiviral vector pWPI-IRES-Bla-Ak-ACE2-TMPRSS2 was acquired from AddGene (plasmid #154983).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pWPI-IRES-Bla-Ak-ACE2-TMPRSS2</div><div>suggested: RRID:Addgene_154983)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">coli TG1 cells (Agilent Technologies Japan, Ltd., Tokyo, Japan) were transformed with the ligated plasmids under chilled conditions (Bio-Rad Laboratories, Inc., Hercules, CA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Bio-Rad Laboratories</div><div>suggested: (Bio-Rad Laboratories, RRID:SCR_008426)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The raw data of reads were trimmed of the adaptor sequence using cutadapt v1.1859, and low-quality reads were subsequently removed using Trimmomatic v0.3960.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Trimmomatic</div><div>suggested: (Trimmomatic, RRID:SCR_011848)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The remaining paired reads were merged using fastq-join61 and then translated to the amino acid sequences using EMBOSS v6.6.0.062.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>EMBOSS</div><div>suggested: (EMBOSS, RRID:SCR_008493)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Finally, unique amino acid sequences in each library were counted using a custom Python script combining seqkit v0.10.163 and usearch v.1164.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Python</div><div>suggested: (IPython, RRID:SCR_001658)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cryo-EM image processing and refinement: The images were processed using RELION 3.169.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RELION</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Movies were motion corrected using MotionCor270, and the contrast transfer functions (CTFs) were estimated using CTFFIND 4.171.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CTFFIND</div><div>suggested: (CTFFIND, RRID:SCR_016732)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data were imported and further processed with non-uniform refinement in cryoSPARC v3.2.072.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After the models were manually fitted into the density using UCSF Chimera v1.1573 and modified in Coot v0.8.9.274, real space refinement was performed in PHENIX v1.19.175.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Figures were prepared using UCSF Chimera73, ChimeraX77, and PyMOL v2.5.0 (Schrödinger, LLC, New York, NY).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMOL</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The program refmac582 in the ccp4 suite83 and the program Phenix-refine75 were used for structural refinement.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ccp4</div><div>suggested: (CCP4, RRID:SCR_007255)</div></div></td></tr></table>
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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sciscore

URL

biorxiv.org/cgi/content/10.1101/2021.10.25.465714
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.11.13.468472

1
1. sciscore 19 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.11.13.468472: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: Animals that lost more than 25% of their initial body weight were euthanized in accordance with our animal ethics protocol.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Two groups of ferrets (5 female and 5 male ferrets in the vaccine group and 3 female and 3 male ferrets in the control group) were immunized intranasally with a single-dose 1×106 PFU of dNS1-RBD and CA04-WT virus respectively diluted in 1640 media to a final 500 μL volume.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Proteins were separated on a 10% gel, and then following transfer, blots were incubated with an anti-influenza A NP protein antibody 19C10 generated by our laboratory (1:1000)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-influenza A NP protein</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">and anti-V5 tag antibody (Thermo,1:5000) and visualized with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Invitrogen, 1:5000)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-V5</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-mouse IgG</div><div>suggested: (LSBio (LifeSpan Cat# LS-C69682-5000, RRID:AB_1653096)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Based on the ELISA results using a sandwich assay with anti-RBD monoclonal antibodies on both sides (Wantai, Beijing, China) and plaque assay results, serial passages 1 to 10 of purified vaccines were confirmed to be stable under current vaccine manufacturing conditions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-RBD IgG measurements: RBD-specific antibody titers in serum samples collected from immunized animals with 1×106 PFU of vaccine were determined by indirect ELISA.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-RBD IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Diluted sera (1:100) were successively diluted in a 2-fold series and applied to each well for 1 h at 37°C, followed by incubation with goat anti-mouse, anti-hamster or anti-human antibodies conjugated with HRP for 1 h at 37°C after 3 washes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-mouse ,</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cells were stained with murine antibodies for phenotype and activation (CD4 [clone GK1.5, APC/Cy7], CD8 [clone 53-6.7, PerCP/Cy5.5], CD11b [clone M1/70, PE], CD11c [clone N418, BV421],</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD4</div><div>suggested: (Miltenyi Biotec Cat# 130-109-536, RRID:AB_2657974)</div></div><div style="margin-bottom:8px"><div>CD8</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD11b</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CD11c</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>BV421</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunohistochemical staining was performed by using a mouse monoclonal anti-SARS-CoV-2 N protein antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 N protein</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Human embryonic kidney cells (293T), African green monkey kidney epithelial cells (Vero E6), and Madin-Darby canine kidney cells (MDCK) were maintained in DMEM-high glucose (Sigma Aldrich, USA) supplemented with 10% low endotoxin FBS (Cegrogen Biotech, Germany) and penicillin-streptomycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MDCK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Generation and passage of dNS1-RBD viruses: Eight pHW2000 plasmids containing the DelNS1 segment and the other seven influenza virus genomic segments, together with an NS1 expression plasmid, pCX-CA04-NS1-Flag, which derived from the parental influenza virus A/California/04/2009(H1N1) (GenBank: MN371610.1-371617.1), were transfected into 293T cells and incubated overnight at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 titration assay: Live virus titers in homogenized lung tissues and cell cultures were measured by the standard TCID50 method in Vero E6 cells seeded in 96-well plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunization and infection of mice: BALB/c mice were immunized intranasally with 50 μL containing 1×106 PFU of the vaccine prepared as indicated above under isoflurane anesthesia, while the control group was administered CA04-WT or CA04-dNS1 virus.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For cellular immune response analyses of PBMCs, splenic lymphocytes, pulmonary lymphocytes and lymph node cells, C57BL/6 mice (6-8 weeks old) were immunized intranasally with 1×106 PFU of the vaccine by the one-dose or two-dose regimen as described above (10 animals in each group).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>C57BL/6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immunization and infection of hACE2-KI/NIFDC mice: hACE2-KI/NIFDC mice (8-10 weeks old) were divided into three groups and treated intranasally with 1×106 PFU of the vaccine by gently adding 50 μL droplets of virus stock for the vaccine-immunized group (5 animals) at two time points (days 0 and 14), and then, the vaccine-immunized group and unvaccinated group (3 animals each) were challenged with 1×104 PFU of SARS-CoV-2 by the intranasal route 30 days post immunization.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>hACE2-KI/NIFDC</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequence encoding the RBD segment was then cloned into the NS1 deletion plasmid pHW2000-DelNS1 as described previously.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHW2000-DelNS1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Generation and passage of dNS1-RBD viruses: Eight pHW2000 plasmids containing the DelNS1 segment and the other seven influenza virus genomic segments, together with an NS1 expression plasmid, pCX-CA04-NS1-Flag, which derived from the parental influenza virus A/California/04/2009(H1N1) (GenBank: MN371610.1-371617.1), were transfected into 293T cells and incubated overnight at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHW2000</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pCX-CA04-NS1-Flag</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data were analyzed by FlowJo V10.6.0 and GraphPad Prism 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis: Statistical significance was assigned when P values were < 0.05 using GraphPad Prism 8.0 (GraphPad Software, Inc.)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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:
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
  </footer>
Visit annotations in context

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sciscore

URL

biorxiv.org/cgi/content/10.1101/2021.11.13.468472
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.10.31.466651

1
1. sciscore 19 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.31.466651: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Field Sample Permit: Sample collection, preparation, and storage: All studies were approved by the Institutional Review Board of Washington University in St Louis.<br>IRB: Sample collection, preparation, and storage: All studies were approved by the Institutional Review Board of Washington University in St Louis.<br>Consent: Written consent was obtained from all participants.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">For selection, where a clone spanned both the GC and LNPC compartments, and/or multiple time points, a compartment and a timepoint were first randomly selected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HRP-conjugated goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch, 109-035-088, 1:2500) was used to detect monoclonal antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (Jackson ImmunoResearch Labs Cat# 109-035-088, RRID:AB_2337584)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HRP-conjugated goat anti-Human IgG Fcγ fragment (Jackson ImmunoResearch, 109-035-190, 1:1500), HRP-conjugated goat anti-human serum IgA α chain (Jackson ImmunoResearch, 109-035-011, 1:2500), and HRP-conjugated goat anti-human IgM (Caltag, H15007, 1:4000) were used to detect plasma antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human serum IgA α chain</div><div>suggested: (Jackson ImmunoResearch Labs Cat# 109-035-011, RRID:AB_2337580)</div></div><div style="margin-bottom:8px"><div>anti-human IgM (Caltag, H15007</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, a mammalian cell codon-optimized nucleotide sequences coding for the soluble version of S (GenBank: MN908947.3, amino acids 1-1,213) including a C-terminal thrombin cleavage site, T4 fold trimerization domain and hexahistidine tag was cloned into the mammalian expression vector pCAGGS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAGGS</div><div>suggested: RRID:Addgene_18926)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry data were analyzed using FlowJo v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">V(D)J gene annotation and genotyping: Initial germline V(D)J gene annotation was performed on the preprocessed BCRs using IgBLAST v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgBLAST</div><div>suggested: (IgBLAST, RRID:SCR_002873)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">1.17.138 with IMGT/GENE-DB release 202113-239.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IMGT/GENE-DB</div><div>suggested: (IMGT/GENE-DB, RRID:SCR_006964)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">BCR analysis: BCR analysis was performed in R v4.1.0 with visualization performed using base R, ggplot2 v3.3.544, and GraphPad Prism v9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ggplot2</div><div>suggested: (ggplot2, RRID:SCR_014601)</div></div><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Clonal overlap between B cell compartments was visualized using circlize v.0.4.1345.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>circlize</div><div>suggested: (circlize, RRID:SCR_002141)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Phylogenetic trees for S+ clones containing BMPCs were constructed on a by-participant basis using IgPhyML v1.1.346 with the HLP19 model47.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgPhyML</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Gene annotation on human reference chromosomes and scaffolds in Gene Transfer Format (‘gencode.v32.primary_assembly.annotation.gtf’) was downloaded (2021-06-02) from GENCODE v3252, from which a biotype (‘gene_type’) was extracted for each feature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GENCODE</div><div>suggested: (GENCODE, RRID:SCR_014966)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Quality control was performed as follows on the aggregate gene expression matrix consisting of 360,803 cells and 36,601 features using SCANPY v1.7.253 and Python v3.8.8.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Python</div><div>suggested: (IPython, RRID:SCR_001658)</div></div></td></tr></table>
  Results from OddPub: Thank you for sharing your data.
  Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
  A potential limitation to our analyses of S-binding clones is that our selection strategy may have excluded some low-abundance or low-affinity S-specific clones. Nonetheless, we were able to account for 45% and 67% of all GC B cell and LNPC clones, respectively identified by scRNA-seq. This is the first study to provide direct evidence for the induction of antigen-specific BMPCs by an mRNA-based vaccine in humans. Notably, none of the 11 participants from whom post-vaccination bone marrow specimens were examined had a history of SARS-CoV-2 infection. BMPCs that recognized contemporary seasonal influenza virus vaccine antigens and diphtheria/tetanus vaccine antigens were present at frequencies roughly 10- and 2-fold greater than those against SARS-CoV-2 S, respectively. This is likely due to both the greater number of antigenic targets contained in the former vaccines and the repeated exposures to influenza and tetanus/diphtheria vaccine antigens our study participants likely experienced in comparison to the initial exposure to the novel SARS-CoV-2 S antigen. There are some epitopes within the S protein that are conserved between human seasonal coronaviruses and SARS-CoV-228,29. Cross-reactive B cells targeting these epitopes participate in PB and GC B cell responses to SARS-CoV-2 vaccination6,30. It is unlikely, however, that a substantial proportion of the SARS-CoV-2 S+ BMPCs we observed six months after immunization were part of a pre-existing pool of BMPCs, as in a previou...
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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  About SciScore
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https://biorxiv.org/cgi/content/10.1101/2021.10.29.466401

1
1. sciscore 19 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.29.466401: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  NIH rigor criteria are not applicable to paper type.
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary antibodies against the His-tag (1:2000, Invitrogen) were followed by secondary HRP conjugated antibodies (1:2000, Dako, Denmark), for detection of both proteins.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>His-tag</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sf9 cells transfected by following were used to express the S protein by following bac-to-bac baculovirus expression system (Thermo Fisher, SG).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Sf9</div><div>suggested: CLS Cat# 604328/p700_Sf9, RRID:CVCL_0549)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 S, SARS-CoV-2 S1 and two variants of SARS-CoV-2 S2 proteins, produced by ACROBiosystems (USA), were used for BN-PAGE, MST and to stimulate THP-1 cells and for in vivo experiments.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>THP-1</div><div>suggested: CLS Cat# 300356/p804_THP-1, RRID:CVCL_0006)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mouse inflammation model and in vivo imaging: BALB/c tg (NF-B-RE-Luc)-Xen reporter mice (Taconic Biosciences, Albany, NY, USA, 10– 12 weeks old) were used to study the immunomodulatory effects of SARS-CoV-2 S protein subunits alone or in combination with LPS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: RRID:IMSR_ORNL:BALB/cRl)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The image was obtained using a Gel Doc Imager (Bio-Rad Laboratories,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Bio-Rad Laboratories</div><div>suggested: (Bio-Rad Laboratories, RRID:SCR_008426)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The initial coordinates of E. coli lipid A and LPS were obtained from CHARMM-GUI LPS modeller,62 while the initial coordinates of the S protein were extracted from the cryo-EM structure of S ECD in the closed state (PDB: 6XR8)23 with missing loops constructed using Modeller version 9.21.63 Protein and lipid were parameterized using the CHARMM36 forcefield.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Modeller</div><div>suggested: (MODELLER, RRID:SCR_008395)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">70 All simulations were performed using GROMACS 201871 and the trajectories visualised in VMD.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GROMACS</div><div>suggested: (GROMACS, RRID:SCR_014565)</div></div></td></tr></table>
  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: Please consider improving the rainbow (“jet”) colormap(s) used on page 31. 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.
  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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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https://biorxiv.org/cgi/content/10.1101/2021.11.11.468228

1
1. sciscore 19 Nov 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.11.11.468228: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  NIH rigor criteria are not applicable to paper type.
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Enzymatic assays: NEMO peptide expression and purification: The constructs of Human NEMO (residues 215-247) and mouse NEMO (residues 221-250) cloned into pGEX-6p-1 vector were transformed into BL21 (DE3) cells and selected using ampicillin-enriched LB media.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGEX-6p-1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">3CLpro expression and purification: 3CLpro WT enzyme for assays was prepared independently from a clone of the SARS-CoV-2 NSP5 gene in pD451-SR (Atum, Newark, CA) according to published procedure 6.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pD451-SR</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Bands were visualized with BullDog Bio Acquastain. 5.2. Crystallography: 3CLpro WT expression and purification: BL21(DE3) cells were transformed with pMCSG53 pDNA containing a 3CLpro WT insert with an autoprocessing-sensitive N-terminal Maltose Binding Protein (MBP) tag and a PreScission protease-sensitive C-terminal His6 tag (provided by Andrzej Joachimiak).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pMCSG53 pDNA</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Iterative refinement was performed manually in Coot 51 and REFMAC 52.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All bonds of the peptide were kept rigid as the goal was to preserve the initial conformation and compute the binding free energy using the AutoDock Vina scoring function.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>AutoDock</div><div>suggested: (AutoDock, RRID:SCR_012746)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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EJ_EvenCinderella.pdf

1
1. yujiao 16 Nov 2021
  
  in Public
  
  girls who sit at the intersections of racism and sexism
  
  Racism happens between different racial groups, but sexism can happen in the same racial group. Black women bear the tag"black" and "women", so they may encounter more discrimination than other groups.
  
  #CI45021
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Another Bullshit Night in Suck City

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1. ethanseow 15 Nov 2021
  
  in Public
  
  One by one DAUGHTERS read thevital stats off each toe tag, and as they read each SANTAsits up briefly, winks, tells story, lays back dead
  
  daughters are the shelther wokers
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📑 Secrets of the Edu-Twitter Influencers | Read Write Collect

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1. chrisaldrich 13 Nov 2021
  
  in Public
  
  https://collect.readwriterespond.com/antennapod/
  
  I feel your pain here Aaron.
  
  Perhaps it helps, perhaps not, but I've been using AntennaPod for a few years now. In particular I love it on Android because I can use the share functionality to share to a custom email address which posts to reading.am for an account that aggregates everything I'm listening to. Then I port the RSS feed of that back into my site. It's a stupid amount of manual work, but it mostly works.
  
  Alternately you could share material you listen to to Huffduffer and pull data out that way as well. My problem here is that Huffduffer is more of a bookmark service than a "listened to this" sort of service, though you could always add a "listened" tag to the things you've heard in the past.
  
  The tougher part of all this is that podcasts have "canonical" links for the podcast episodes (sometimes) and an entirely different link for the audio file which has no meta data attached to it (presuming you can even find the URL for the audio file to begin with.)
  
  AntennaPod allows you to pick and choose what you want to share, so usually I default to the audio file to get that in to the workflow and finding/adding the data for the particular episode is a bit easier.
  
  I will say that this is one of the ugliest and most labor intensive workflows I've got for social posts, so I'm usually only doing it and posting publicly for things that I really think are worth the time that make for interesting notes/observations that go along with the post.
  
  I'm curious to see what others come up with for this workflow.
  
  read podcatchers AntennaPod Huffduffer IndieWeb listen posts RSS reply
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Untitled document

1
1. Public_Reviews 10 Nov 2021
  
  in eLife
  
  Reviewer #3 (Public Review):
  
  The emergence of AR-V7 and other AR splice variants in patient tumors has been shown to track with inferior response to AR targeted and chemotherapies in prostate cancer. Using a series of sophisticated imaging techniques the authors have determined that the AR-V7 uses a mechanism for nuclear import distinct from the FL-AR and the AR-V567 variant. AR-V7 also appears to have very fast intranuclear mobility, a characteristic shown to be associated with antagonist bound AR and yet AR-V7 efficiently activates transcription of a reporter gene and at least a subset of AR target genes. This study provides new insights into how AR-V7 may contribute to the pathology of CRPC.<br> Although it is well accepted that the AR-Vs serves as a strong biomarker for resistance to antiandrogen treatment, it is still being debated as to whether and how AR-Vs contribute to the pathology of castration resistance prostate cancer. In this regard, significant efforts have been invested to understand how these splice variants work and how to best target them. Several models of have been proposed, some of which suggested that the AR-Vs can dimerize with the full-length (FL) receptor and require FL-AR for activity, others indicated that AR-V has unique activities independent of FL-AR. Using a series of sophisticated imaging techniques, the authors have addressed some of these unresolved issues in the field, in particular determining that AR-V7 uses a mechanism for nuclear import distinct from the FL-AR and the AR-V567 variant. AR-V7 also appears to have very fast intranuclear mobility, a characteristic shown to be associated with antagonist bound AR; and yet, AR-V7 efficiently activates transcription of a reporter gene and at least a subset of AR target genes. Overall, this is a valuable study, and the authors are to be commended for the high-quality figures and illustrations. Specific strengths include the following points:
  
  1. It has been observed that the AR-V7 predominantly resides in the nucleus; however, the mechanism(s) by which AR-V7 used for nuclear import is not clear. Using live imaging combined with pharmacological and genetic approaches the authors have determined that the importin / complex is required for FL-AR, but not AR-V7 nuclear import. However, nucleoporin complex and Ran-GTP activity are required for FL-AR import, as expected, and are at least partially required for AR-V7 nuclear accumulation. These series of studies have confirmed previously finding that AR-V7 uses a mechanism for nuclear import, distinct from the FL-AR and the AR-V567 variant.
  
  2. Using mutagenesis of the D-box and DNA binding mutants, the authors have also contrasted the structural requirements within FL-AR vs AR-V7 for nuclear localization and transcription. It was determined that the dimerization surface is required for AR-V7 nuclear retention but is dispensable for FL-AR. However, disruption of DNA binding with a single point mutation has demonstrated that DNA binding is not an obligatory step for FL-AR and AR-V7 nulcear retention. This series of experiments have enhanced our understanding of the nuclear cytoplasmic dynamics of AR-V7 and how it differs from FL-AR and suggests that interfering with the dimerization interface may be a means by which AR-V7 can be targeted.
  
  3. Using FRAP and photoconvertible fluorescent protein tag, the authors were able to track the intranuclear dynamics of AR and AR-V7 in real time, which is a strength of this study. They have determined that AR-V7 has higher sub-nuclear mobility compared to FL-AR and that DNA binding is required for even the short residence time of this mutant AR on the chromatin. Together these data suggested that AR-V7 and FL-AR may use different means to activate transcription.
  
  There are some weaknesses that could be addressed to improve the work:
  
  1. Most of the studies were done in PC3 AR negative cell line. It would be helpful to confirm some the key findings in AR positive cell line as the import mechanism may not be the same in AR negative vs AR-positive cell lines.
  
  2. In Figure 2 and 3 where authors used mutagenesis to determine the structural requirements of FL-AR and AR-V7 for nuclear import/retention. These studies used nuclear:cytoplasmic ratios as readouts, not transport kinetics, and thus the observed changes in N/C ratios could be the results of changes in nuclear export and should be discussed appropriately.
  
  3. The observation that co-expression of AR-V7 increased nulcear FL-AR in the absence of ligand is interesting. The fact that IPZ interferes with nuclear accumulation of FL-AR in the presence of AR-V7 indicated that FL-AR import still requires importin but does not rule out the possibility that FL-AR via its dimerization with AR-V7 within the nucleus could lead to increased retention of FL-AR within the nucleus, a possibility that the authors should consider.
  
  4. The authors used ChIP assays to confirm the fast chromatin mobility of AR-V7 they have observed using FRAP, however ChIP efficiency could differ significantly using different antibodies and the results should be discussed with caution. Although the authors tried to confirm the same using chromatin bound fraction as another readout for transient chromatin binding of AR-V7, it was unclear why the authors didn't use endogenous AR-V7 in 22RV1 cells to look at chromatin bound fraction, as overexpressed protein may have different behavior compared to endogenous protein.
  
  peerReview
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Designing better file organization around tags, not hierarchies

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1. prnc 10 Nov 2021
  
  in Public
  
  The solution is to create a tag file that points to the original and edited photo, like DerivedWork(original=(some hash), derived=(some hash)).
  
  Relational tags
  
  knowledge+graph
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How Secure Is Your Cloud? Take The Mud Puddle Test

1
1. TylerRick 05 Nov 2021
  
  in Public
  
  It's all too complex for our little brains to handle. And like any situation of excess complexity, we collapse dimensions until we have a structure we can comprehend. The problem, in this case, is that our simplifications create tunnels large enough for the trucks of hacker to drive through—with ease.
  
  security annotation meta: may need new tag
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forbes.com/sites/anthonykosner/2012/08/05/how-secure-is-your-cloud-take-the-mud-puddle-test/
hypothes.is hypothes.is

Hipótesis

1
1. Francisco_corona 04 Nov 2021
  
  in Public
  
  la omt en 1994 definió turismo como “las actividades que realizan las personas durante sus viajes y estancias en lugares distintos al de su entorno habitual, por un período de tiempo consecutivo inferior a un año con fines de ocio, por negocios y otros” (Sancho, 1998, p. 11).<
  
  MI COMENTARIO:
  
  #UTLP #EOE1 #GAS1a
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www.reddit.com www.reddit.com

r/pop_os - Time for System76 to abandon Ubuntu?

1
1. TylerRick 03 Nov 2021
  
  in Public
  
  They wanna be to Linux what the Play Store is to Android, what the App Store is to iOS.But we don't do that around here. We use Flatpak round 'ere.
  
  annotation meta: may need new tag: company [aspiring] to be bigger / take over the world
  
  Snap: bad Canonical annotation meta: may need new tag big/grand/lofty goals app stores: bad app stores
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reddit.com/r/pop_os/comments/ppk7kn/time_for_system76_to_abandon_ubuntu/
beepb00p.xyz beepb00p.xyz

The sad state of personal data and infrastructure | beepb00p

1
1. ankostis 03 Nov 2021
  
  in Public
  
  Why does it bother me?
  
  An important list of pk2b & web-tag problems:
  
  cannot search user activity & annotations; less so when offline
  
  half-baked UX
  
  cannot integrate apps, across platforms, desktop/mobile, cloud/local.
  
  What if sites disappear (next chapter)?
  
  pkdb web_tag
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beepb00p.xyz/sad-infra.html
web.hypothes.is web.hypothes.is

Annotating the law | Hypothes.is

1
1. suzannah 03 Nov 2021
  
  in Public
  
  Also apparently by group!
  
  Look for your group ID code and stick it in the equation. https://hypothes.is/stream.rss?group=[yourgroupscode]
  
  or combine them!! https://hypothes.is/stream.rss?user=[yourusername]&tag=[tagnameyouresearchingfor]
  
  Hypothesis RSS
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web.hypothes.is/blog/introducing-math/
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Web Annotation Data Model

1
1. zhurovandrew 02 Nov 2021
  
  in Public
  
  Hannah associates a tag and a description with two images using a single annotation
  
  How, in this case, Hannah would refer to an image from within the description?
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meta.stackoverflow.com meta.stackoverflow.com

What is the recommended way of dealing with old (then on-topic) question with a bounty?

1
1. TylerRick 01 Nov 2021
  
  in Public
  
  I posted a question about MD5 hash collision back in 2014. As far as I know questions about algorithms are on-topic on Stack Overflow, and the cryptography tag did not have the warning "CRYPTOGRAPHY MUST BE PROGRAMMING RELATED" back then.
  
  is it on topic? changes to policy/terms
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meta.stackoverflow.com/questions/412697/what-is-the-recommended-way-of-dealing-with-old-then-on-topic-question-with-a
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TypeScript: pick properties with a defined type

1
1. TylerRick 01 Nov 2021
  
  in Public
  
  I arrived at this question searching for the answer as well
  
  annotation meta: may need new tag
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stackoverflow.com/questions/46583883/typescript-pick-properties-with-a-defined-type
docdrop.org docdrop.org

Note Taking as an Art of Transmission

1
1. chrisaldrich 01 Nov 2021
  
  in Public
  
  wn written cultures material is typically sorted alphabeticallySor by some other method of linguistic ordering such as the number ofstrokes in qhinese charactersTW or systematicallyW according to various sysXtems that strive to map or hierarchize the relations between the items storedSincluding those of uoogle or ffiahooTW or miscellaneouslyY
  
  What about the emergence of non-hierarchal methods? (Can these logically be sorted somehow without this structure?)
  
  With digital commonplacing methods, I find that I can sort and search for things temporally by date and time as well as by tag/heading.
  
  Cross reference:
  
  https://www.heise.de/newsticker/meldung/Missing-Link-Luhmanns-Denkmaschine-endlich-im-Netz-4364512.html#annotations:ed1DZDnAEeyWdbsat73FrA
  
  http://takingnotenow.blogspot.com/2007/12/luhmanns-zettelkasten.html#annotations:IBHItPaBEeuVb1Nwojnj3Q
  
  hierarchies discovery search sorting commonplace books
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docdrop.org/download_annotation_doc/Note-Taking-as-an-Art-of-Transm---Blair-Ann-M_-1ht5j.pdf
Oct 2021
docdrop.org docdrop.org

Dear_Future_President_of_the_United_Stat.pdf

1
1. mehsodpaw 31 Oct 2021
  
  in Public
  
  students generated a total of 1,636 dif-ferent issue tags, including a large proportion of redundant and related tag
  
  Since they give the students no restrictions on what to write about, it resulted in having many "thematic tags. It is amazing how the types were varied.
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docdrop.org/download_annotation_doc/Dear_Future_President_of_the_United_State-FA21-d5t2u.pdf
www.amazon.com www.amazon.com

The Zettelkasten Notebook: 8.5 x 11" soft cover book, 200 pages - one Zettel Note per page with NARROW RULED, quick note-taking section. Record notes now and update your Zettelkasten system later.: Press, Essenjey: 9781798844731: Amazon.com: Books

1
1. chrisaldrich 30 Oct 2021
  
  in Public
  
  Of course someone has published a zettelkasten notebook for taking notes to be moved into one's zettelkasten at a later date.
  
  It includes space for notes as well as meta data box which includes labels and spaces for the following:
  
  UID
  
  Parent UID
  
  Zettel
  
  tags
  
  refs
  
  It's also got (at least one) page for an index in the end titled "tag list" with a two column ruling
  
  This follows the general pattern of pre-printed commonplace books which was common with at least John Locke's index pre-printed.
  
  Other examples include published bullet journals with custom formatting.
  
  zettelkasten publishing John Locke bullet journals
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amazon.com/Zettelkasten-Notebook-Zettel-note-taking-section/dp/1798844737
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Untitled document

1
1. pbk1 29 Oct 2021
  
  in Public
  
  SNAP-Capture Magnetic Beads are used to selectively immobilize and magnetically separate a SNAP-tag fusion protein from solution using magnetic agarose beads
  
  surface-display pull-down
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neb.com/products/s9145-snap-capture-magnetic-beads
www.medrxiv.org www.medrxiv.org

https://medrxiv.org/cgi/content/10.1101/2021.10.25.21265476

1
1. sciscore 29 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.25.21265476: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Participant recruitment and study approval – Toronto cohorts: Negative control serum samples were from patients enrolled in cancer studies pre-COVID-19 (prior to November 2019; Mount Sinai Hospital (MSH) Research Ethics Board (REB) studies #01-0138-U and #01-0347-U), which were archived and frozen in the Lunenfeld-Tanenbaum Research Institute (LTRI</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Recombinant antibody production: The llama single domain antibody (VHH) VHH72-hFc1X7 (VHH72-Fc) was described previously (PDB entry 6WAQ_1) (17); additional VHHs (NRCoV2-04 and NRCoV2-20) were isolated in-house from llamas immunized with recombinant SARS-CoV-2 trimeric spike ectodomain SmT1 (Supplementary Figure 2).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NRCoV2-20</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VHH sequences were fused to an antibody-dependent cell-mediated cytotoxicity (ADCC)-attenuated human IgG1 Fc domain (hFc1X7, from patent US 2019 352 383A1).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human IgG1 Fc domain ( hFc1X7</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The anti-human-IgG monoclonal antibodies (mAbs) IgG#5 and IgG#6 were derived from mice immunized with human IgG; heavy chain (HC) and light chain (LC) variable domain sequences (VH and VL) were fused to mouse IgG2a and mouse kappa LC constant sequences, respectively, to express full-length mAbs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human-IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>mouse IgG2a</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 antibody-negative blood was spotted directly from EDTA Vacutainer tubes onto DBS cards.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All matched plasma and contrived DBS samples were tested using the anti-SARS-CoV-2 ELISA IgG kit (EUROIMMUN, Lübeck, Germany), according to the manufacturer’s instructions, to verify that donors were either positive or negative for SARS-CoV-2 antibodies prior to shipping to Toronto and Ottawa.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 ELISA IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For spike and its RBD, the recombinant antibodies used were VHH72-Fc IgG (NRC; see above), human anti-spike S1 IgG (clone HC2001, GenScript, #A02038), human anti-Spike S1 IgM (clone hIgM2001, GenScript, #A02046), and human anti-spike IgA (clone CR3022, Absolute Antibody, Oxford, United Kingdom, #Ab01680-16.0).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-spike S1 IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-Spike S1 IgM</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-spike IgA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For N, the antibodies used were human anti-nucleocapsid IgG (clone HC2003, GenScript, #A02039)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-nucleocapsid IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">anti-nucleoprotein IgM (CR3018 (03-018), Absolute Antibody, #Ab01690 -15.0), and anti-nucleoprotein IgA (CR3018 (03-018), Absolute Antibody, #Ab01690 -16.0).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-nucleoprotein IgM</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-nucleoprotein IgA</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>CR3018</div><div>suggested: (Imported from the IEDB Cat# CR3018, RRID:AB_2833185)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Negative control antibodies purified from human serum (final 1 µg/mL; human IgG, Sigma-Aldrich, Oakville, ON, Canada,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Anti-human secondary antibodies (recombinant anti-human IgG#5-HRP, goat anti-human IgG Fcy-HRP (Jackson ImmunoResearch Labs, West Grove, PA, USA, #109-035-098)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-human secondary</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgG#5-HRP</div><div>suggested: (Jackson ImmunoResearch Labs Cat# 109-035-098, RRID:AB_2337586)</div></div><div style="margin-bottom:8px"><div>anti-human IgG Fcy-HRP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, 100 μL samples of titrations of anti-SARS-CoV-2 S CR3022 Human IgG1 (Absolute Antibody, Ab01680-10.0), anti-SARS-CoV-2 S CR3022 Human IgA (Absolute Antibody, Ab01680-16.0), or anti-SARS-CoV-2 S CR3022 Human IgM (Absolute Antibody, Ab01680-15.0) were diluted in 1% w/v skim milk in PBST.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 S CR3022 Human IgG1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Human</div><div>suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 S CR3022 Human IgA</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 S CR3022 Human IgM</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The final isotype-specific secondary antibodies used were anti-human IgG#5-HRP (Supplementary Figure 3), anti-human IgA-HRP (Jackson ImmunoResearch Labs, 109-035-011), and anti-human IgM-HRP (Jackson ImmunoResearch Labs, 109-035-129).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgA-HRP</div><div>suggested: (SouthernBiotech Cat# 2050-05, RRID:AB_2687526)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two concentrations of secondary antibody IgG#5-HRP (0.09 and 0.18 μg/mL) were assessed using dilution curves of the VHH72-Fc antibody (to detect spike and its RBD) or an anti-N antibody (to detect N; Supplementary Figure 7), and the best concentration (0.18 μg/mL) was further tested on a dilution series of 32 serum samples provided by CBS (Supplemental Figure 8).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VHH72-Fc</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-N</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We also tested anti-RBD NRCoV2-04 and NRCoV2-20 recombinant calibration antibodies, which were comparable to VHH72-Fc in reference curves (Supplementary Figure 7).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>NRCoV2-04</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For chemiluminescent assays, 10 µL of goat anti-human IgM-HRP (1:10,000; 0.80 ng/well) or goat anti-human IgA-HRP (1:12,000, 0.66 ng/well) were used as secondary antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgM-HRP</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Protein production: Spike trimer: The SARS-CoV-2 spike ectodomain construct (SmT1) with S1/S2 furin site mutations, K986P/V987P prefusion-stabilizing mutations, and human resistin as a trimerization partner (15) was produced using stably transfected Chinese Hamster Ovary (CHO) pools (CHOBRI/2353™ cells) and purified as described (8).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHOBRI/2353™</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The construct was expressed by transient gene expression in CHOBRI/55E1™ cells as described above (15).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHOBRI/55E1™</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VHH and mAb sequences were synthesized by GenScript using C. griseus codon bias for expression in CHO cells and cloned into the pTT5™ plasmid.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO</div><div>suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The ACE2-BAP cDNA was expressed by transient gene expression in CHOBRI/55E1 cells as described (15) with the addition of 5% (w/w) pTT5™-BirA (an Escherichia coli biotin ligase) expression plasmid as described previously (18).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHOBRI/55E1</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Nucleocapsid: N cDNA (corresponding to amino acids 1–419 of YP_009724397) was synthesized by GenScript (Piscataway, NJ, USA; using Cricetulus griseus codon bias) with a C-terminal FLAG-Twin-Strep-tag-(His)6 tag and cloned into the pTT5 expression plasmid (NRC) to create NCAP (16).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTT5</div><div>suggested: RRID:Addgene_52326)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Amino acids 331–521 of the SARS-CoV-2 spike protein (YP_009724390.1) corresponding to the RBD were cloned into the pTT5™ vector using EcoRI and BamHI.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTT5™</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The ACE2-BAP cDNA was expressed by transient gene expression in CHOBRI/55E1 cells as described (15) with the addition of 5% (w/w) pTT5™-BirA (an Escherichia coli biotin ligase) expression plasmid as described previously (18).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pTT5™-BirA</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Ottawa: Samples from DBS cards were punched manually or in a semi-automated manner using a PerkinElmer DBS puncher (PerkinElmer, Woodbridge, ON, Canada; 3.2 mm discs) or a BSD600 Ascent puncher (BSD Robotics; 3 mm discs) and eluted in 100 μL/disc PBS + 1% Triton X-100 for up to 16 h (minimum 4 h) in 96-well U-bottom plates on a shaker at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Canada</div><div>suggested: (Brain Canada, RRID:SCR_005053)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Other data analyses: Plots were generated in R using the ggplot2, lattice, latticeExtra, grid, and gridExtra packages.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ggplot2</div><div>suggested: (ggplot2, RRID:SCR_014601)</div></div></td></tr></table>
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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The histone demethylase KDM6B regulates temperature-dependent sex determination in a turtle species | Science in the Classroom

3
1. klcates 28 Oct 2021
  
  in Public
  
  Lentiviral treatment of two different shRNAs led to a 73 to 82% reduction of Kdm6b transcripts in 26°C gonads from early stage 15 onward (fig. S4), as compared with treatment with nonsilencing scrambled virus
  
  The authors validated the shRNAs using multiple methods.
  
  The shRNA was infected with a fluorescence tag, called green fluorescent protein or GFP. The infected embryos were then visualized at multiple developmental stages. The treated embryos fully fluoresced green, including the gonads, which indicated that the virus successfully introduced the shRNA to the embryos.
  
  The authors also checked the levels of transcript between the control shRNA- (a non-specific sequence) and Kdm6b shRNA-treated embryo gonads. In the green, GFP+ gonads, the shRNAs specifically reduced Kdm6b by up to 80% when compared to the controls.
  
  AuthorsExperiments
2. klcates 27 Oct 2021
  
  in Public
  
  in situ hybridization
  
  A common technique to visualize nucleotide (DNA or RNA) in cells.
  
  A chemical or radioactive label is added to a nucleotide sequence that is complimentary to the sequence of interest. When added to the cells, this complimentary nucleotide sequence will bind to and tag the sequence of interest, allowing scientists to visualize the DNA or RNA of interest within the cell.
  
  You can read more about in situ hybridization methods here: https://www.nature.com/scitable/topicpage/fluorescence-in-situ-hybridization-fish-327/
  
  Glossary
3. klcates 27 Oct 2021
  
  in Public
  
  Immunofluorescence
  
  A widely-used technique used to image proteins on or within cells. It uses antibodies to bind and tag a protein of interest.
  
  The first antibody specifically binds the protein of interest, for example KDM6B. Then, a second antibody carrying a fluorescent dye attaches to the first antibody.
  
  These proteins are visualized using a microscope with lasers. The lasers excite the fluorophore, which in turn emits specific light waves. Here, the KDM6B is marked by the emission of light waves in the green spectrum and beta catenin produces waves in the red spectrum.
  
  Here is a brief introduction to immunofluorescence techniques: https://oni.bio/nanoimager/super-resolution-microscopy/immunofluorescence/
  
  AuthorsExperiments
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web.hypothes.is web.hypothes.is

Annotating the law | Hypothes.is

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1. AnastasiiaNovozhilova 28 Oct 2021
  
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  Have students highlight, tag, and annotate words or passages that are confusing to them in their readings
  
  that's nice!
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web.hypothes.is/blog/introducing-math/
wagtailmenus.readthedocs.io wagtailmenus.readthedocs.io

Overview and key concepts — wagtailmenus 3.0.2 documentation

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1. deleyva 26 Oct 2021
  
  in Public
  
  Use settings to change the default templates used for each tag Specify templates using template and sub_menu_template arguments for any of the included menu tags (See Specifying menu templates using template tag parameters). Put your templates in a preferred location within your project and wagtailmenus will pick them up automatically (See Using preferred paths and names for your templates).
  
  Dónde especificar las plantillas para los menús. Si no usas las tuyas, el paquete usa plantillas por defecto usando bootstrap3
  
  python wagtail wagtailmenus
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wiki.elte-dh.hu wiki.elte-dh.hu

Az összes lap listája – wikibase-docker

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1. banfidavid 25 Oct 2021
  
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  Címkefelhő
  
  A címkefelhő (tag cloud) egy adott weboldal tartalmára jellemző kulcsszavak látványos, vizuális ábrázolása. Az ábrázolásmódot befolyásolja az adott szó vagy címke valamely tulajdonsága, például a szövegbeli gyakorisága vagy az olvasói használat gyakorisága.
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www.johndcook.com www.johndcook.com

Literate programming: presenting code in human order

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1. gyuri 25 Oct 2021
  
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  Literate programming: presenting code in human order
  
  about : literate programming
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johndcook.com/blog/2016/07/06/literate-programming-presenting-code-in-human-order/
medium.com medium.com

How to Finally Beat Procrastination and Take Action On Your Life Goals

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1. wasique1146 24 Oct 2021
  
  in Public
  
  You can tell your friends and family of your plans to become healthier so they can give you encouragement and keep you accountable. There are no real consequences to breaking your pledge other than letting other people down.But a commitment that comes with only a psychological price tag for failure is surprisingly effective for two reasons:We don’t want to be seen contradicting ourselves in front of people we respect and love.We want to be consistent with our past pronouncements. Cognitive dissonance, where we say one thing but do another, can cause unpleasant feelings within us.Through soft commitment devices, you are using the power of social pressure to promote behaviors you want to change in your life. And if your goals are made to show, they are made to grow.
  
  this is just leveraging the basic desires of human for social bonding and recognition and also satisfying the ego, we will not break the commitments for the fear of being ostracized by the public
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medium.com/better-advice/how-to-finally-beat-procrastination-and-take-action-on-your-life-goals-f155e9b8bc3a
www.medrxiv.org www.medrxiv.org

https://medrxiv.org/cgi/content/10.1101/2021.10.16.21265096

1
1. sciscore 23 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.16.21265096: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  NIH rigor criteria are not applicable to paper type.
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Some cleaned Fasta files were also uploaded to SnapGene (version 5.3.2) for multisequence analysis via the MAFFT tool.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SnapGene</div><div>suggested: (SnapGene, RRID:SCR_015052)</div></div><div style="margin-bottom:8px"><div>MAFFT</div><div>suggested: (MAFFT, RRID:SCR_011811)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">With these factors all considered, an optimal tree was selected from the 21 trees for re-rooting via FigTree (https://github.com/rambaut/figtree/releases/tag/v1.4.4).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FigTree</div><div>suggested: (FigTree, RRID:SCR_008515)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PyMol structural modeling: The PyMol molecular graphics system (version 2.4.2, https://pymol.org/2/) from Schrödinger, Inc. was used for downloading structure files from the PDB database for further analysis and image export.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The images were cropped via Adobe Photoshop and further presentation using Illustrator.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Adobe Photoshop</div><div>suggested: (Adobe Photoshop, RRID:SCR_014199)</div></div><div style="margin-bottom:8px"><div>Illustrator</div><div>suggested: (Adobe Illustrator, RRID:SCR_010279)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pandemic and vaccination data: Pandemic and vaccination data were downloaded from the Our World in Data website (https://ourworldindata.org/explorers/coronavirus-data-explorer?zoomToSelection=true&time=2020-03-01..latest&facet=none&pickerSort=desc&pickerMetric=total_cases&Metric=Confirmed+cases&Interval=Cumulative&Relative+to+Population=false&Align+outbreaks=false&country=~OWID_WRL) as a .csv file for further processing via Excel and figure generation through Prism 9.0 and Adobe Illustrator.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Excel</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div><div style="margin-bottom:8px"><div>Adobe Illustrator</div><div>suggested: (Adobe Illustrator, RRID:SCR_010279)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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medrxiv.org/cgi/content/10.1101/2021.10.16.21265096
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.10.13.464050

1
1. sciscore 22 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.13.464050: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells and expression vectors: HEK293T cells and SiHa cells were procured from the central cell line repository of RGCB (CCL</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SiHa</div><div>suggested: KCB Cat# KCB 2013026YJ, RRID:CVCL_0032)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">CaCo-2 cells were obtained from ATCC.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CaCo-2</div><div>suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">DLD-1 cells were obtained from Sigma (Merck #90102540).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>DLD-1</div><div>suggested: ECACC Cat# 90102540, RRID:CVCL_0248)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were transfected with lenti-ZsGreen (Addgene) and 10 µg of psPAX2 (Addgene) and SARS-CoV-2-S variant (pCDNA 3.1_Spike_Del19, Addgene) at a ratio of 1:2:1 using the transfection reagent (Lipofectamine 3000, Thermo Fisher Scientific #L3000001).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mixture was added to the HEK293T-HA-ACE2 cells for 4 hours, followed by fresh medium, and wells with IgG served as control.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-HA-ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mouse immunization and serum preparation: BALB/c mice were immunized with purified RBD-His protein expressed in E. coli as per the standard protocol approved by IAEC.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: RRID:IMSR_ORNL:BALB/cRl)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">) Spike S1 Gene with C terminal OFP Spark fluorescent tag (pCMV3-C-OFPSpark) was obtained from Sino biological (#VG40591-ACR)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV3-C-OFPSpark</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Full-length DNA clone of Human angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 with C terminal GFPSpark tag (pCMV3-hACE2-GFPSpark) was obtained from Sino biological (#HG10108-ACG).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV3-hACE2-GFPSpark</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mammalian expression vector for soluble ACE2 pcDNA3-sACE2 (WT)-sfGFP (#145171) was obtained from Addgene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3-sACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The packaging vectors and Lenti ZsGreen were from Addgene (pHIV-Zsgreen #18121</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHIV-Zsgreen</div><div>suggested: RRID:Addgene_18121)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Immobilization of GFP Nanobody to Agarose beads: The bacterial expression vector for anti-GFP nanobody pGEX-6P-1 was procured from Addgene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pGEX-6P-1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T cells were transfected with lenti-ZsGreen (Addgene) and 10 µg of psPAX2 (Addgene) and SARS-CoV-2-S variant (pCDNA 3.1_Spike_Del19, Addgene) at a ratio of 1:2:1 using the transfection reagent (Lipofectamine 3000, Thermo Fisher Scientific #L3000001).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>pCDNA 3.1_Spike_Del19</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The images were acquired and analyzed using NIS-Elements software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NIS-Elements</div><div>suggested: (NIS-Elements, RRID:SCR_014329)</div></div></td></tr></table>
  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 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 22. 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.
  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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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sciscore

URL

biorxiv.org/cgi/content/10.1101/2021.10.13.464050
www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.10.13.464307

1
1. sciscore 22 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.13.464307: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  NIH rigor criteria are not applicable to paper type.
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The anti-His tag antibodies, diluted at 50 μg/mL in 10 mM sodium acetate, pH 4.5, were immobilized on both the active and reference flow cells surface of the activated CM5 sensor chip using amine coupling method.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His tag</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Approximately 200 RU of His-tagged SARS-CoV-2 and SARS-CoV spike trimers and RBDs were captured onto the chip for the active surface, and anti-His antibody alone served as the reference surface.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines: HEK293T/17 (cat# CRL-11268) and Vero E6 cells (cat# CRL-1586) were from ATCC, 293T-ACE2 cells were kindly provided by J. Sodroski of Harvard Medical School, and they were cultured in 10% fetal bovine serum (FBS, GIBCO cat# 16140071) supplemented Dulbecco’s Modified Eagle Medium (DMEM, ATCC cat# 30-2002) at 37°C, 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>293T-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK293T cells were grown to 80% confluency before transfection with the spike gene using Lipofectamine 3000 (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The proteins were expressed in HEK293 Freestyle cells (Invitrogen) in suspension culture using serum-free media (Invitrogen) and transfected into HEK293 cells using polyethyleneimine (Polysciences).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The results were then converted into percentage neutralization at a given sample dilution or mAb concentration, and the averages ± SEM were plotted using a five-parameter dose-response curve in GraphPad Prism v.8.4.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data processing was performed using cryoSPARC v2.15 [14].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>cryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Model building and refinement: The 2-36-RBD complex model was built starting from template PDB structures 6BE2 (Fab) and 7BZ5 (RBD) using Phenix Sculptor.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phenix Sculptor</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Automated and manual model building were iteratively performed using real space refinement in Phenix [16] and Coot [17].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Phenix</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Geometry validation and structure quality assessment were performed using Molprobity [18].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Molprobity</div><div>suggested: (MolProbity, RRID:SCR_014226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The visualization of sequence entropy was displayed by PyMol version 2.3.2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr></table>
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2021.10.13.464307
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https://biorxiv.org/cgi/content/10.1101/2021.10.13.464254

1
1. sciscore 22 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.13.464254: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">In brief, female Syrian hamsters (Mesocricetus auratus) of 6-8 weeks old were anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 μL containing 1×104 TCID50 Beta B.1.351 (derived from hCoV-19/Belgium/rega-1920/2021; EPI_ISL_896474, 2021-01-11).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody isolation and recombinant production: Antigen specific IgG+ memory B cells were isolated and cloned from PBMC of SARS-CoV-2 convalescent individuals.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Antigen specific IgG+</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">goat anti-human IgG secondary antibody (Southern Biotech, 2040-04) was added and incubated for 45 min at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>goat anti-human IgG secondary antibody</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (SouthernBiotech Cat# 2040-04, RRID:AB_2795643)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 2 h, infected cells were washed an additional five times with DMEM prior to adding media supplemented with anti-VSV-G antibody (I1-mouse hybridoma supernatant diluted 1:25, from CRL-2700, ATCC) to reduce parental background.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-VSV-G</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were fixed with 4% PFA (Electron Microscopy Sciences, #15714S), permeabilized with Triton X-100 (SIGMA, #X100-500ML) and stained with an antibody against the viral nucleocapsid protein (Sino Biologicals, #40143-R001) followed by a staining with the nuclear dye Hoechst 33342 (Fisher Scientific, # H1399) and a goat anti-rabbit Alexa Fluor 647 antibody (Invitrogen, #A-21245).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>#X100-500ML</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit</div><div>suggested: (Molecular Probes Cat# A-21245, RRID:AB_141775)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T-ACE2, Vero-TMPRSS2) (32)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudotyped viruses were prepared using Lenti-X 293 cells seeded in 15-cm dishes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For neutralization experiments, HEK-293T cells expressing hACE2 (Crawford et al. 2020) in DMEM supplemented with 10% FBS and 1% PenStrep were seeded at 20,000 cells per well into clear bottom, white manually poly-D-lysine coated 96 well plates and incubated at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Neutralization of authentic SARS-CoV-2 viruses: Vero-TMPRSS2 cells were seeded into black-walled, clear-bottom 96-well plates at 2 × 104 cells/well and cultured overnight at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell-surface mAb-mediated S1 shedding: CHO cells stably expressing the prototypic SARS-CoV-2 Spike protein were harvested, washed in wash buffer (PBS 1% BSA 2 mM EDTA) and resuspended in PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO</div><div>suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral replication fitness assays: Vero E6 cells (ATCC, CRL-1586) were seeded at 1×106 cells per well in 6-well plates.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After a 15-minute incubation, Jurkat cells stably expressing FcγRIIIa receptor (V158 variant) or FcγRIIa receptor (H131 variant) and NFAT-driven luciferase gene (effector cells) were added at an effector to target ratio of 6:1 for FcγRIIIa and 5:1 for FcγRIIa.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">fold-on trimerization motif, and an 8× His tag in the pCMV vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV</div><div>suggested: RRID:Addgene_20783)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Using the Database IMGT (http://www.imgt.org), the VH and VL gene family and the number of somatic mutations were determined by analyzing the homology of the VH and VL sequences to known human V, D and J genes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>http://www.imgt.org</div><div>suggested: (IMGT - the international ImMunoGeneTics information system, RRID:SCR_012780)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">UCA sequences of heavy and light variable regions were constructed using IMGT/V-QUEST.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IMGT/V-QUEST</div><div>suggested: (IMGT/V-QUEST, RRID:SCR_010749)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 45 min incubation, absorbance at 405 nm was measured by a plate reader (Biotek) and data plotted using Prism GraphPad.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After a 30 sec stabilization step in KB, biosensors were moved in SARS-CoV or SARS-CoV-2 :2 dilution series (starting concentration: 18.5 nM) for the 600 sec association step, and then moved back in KB to record dissociation signals for 540 sec.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: (BioLegend Cat# 946101, RRID:AB_2892515)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data were baseline subtracted, results fitted using the Pall FortéBio/Sartorius analysis software (version 12.0) and plotted using GraphPad Prism (version 9.1.1</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed and visualized with Prism (Version 9.1.1).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Micrographs were recorded using the Leginon software on a 120 kV FEI Tecnai G2 Spirit with a Gatan Ultrascan 4000 4k</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Leginon</div><div>suggested: (Leginon, RRID:SCR_016731)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">3D refinements were carried out using non-uniform refinement along with per-particle defocus refinement in CryoSPARC (57).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CryoSPARC</div><div>suggested: (cryoSPARC, RRID:SCR_016501)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Model building and refinement: UCSF Chimera (61) and Coot (62) were used to fit atomic models into the cryoEM maps.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This variant was originally isolated in house from nasopharyngeal swabs taken from travelers returning to Belgium (baseline surveillance) and were subjected to sequencing on a MinION platform (Oxford Nanopore) directly from the nasopharyngeal swabs (65).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MinION</div><div>suggested: (MinION, RRID:SCR_017985)</div></div></td></tr></table>
  Results from OddPub: Thank you for sharing your code and 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 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: Please consider improving the rainbow (“jet”) colormap(s) used on page 24. 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.
  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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2021.10.13.464254
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https://biorxiv.org/cgi/content/10.1101/2021.10.16.464644

1
1. sciscore 22 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.16.464644: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  NIH rigor criteria are not applicable to paper type.
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cleaned Fasta file was then uploaded to SnapGene (version 5.3.2) for multisequence analysis via the MAFFT tool.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SnapGene</div><div>suggested: (SnapGene, RRID:SCR_015052)</div></div><div style="margin-bottom:8px"><div>MAFFT</div><div>suggested: (MAFFT, RRID:SCR_011811)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">With these factors all considered, an optimal tree was selected from the 21 trees for re-rooting via FigTree (https://github.com/rambaut/figtree/releases/tag/v1.4.4).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FigTree</div><div>suggested: (FigTree, RRID:SCR_008515)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting tree was exported for image processing via Adobe Photoshop and subsequent presentation via Illustrator.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Adobe Photoshop</div><div>suggested: (Adobe Photoshop, RRID:SCR_014199)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PyMol structural modeling: The PyMol molecular graphics system (version 2.4.2, https://pymol.org/2/) from Schrödinger, Inc. was used for downloading structure files from the PDB database for further analysis and image export.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The images were cropped via Adobe Photoshop and further presentation using Illustrator.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Illustrator</div><div>suggested: (Adobe Illustrator, RRID:SCR_010279)</div></div></td></tr></table>
  Results from OddPub: Thank you for sharing your code.
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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https://medrxiv.org/cgi/content/10.1101/2021.10.13.21264916

1
1. sciscore 22 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.13.21264916: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Deidentified data from staff testing and patients were obtained from the Infections in Oxfordshire Research Database (IORD) which has generic Research Ethics Committee, Health Research Authority and Confidentiality Advisory Group approvals (19/SC/0403, ECC5-017(A)/2009).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Secondary antibody rabbit anti-human whole IgG conjugated to alkaline phosphatase (Sigma, USA) or a secondary antibody mouse anti-human IgA conjugated to horse radish peroxidase (Sigma, USA) was added at a dilution of 1:1000 in casein–PBS solution and incubated for 1 hour at RT after which a final wash was performed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human whole IgG</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For detection of the IgA secondary antibody, plates were developed by adding Tetramethylbenzidine (TMB) (Thermo scientific USA) to visualize and Sulfuric acid to stop the reaction.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A 1x working concentration of the SULFO-TAG anti-human IgG Detection Antibody was prepared in Diluent 100.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: (RevMAb Biosciences Cat# 31-1019-MK, RRID:AB_2783627)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Microplates were coated with RBD in Dulbecco’s Phosphate Buffered Saline (DPBS) to bind corresponding ACE2 or blocking antibodies of the sample.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MSD V-PLEX assay: IgG antibody responses to SARS-CoV-2 spike, RBD, NTD and nucleocapsid and the spike proteins of SARS-CoV-1, HCoV-229E, HCoV-NL63, HCoV-HKU1 and HCoV-OC43 were assessed using the Meso Scale Diagnostics (MSD) Multi-Spot Assay System (MSD, USA)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HCoV-NL63</div><div>suggested: RRID:CVCL_RW88)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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https://biorxiv.org/cgi/content/10.1101/2021.10.17.464700

1
1. sciscore 22 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.17.464700: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Patient Samples and Collection: Use of human samples for this study was approved by the University of Ottawa Ethics Review Board: Certificates H-04-20-5727, H-04-21-6643 and H-07-20-6009.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In initial experiments, aliquots of each fraction were visualized via immunoblotting (anti-His antibody; Cat: SAB2702218</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In conjunction, titration curves of conformation-dependent monoclonal IgM (Absolute Antibody, Ab01680-15.0), IgA (Absolute Antibody, Ab01680-16.0), and IgG (Absolute Antibody, Ab01680-10.0) CR3022 antibodies were used as reference material to assess protein folding.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>titration curves of conformation-dependent monoclonal IgM ( Absolute Antibody , Ab01680-15.0) , IgA ( Absolute Antibody , Ab01680-16.0)</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgG ( Absolute Antibody , Ab01680-10.0 ) CR3022 antibodies</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After blocking, plates were washed thrice with PBS-T, and followed by addition of 100 μL of the respective diluted serum samples and CR3022 antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CR3022</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The plates were incubated for two hours on a shaker at room temperature, washed thrice with PBS-T followed by the addition of 50 μL of the respective secondary-HRP antibody at specified dilutions (1:4000 secondary anti-human IgG-HRP (NRC anti-hIgG#5-HRP</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG-HRP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Surrogate Neutralization ELISA (snELISA) assay for evaluation of neutralization in serum samples: The described methodology was adapted from the surrogate neutralization ELISA assay as shown in Abe et al. 2020, for the evaluation of the relative inhibition of neutralizing antibodies to RBD protein from binding to soluble ACE2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD Production in mammalian cells: For comparison, a mammalian RBD was produced in HEK 293F cells and purified.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293F</div><div>suggested: RRID:CVCL_6642)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">(Mount Sinai, NYC) encoding the Whuhan-Hu-1 RBD (MN908947) sequence coding for the amino acid 319-541 and fused with the N-terminal SARS-CoV-2 spike secretory signal and a C-terminal hexa-histidine tag was transfected into 293F cells cultivated in Freestyle 293 expression media (Thermo Fisher, #12338018) at 37°C, 7% CO2, while shaking (125rpm).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Whuhan-Hu-1 RBD</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The construct was codon-optimized for N. benthamiana expression and was synthesized by GenScript into the pHREAC vector (Peyret et al., 2019) via BsaI sites</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHREAC</div><div>suggested: RRID:Addgene_134908)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lectin-binding human chaperone calreticulin (NP_004334.1) was codon-optimized for N. benthamiana and synthesized in the pHRE vector (Peyret et al., 2019).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHRE</div><div>suggested: RRID:Addgene_134909)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Transient expression in Nicotiana benthamiana: Prior to infiltration, pHREAC-RBD and pHRE-calreticulin were freshly transformed into Agrobacterium tumefaciens strains AGL1 and Gv2260, respectively, via electroporation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHREAC-RBD</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pHRE-calreticulin</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Dissociation constant (KD) was determined using 4-parameter curve fitting with GraphPad Prism 9.1.2 software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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docdrop.org docdrop.org

Microsoft Word - TIP 525 Syllabus.docx

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1. rebecca.saunders 21 Oct 2021
  
  in Public
  
  COURSE CONTENT
  
  I love the verbs chosen for each page/discussion/assignment. It is a great call to action! The structure is outstanding. The only issue I see with this display is that the image raises an equity issue - students who require screen readers will not be able to access this information unless there is a really long alt tag associated with the image.
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docdrop.org/download_annotation_doc/TIP-525-Syllabus-Fall-21-d7d57.pdf
psyarxiv.com psyarxiv.com

Practical behavioural solutions to COVID-19: Changing the role of behavioural science in crises

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1. jasminehollingworth 21 Oct 2021
  
  in BehSci
  
  For a very long time in the COVID-19 crisis, behavioural change leading to physical distancing behaviour was the only tool at our disposal to mitigate virus spread. In this large-scale naturalistic experimental study we show how we can use behavioural science to find ways to promote the desired physical distancing behaviour. During seven days in a supermarket we implemented different behavioural interventions: (i) rewarding customers for keeping distance; (i) providing signage to guide customers; and (iii) altering shopping cart regulations. We asked customers to wear a tag that measured distances to other tags using ultra-wide band at 1Hz. In total N = 4,232 customers participated in the study. We compared the number of contacts (< 1.5 m, corresponding to Dutch regulations) between customers using state-of-the-art contact network analyses. We found that rewarding customers and providing signage increased physical distancing, whereas shopping cart regulations did not impact physical distancing. Rewarding customers moreover reduced the duration of remaining contacts between customers. These results demonstrate the feasibility to conduct large-scale behavioural experiments that can provide guidelines for policy. While the COVID-19 crisis unequivocally demonstrates the importance of behaviour and behavioural change, behaviour is integral to many crises, like the trading of mortgages in the financial crisis or the consuming of goods in the climate crisis. We argue that by acknowledging the role of behaviour in crises, and redefining this role in terms of the desired behaviour and necessary behavioural change, behavioural science can open up new solutions to crises and inform policy. We believe that we should start taking advantage of these opportunities.
  
  ann:summary
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psyarxiv.com/q349k/
clojure.org clojure.org

Clojure - tools.build Guide

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1. crowbrammer 21 Oct 2021
  
  in Public
  
  {:tag "TAG" :sha "SHA"}
  
  Go here https://github.com/clojure/tools.build
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tutorial.eyehunts.com tutorial.eyehunts.com

HTML a tag | html anchor tag | name, link, href, target, onClick - EyeHunts

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1. gyuri 20 Oct 2021
  
  in Public
  
  HTML a tag | html anchor tag | name, link, href, target, onClick Posted April 8, 2019May 15, 2021 by Rohit
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tutorial.eyehunts.com/html/html-a-tag-html-anchor-tag-name-link-href-target-onclick/
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Chemoreceptor Co-Expression in Drosophila Olfactory Neurons

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1. Public_Reviews 19 Oct 2021
  
  in eLife
  
  Reviewer #1 (Public Review):
  
  In this manuscript, the authors challenge the long-standing conclusion that Orco and IR-dependent olfactory receptor neurons are segregated into subtypes such that Orco and IR expression do not overlap. First, the authors generate new knock-in lines to tag the endogenous loci with an expression reporter system, QF/QUAS. They then compare the observed expression of these knock-ins with the widely used system of enhancer transgenes of the same receptors, namely Orco, IR8a, IR25a, and IR76b. Surprisingly, they observe an expansion of the expression of the individual knock-in reporters as compared to the transgenic reporters in more chemosensory neurons targeting more glomeruli per receptor type than previously reported. They verify the expression of the knock-in reporters with antibody staining, in situ hybridization and by mining RNA sequencing data.
  
  Finally, they address the question of physiological relevance of such co-expression of receptor systems by combining optogenetic activation with single sensillum recordings and mutant analysis. Their data suggests that IR25a activation can modulate Orco-dependent signaling and activation of olfactory sensory neurons.
  
  The paper is well written and easy to follow. The data are well presented and very convincing due in part to the combination of complementary methods used to test the same point. Thus, the finding that co-receptors are more broadly and overlappingly expressed than previously thought is very convincing and invites speculation of how this might be relevant for the animal and chemosensory processing in general. In addition, the new method to make knock-ins and the generated knock-ins themselves will be of interest to the fly community.
  
  The last part of the manuscript, although perhaps the most interesting, is the least developed compared to the other parts. In particular, the following points could be addressed:
  
  - It would be good to see a few more traces and not just the quantifications. For instance, the trace of ethyl acetate in Fig. 6C, and penthyl acetate for 6G.
  
  - In Fig. 4D, the authors show the non-retinal fed control, which is great. An additional genetic control fed with retinal would have been nice.
  
  - It appears that mostly IR25a is strongly co-expressed with other co-receptors. The provided experiments suggest a possible modulation between IR25a and Orco-dependent neuronal activity. However, what does this mean? How could this be relevant? And moreover, is this a feature of Drosophila melanogaster after many generations in laboratories?
  
  peerReview
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biorxiv.org/content/10.1101/2020.11.07.355651v2
oer.pressbooks.pub oer.pressbooks.pub

Recommendation on Open Educational Resources (OER)

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1. alanoeg 18 Oct 2021
  
  in Public
  
  effective use of high quality educational and research materials
  
  Noting here University of Edinburgh's collection of OERs that support SD4 including
  
  Foundations for All - supporting refugee scholars
  
  Wikimedia in Education published in cooperation with Wikimedia UK "bringing together a collection of case studies from across the UK in order to provide insight into the use of the Wikimedia projects in education."
  
  Critical Thinking in Global Challenges - a series of open licensed videos "to better understand what critical thinking is, and to practice and enhance critical thinking skills" and how they were applied in course activities designed around global issues
  
  There's much more here https://open.ed.ac.uk/tag/sdg4/
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oer.pressbooks.pub/oeg2021/chapter/english/
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2021-10-15: More WYSIWYG Functionality & Some Really Nice Documentation

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1. waynemsmith 17 Oct 2021
  
  in Public
  
  Highlightr adds a menu with pre-defined colors for the <mark> html tag to allow for an easier multi-color highlighting experience.
  
  Here is a way to make better highlights
  
  obsidian
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https://biorxiv.org/cgi/content/10.1101/2021.10.12.464150

1
1. sciscore 17 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.12.464150: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Informed consent was obtained from all patients, and all patient samples were collected after a full recovery from illness and under IRB approval.<br>IACUC: Animal work was performed at Lovelace Biomedical Research Institute (LBRI), with approval from the Institutional Animal Care and Use Committee (IACUC) and within Animal Biosafety Level 3 (ABSL3) containment.<br>Euthanasia Agents: All animals were euthanized with an euthanasia solution consisting of 390 mg of sodium pentobarbital and 50 mg of phenytoin per mL.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">The FPF of delivered dose was calculated as the total amount of sugar or sugar alcohol (e.g., trehaose, mannitol) collected with an aerodynamic diameter below 5 µm as a percentage of the total amount of sugar or sugar alcohol deposited on the adapter, the induction port, stages 1–7 and Micro-Orifice Collector. 2.8. Efficacy of AUG-3387 in and in vivo model of SARS-CoV-2 infected Syrian Hamsters: An in vivo efficacy study was performed with male Syrian Hamsters (Mesocricetus auratus) approximately 9 weeks of age with a weight range of 110-134 g, at time of randomization, were sourced from Charles River Laboratory.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The FPF of delivered dose was calculated as the total amount of sugar or sugar alcohol (e.g., trehaose, mannitol) collected with an aerodynamic diameter below 5 µm as a percentage of the total amount of sugar or sugar alcohol deposited on the adapter, the induction port, stages 1–7 and Micro-Orifice Collector. 2.8. Efficacy of AUG-3387 in and in vivo model of SARS-CoV-2 infected Syrian Hamsters: An in vivo efficacy study was performed with male Syrian Hamsters (Mesocricetus auratus) approximately 9 weeks of age with a weight range of 110-134 g, at time of randomization, were sourced from Charles River Laboratory.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A fluorescently labelled anti-human IgG/IgA/IgM secondary antibody was also added to each well.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG/IgA/IgM</div><div>suggested: (Sigma-Aldrich Cat# S4893, RRID:AB_10625701)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For identification of surface bound antibodies binding to SARS-CoV-2, SARS-CoV-2 antigens conjugated to Alexa Fluor 488 stained SARS-CoV-2 antigen was used as a staining agent for single cell sorting of antigen reactive memory B cells into 96 well plates on a Sony SH-800 Cell Sorter.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: (Thermo Fisher Scientific Cat# 53-6490-82, RRID:AB_2884046)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">AUG-3705 was attached to the LSA flow cell via interaction with its V5 tag and a surface bound anti-V5 antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-V5</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The cells were enriched 4 times until 97% of the cells showed signal above the negative control as read out by staining with anti-ACE-2 and secondary antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-ACE-2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Assay: 2.4.1 ACE-2 Expressing HEK293T Cell Line Construction: An ACE2 expressing HEK293T cell line (“LentiX ACE2.S4”) was constructed by packaging pCMV-AC-GFP (Origene) into lentivirus and transducing HEK293T’s (ATCC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Infected cell culture supernatant was diluted with 950 µL D10 media, and then serial diluted before 50 µL of each dilution was added to 8 wells of Vero E6 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 96 hours, 100 µL CellTiterGlo reagent was added to each well of the infected Calu-3 cells to assay for live cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Calu-3</div><div>suggested: BCRJ Cat# 0264, RRID:CVCL_0609)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Assay: 2.4.1 ACE-2 Expressing HEK293T Cell Line Construction: An ACE2 expressing HEK293T cell line (“LentiX ACE2.S4”) was constructed by packaging pCMV-AC-GFP (Origene) into lentivirus and transducing HEK293T’s (ATCC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV-AC-GFP</div><div>suggested: None</div></div></td></tr></table>
  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: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04872231</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Completed</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Single Ascending Dose and Multiple Ascending Dose Study of V…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04576325</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Pharmacokinetic Profile of Voriconazole Inhalation Powder in…</td></tr></table>
  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:
  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.
  Results from scite Reference Check: We found no unreliable references.
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  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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https://biorxiv.org/cgi/content/10.1101/2021.10.12.464114

1
1. sciscore 17 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.12.464114: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Description of cohort and ethics statement: Samples were collected from participants enrolled in a prospective cohort study approved by the Biomedical Research Ethics Committee (BREC) at the University of KwaZulu–Natal (reference BREC/00001275/2020).<br>Consent: Written informed consent was obtained from each participant.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Four were males and 5 were females.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For RBD expression experiments, 45 OD units of yeast were labeled in 1:100 diluted chicken-anti-Myc-FITC antibody (Immunology Consultants CMYC45F) to detect the RBD’s C-terminal Myc tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Myc tag .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After the plasma incubations, the libraries were secondarily labeled for 1 hour with 1:100 fluorescein isothiocyanate-conjugated anti-MYC antibody (Immunology Consultants Lab, CYMC-45F) to label for RBD expression and 1:200 Alexa Fluor-647-conjugated goat anti-human-IgA+IgG+IgM (Jackson ImmunoResearch 109-605-064) to label for bound plasma antibodies.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-MYC</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human-IgA+IgG+IgM</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, version 0.8.10) to process Illumina sequences into counts of each barcoded RBD variant in each pre-selection and antibody-escape population.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>antibody-escape population .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Magnetic Separation Rack, Thermo Fisher Scientific, CS15000) was used to separate antibodies that bind RBD from the supernatant, and the supernatant (the post-RBD antibody depletion sample) was removed.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>post-RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Note that these assays were performed in 293T cells over-expressing human ACE2, which may underestimate contributions of non-RBD-binding antibodies to viral neutralization (7, 35, 60).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Dilution series of the synthetic sera comprised of the anti-RBD antibody REGN10987 (72), which binds to both Wuhan-1-like RBD and B.1.351 RBD, and pooled pre-pandemic human serum from 2017-2018 (Gemini Biosciences; nos. 100–110, lot H86W03J; pooled from 75 donors) were performed such that the anti-spike antibody was present at a highest concentration of 0.25 µg/mL.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-spike</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pre-pandemic serum alone, without anti-RBD antibody depletion, was used as a negative control, averaged over 2 replicates</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody binding was detected with TMB/E HRP substrate (Millipore Sigma, ES001) and 1 N HCl was used to stop the reaction.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ES001</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Generation of pseudotyped lentiviral particles: HEK-293T (American Type Culture Collection, CRL-3216) cells were used to generate SARS-CoV-2 spike-pseudotyped lentiviral particles and 293T-ACE2 cells (Biodefense and Emerging Infectious Research Resources Repository (BEI Resources), NR-52511) were used to titer the SARS-CoV-2 spike-pseudotyped lentiviral particles and to perform neutralization assays (see below).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To generate spike-pseudotyped lentiviral particles (70), 6e105 HEK-293T (ATCC CRL-3216) cells per well were seeded in 6-well plates in 2 mL D10 growth media (Dulbecco’s Modified Eagle Medium with 10% heat-inactivated fetal bovine serum, 2 mM l-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: ATCC Cat# CRL-3216, RRID:CVCL_0063)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Note that these assays were performed in 293T cells over-expressing human ACE2, which may underestimate contributions of non-RBD-binding antibodies to viral neutralization (7, 35, 60).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For experiments involving D614G spike, we used spike-pseudotyped lentiviral particles that were generated essentially as described in (70), using a codon-optimized SARS-CoV-2 spike from Wuhan-Hu-1 strain that contains a 21-amino-acid deletion at the end of the cytoplasmic tail (27) and the D614G mutation that is now predominant in human SARS-CoV-2 (30).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Wuhan-Hu-1</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">AWY101 yeast containing a negative control (containing an empty vector pETcon plasmid) were grown overnight at 30°C in galactose-containing media.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pETcon</div><div>suggested: RRID:Addgene_41522)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting CCSs are available on the NCBI Sequence Read Archive, BioProject PRJNA770094,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NCBI Sequence Read Archive</div><div>suggested: (NCBI Sequence Read Archive (SRA, RRID:SCR_004891)</div></div><div style="margin-bottom:8px"><div>BioProject</div><div>suggested: (NCBI BioProject, RRID:SCR_004801)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical Analysis: The percent of neutralizing activity of early-2020 and B.1.351-convalescent plasmas due to RBD-binding antibodies is plotted with the plotnine python package, version 0.8.0 (https://plotnine.readthedocs.io/en/stable/index.html), shown as a Tukey boxplot (middle line indicating median, box limits indicating interquartile range) with individual measurements overlaid as points.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>python</div><div>suggested: (IPython, RRID:SCR_001658)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All raw sequencing data are available on the NCBI Short Read Archive at BioProject PRJNA770094, BioSample SAMN22208699, SAMN22208700.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>NCBI Short Read Archive</div><div>suggested: None</div></div></td></tr></table>
  Results from OddPub: Thank you for sharing your code and data.
  Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
  Our study has several limitations. The cohorts of individuals infected with early 2020 and B.1.351 viruses are small, and are geographically and temporally distinct. Nevertheless, the two cohorts are relatively well-matched with respect to age, sex, and days-post symptom onset of sample collection (Table 1) and assays were performed under comparable conditions. Our deep mutational scanning measured binding to yeast-displayed RBD, which may not capture all relevant features of full-length spike in the context of virus. Finally, our neutralization assays used pseudotyped lentiviral particles and ACE2-overexpressing cells, and some recent works suggest that the relative importance of different spike epitopes for neutralization can depend on the viral system and target cell line used (7, 35, 36, 60). Although the B.1.351 variant has now been displaced by the Delta variant, our results illustrate the need to understand immunity elicited by different SARS-CoV-2 variants. As population immunity due to infection or vaccination increases, preexisting immunity is becoming an increasingly important driver of SARS-CoV-2 evolution (61), as has shown to be the case for seasonal coronaviruses (62, 63). Moreover, as individuals begin to accumulate more complex SARS-CoV-2 immune histories due to multiple infections and/or vaccinations, the effects of immune imprinting or original antigenic sin (64, 65) may start to interact with the variant-specific immunodominance hierarchies we have describ...
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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publish.obsidian.md publish.obsidian.md

011.02 - NCM - Thesis notes - Obsidian Publish

1
1. ncmthesis2021 15 Oct 2021
  
  in Public
  
  .
  
  Are you aware of work by Jane Lugea and Dan Mcintyre at Huddersfield who also developed some software (called Worldbuilder) that can tag and annotate texts using TWT principles? They discuss it in this article but there may be other publications about it too: link to article The software has mixed reviews from TW theorists... some don't see it as necessary for TWT analysis. I'm not sure how often Worldbuilder is used now.
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augmentingcognition.com augmentingcognition.com

Augmenting Long-term Memory

1
1. masuuni 15 Oct 2021
  
  in Public
  
  I said above that I typically spend 10 to 60 minutes Ankifying a paper, with the duration depending on my judgment of the value I'm getting from the paper. However, if I'm learning a great deal, and finding it interesting, I keep reading and Ankifying. Really good resources are worth investing time in.
  
  How to tag and store the "really good sources" for later review? I wouldn't want to have thousands of really good sources because then I wouldn't have any bandwidth for new material. Always pop something out if something goes in?
  
  anki study
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https://biorxiv.org/cgi/content/10.1101/2021.10.11.463956

1
1. sciscore 14 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.11.463956: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">No statistical methods were used to predetermine sample size.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell Lines: 293T (or HEK293T) cells, 293T-ACE2, and Expi293F cells were cultured in DMEM media, supplemented with 10% FBS, 100 U/ml penicillin/ streptomycin, 2 mM L-glutamine, and in the presence of 5% CO2.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Expi293F</div><div>suggested: RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">293T-ACE2 cells, derived from 293T, stably express human ACE2 71.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T-ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expi293F cells, derived from the 293 cells, were purchased from ThermoFisher Scientific (cat # A14528; RRID: CVCL_D615).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Expi293F</div><div>detected: ( RRID:CVCL_D615)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral infectivity measurements: The infectivity of lentivirus particles carrying S proteins (including variants) on the surface was evaluated using a vector containing an HIV-1 long terminal repeat (LTR) that expresses a Gaussia luciferase reporter (HIV-1-inGluc) 72,73. 293T cells were transfected at 60–80 % confluency with the plasmid encoding indicated full-length SARS-CoV-2 S glycoproteins, the plasmid encoding an intron-regulated Gluc (HIV-1-inGluc), and a plasmid pCMV delta R8.2 encoding HIV-1 GagPol (Addgene, plasmid # 12263) using FuGENE 6 (Promega, # E2311).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Untagged and double-tagged SARS-CoV-2 spike variants were generated based on a template full-length pCMV3-SARS-CoV-2 Spike (codon-optimized, Sino Biological, cat # VG40589-UT) plasmid that has translated amino acid sequence identical to QHD43416.1 (GenBank).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV3-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">D614G point mutation was introduced into both untagged full-length pCMV3 SD614 and double-tagged SD614 Q3/A4 constructs to generate both untagged and tagged SG614 variants.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV3</div><div>suggested: RRID:Addgene_161029)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">E484K point mutation was introduced into both untagged and tagged pcDNA3.1 SAlpha by site-specific mutagenesis.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.1</div><div>suggested: RRID:Addgene_79663)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Viral infectivity measurements: The infectivity of lentivirus particles carrying S proteins (including variants) on the surface was evaluated using a vector containing an HIV-1 long terminal repeat (LTR) that expresses a Gaussia luciferase reporter (HIV-1-inGluc) 72,73. 293T cells were transfected at 60–80 % confluency with the plasmid encoding indicated full-length SARS-CoV-2 S glycoproteins, the plasmid encoding an intron-regulated Gluc (HIV-1-inGluc), and a plasmid pCMV delta R8.2 encoding HIV-1 GagPol (Addgene, plasmid # 12263) using FuGENE 6 (Promega, # E2311).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCMV</div><div>suggested: RRID:Addgene_20783)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, DNA sequence encoding monomeric hACE2 followed by an HRV3C cleavage site, monomeric Fc tag, and 8xHisTag at the 3’-end were synthesized and cloned into the pVRC8400 vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVRC8400</div><div>suggested: RRID:Addgene_63164)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Where indicated, the conformational effects of hACE2 on S proteins were conducted by pre-incubating fluorescently labeled viruses with 200 μg/ml hACE2 for 90 mins at room temperature before imaging and smFRET imaging data were taken in the continued presence of 200 μg/ml hACE2. smFRET quantification and statistical analysis: The analysis of smFRET data was performed using a MATLAB-based customized SPARTAN software package 78.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPARTAN</div><div>suggested: (SPARTAN, RRID:SCR_014901)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Based on visual inspection of fluorescence and FRET traces that revealed direct observations of state-to-state (conformation-to-conformation) transitions, FRET histograms were fitted into the sum of four Gaussian distributions using the least-squares fitting algorithm in MATLAB.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MATLAB</div><div>suggested: (MATLAB, RRID:SCR_001622)</div></div></td></tr></table>
  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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 20 and 22. 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.
  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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Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans

1
1. Public_Reviews 12 Oct 2021
  
  in eLife
  
  Reviewer #1 (Public Review):
  
  Cytoplasmic germ granules are a common feature of germ cells across species. Great effort has gone into trying to (1) identify proteins that localize to these condensates, (2) gain insights into how these granules assemble, and (3) characterize their functional significance. Pioneering studies on P granules in C. elegans have greatly expanded our understanding of these structures.
  
  In this paper, Price et al. use proximately labeling to identify previously unknown P granule components. They tag several known P granule protein genes with TurboID at their endogenous loci and perform biotinylation reactions in two different ways. Labeled proteins were subsequentially pulled down and identified using mass spectrometry. This approach successfully identified known P granule components and many new potential candidates. The authors focus their efforts on characterizing two related proteins EGGD-1 and EGGD-2 (also known as MIP-1 and MIP-2, based on recently published work). Knockdown of EGGD-1 and EGGD-2 using RNAi results in P granule defects. Cas-9 induced mutations confirm and extend this genetic analysis. The authors also perform structure/function analysis on EGGD-1 and define specific roles for its LOTUS and Intrinsically Disordered Region (IDR) domains in perinuclear P granule formation and function. Epistasis analysis shows that EGGD-1 acts upstream of GLH-1 in P granule assembly, and overexpression of EGGD-1 can drive granule formation outside of germ cells.
  
  Strengths
  
  The P granule proteome data presented here provides a useful resource for the community. The genetic analysis on eggd-1 provides important insights into the function of a new P granule component. The data are convincing, and the experiments are well-controlled.
  
  Weaknesses
  
  There are relatively few weaknesses and the general conclusions are supported by the data.
  
  peerReview
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Research: Visual and auditory brain areas share a representational structure that supports emotion perception (Current Biology, 2021) - Neuroscience, Psychology, and Health - Art of Memory Forum

1
1. chrisaldrich 12 Oct 2021
  
  in Public
  
  I just bookmarked this article published today in Current Biology for later reading and annotation. While the article isn't specifically focused on memory, the fact that it touches on visual structures, emotion, music, and movement (dance) which are core to some peoples' memory toolkits, I thought that many here would find it to be of interest.
  
  One of the authors provided the following tl;dr synopsis:
  
  "Across the world, people express emotion through music and dance. But why do music and dance go together?
  
  We tested a deceptively simple hypothesis: Music and movement are represented the same way in the brain."
  
  Article: Visual and auditory brain areas share a representational structure that supports emotion perception01283-5) (Current Biology, 2021)
  
  Preprint: https://www.biorxiv.org/content/10.1101/254961v4
  
  For those who haven't integrated song or dance into their practices, searching around for the idea of songlines will give you some background on their possible uses.
  
  cc: @LynneKelly
  
  dance music movement memory psychology neuroscience perception songlines
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The pathogen-encoded signaling receptor Tir exploits host-like intrinsic disorder to assist infection

1
1. EMBOpress 11 Oct 2021
  
  in Review Commons
  
  Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
  
  Learn more at Review Commons
  
  Reply to the reviewers
  
  Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
  
  Learn more at Review Commons
  
  1. General Statements
  
  We want to thank all three reviewers for their positive feedback, constructive comments, and suggestions for clarity and improvement. We are delighted to find their consensus that the manuscript represents a contribution to the field.
  
  Accordingly, we made changes in the text (all highlighted in blue in the revised manuscript) and added a new figure as detailed in the point-by-point response.
  
  2. Point-by-point description of the revisions
  
  Reviewer #1 (Evidence, reproducibility and clarity (Required)):
  
  The authors describe results of the comprehensive analysis of the prevalence and functionality of intrinsically disordered regions of the pathogen-encoded signaling receptor Tir, which serves as an illustrative example of the bacterial effector proteins secreted by Attaching and Effacing (A/E) pathogens. This is an interesting and important study that represents an impressive amount of data generated computationally and using a broad spectrum of biophysical techniques. The work serves as a model of the well-designed and perfectly conducted study, where intriguing conclusions are based on the results of the comprehensive experiments. The manuscript is well-written and concise, and I have a real pleasure reading it. The text and figures are clear and accurate.
  
  We thank the Reviewer for these positive comments on our work.
  
  Although, in general, prior studies are referenced appropriately, the authors should mention that the pre-formed structural elements they found in Tir are in line with the concept of "PreSMos" (pre-structured motifs) previously introduced and described in several important studies from the laboratory of Kyou-Hoon Han.
  
  We thank the Reviewer for this suggestion. We have added a sentence to acknowledge the presence of “PreSMos” in the target-free state of Tir as putative signatures for target-binding, referring to a review article summarizing several local structural elements in unbound IDPs:
  
  “This supports the presence of pre-structured motifs (PreSMos) as pre-existing signatures for target binding and function within target-free Tir (72)**.”
  
  Please, note that we decided to keep this discussion to a minimum, as we cannot rule out the contribution of the induced fit model to the binding mechanism (i.e., disorder-to-order transition upon binding).
  
  Reviewer #1 (Significance (Required)):
  
  Solid evidence is provided that structural disorder and short linear motifs represent common features of A/E pathogen effectors. In fact, using a set of bioinformatics tools, the authors first show that although prokaryotic proteins typically contain significantly less intrinsic disorder than eukaryotic proteins, A/E pathogen effectors are as disordered as eukaryotic proteins. Using the translocated intimin receptor (Tir) as a subject of focused study, the authors then utilized a number of biophysical techniques to draw an impressive picture of disorder-based functionality. This study clearly represents a major advancement in the field of functional intrinsic disorder in general and in disorder-based functionality of proteins expressed by pathogenic bacteria. This was adds significantly to the field and will have a noticeable impact.
  
  Again, reading this manuscript was a real joy. Finally, this work perfectly fits in the area of my expertise, since for the past 25 years or so I am working on the different aspects of intrinsically disordered proteins.
  
  Thank you for this encouraging assessment.
  
  **Referee Cross-commenting**
  
  I agree with the amended recommendation of reviewer #3 to add in the manuscript EPEC O127.
  
  According to the suggestion of Reviewer #3, we have now included EPEC O127:H6 in the manuscript.
  
  I completely agree with comments of reviewer #2 and partially agree with reviewer #3. In my view, comparison of various strains as references for EPEC represents an interesting but independent project. It can be recommended to the authors as one of the potential future developments of their work.
  
  Thanks for the suggestion. We are pursuing that line of research.
  
  Reviewer #2 (Evidence, reproducibility and clarity (Required)):
  
  The general impression is that this is an excellent study that establishes
  
  The C-terminal intracellular region of Tir called C-Tir spanning residues 338 to 550 is largely disordered, however, observe helical structural elements involved with lipid interactions; multi-phosphorylation. The intracellular N-terminal part of Tir called N-Tir spanning residues 1 to 233 is also partially disordered but include a folded domain that is shown to assemble into a dimer
  
  The only major concern is that no SDS-PAGE gels or size exclusion chromatograms have been included to verify purity and monodispersed of the various constructs worked on. In particular, the SAXS and CD measurement is highly sensitive to purity, and the level of degradation as IDPs are notorious for being difficult to handle in solution. it would strengthen the arguments made based that
  
  We produced N-Tir and C-Tir as fusion proteins with a cleavable N-terminal thioredoxin tag (Trx-His6) and C-terminal Strep-tag. The latter allowed us to purify them via Strep-tag affinity chromatography as indicated by SDS-PAGE (please see Fig. S1).
  
  We agree with the Reviewer that even small amounts of impurities (i.e., higher oligomers/degradation) can interfere with the data analysis and make interpretation of the resulting data difficult and potentially misleading. So, to avoid such problems, all samples were purified in monodispersed forms by size-exclusion chromatography (SEC) before any biophysical study.
  
  Following the Reviewer's suggestion, we added a new supplementary figure (Fig. S5) showing the SEC-SAXS chromatogram profiles of C-Tir, N-Tir, and NS-Tir. Briefly, in the inline SEC-SAXS experiment, the sample eluates from an HPLC system directly and continuously into a BioSAXS flow cell for subsequent X-ray interrogation. Under our experimental conditions, C-Tir elutes as a single peak with Rg-values and mass compatible with a disordered monomeric protein, providing an excellent fit to the experimental SAXS curves. For N-Tir and NS-Tir, by SEC-SAXS, we separated the dimer from small amounts of high-order oligomers to yield the experimental SAXS curves of the pure dimers.
  
  “Fig. S5. SEC-SAXS chromatograms of (A) C-Tir, (B) N-Tir, and (C) NS-Tir. Each plane shows normalized total scattering intensity I(s), over the entire s range, from each frame acquired along elution volume and respective Rg-value (black circles). The flat variation of Rg reflects a pure monodisperse sample. The column type for size exclusion chromatography and sample concentrations are on the top left of each panel. For reference, the retention volume for monomeric BSA (66.4 kDa) is displayed by red triangles.”
  
  **Minor Comments**
  
  Read through the manuscript to remove passages with spoken language
  
  We thank the Reviewer for this suggestion. We went through the manuscript and improved the writing to reduce passages with spoken language.
  
  Line 263, "To do so", should be removed
  
  Line 290 "Our data thus" replaced with "this"
  
  We have amended the manuscript accordingly.
  
  Line 292 "lipid bilayers that might potentially fine-tune Tir's activity in the host cell." Weak sentence and the word fine-tune is slang. Rewrite the sentence. The interaction with lipids is fascinating!
  
  Thanks for the suggestion. The sentence has now been changed to “**This shows that C-Tir can undergo multivalent and tunable electrostatic interaction with lipid bilayers via pre-structured elements, suggesting that membrane-protein interplay at the intracellular side might control the activity and interactions of Tir in host cells.**”
  
  We also reinforce this fascinating message in the abstract by adding the sentence: “Membrane affinity is residue-specific and modulated by lipid composition, suggesting a previously unrecognized mechanism for interaction with the host.”
  
  Line 192 "In figure Fig. 3A," remove the Fig
  
  Fixed.
  
  Line 326, "In a similar fashion," is redundant. Rewrite the sentences below.
  
  We have modified the sentence as follows: “We evaluated whether the N-terminal cytosolic region of Tir (N-Tir; Fig S1) was also intrinsically disordered ...”
  
  Line 342 add spaces between digit and SI unit "52kDa" there are more cases of this.
  
  Thank you for pointing this out. This has now been corrected to 52 kDa.
  
  Reviewer #2 (Significance (Required)):
  
  I expect this study to have broad relevance to microbiologists working with the intimin and translocated intimin receptor, in particular the lipid interaction is likely to be followed up by the community.
  
  We thank the reviewer for this comment. Indeed, we believe that further studies on Tir's lipid-binding ability as a novel molecular strategy in host-pathogen interactions, will potentially provide new insights on virulence, transmembrane signaling in general, and disorder-mediated functions.
  
  **Referee Cross-commenting**
  
  What reviewer 3 suggested in the comments sounds like added value and should be included.
  
  I agree with reviewer 1, that the strain comparison potentially is beyond the scope presented in this manuscript.
  
  We have now included EPEC O127:H6 in the manuscript.
  
  Reviewer #3 (Evidence, reproducibility and clarity (Required)):
  
  **Summary:**
  
  This interesting manuscript look at the structure of the Nter and Cter of the effector Tir from enteropathogenic E. coli. The authors confirmed previous study highlighting the "disordered" part of the Cter. However, the extended experimental work (NMR, Small-angle X-ray scattering and CD spectroscopy) from this study also reveals the connection between different area of Tir and its implication during Tir phosphorylation and its interactions with SH2 domain.
  
  We thank the Reviewer for this positive remark. Indeed, in our work, we highlight the structural features of the SH2-mediated interaction between Tir and host SHP-1 protein, and we also show that C-Tir is capable of lipid interaction via pre-structured motifs and that N-Tir is disordered but assembled into a dimer. Overall, we provide an updated and wide picture of Tir's intracellular side that goes beyond the scrutiny of previously described disorder features.
  
  **Major Comments:**
  
  The authors used E2348/69 (O127:H7) strain as a reference for EPEC. However, this strain are the least effectors of all the EPEC sequences and may over estimated the PDR in EPEC. It would be wiser to use a strain like B171 as a reference for EPEC to be able to conclude "Disordered Proteins (PDR) with long disordered regions occur in EPEC effectors similar to the human proteome". I believe that the PDR in EPEC is similar to EHEC and CR. I do not have any major concern for the rest of the work.
  
  We thank the Reviewer for this comment. So, to clarify, we amended “EPEC” with “EPEC O127:H6” in text and figures.
  
  We also added a paragraph at the beginning of the Discussion section to acknowledge that our prediction analysis concerns EPEC O127:H6 and two additional representative A/E bacteria strains:
  
  “Among the enteropathogenic Escherichia coli strains EPEC O127:H6 (E2348/69) is commonly used as a prototype strain to study EPEC biology, genetics, and virulence (69). Here, we have determined the structural disorder propensity of EPEC O127:H6 sequences and two additional representatives of A/E bacteria: EHEC O157:H7 and CR ICC168.”
  
  Finally, the Reviewer suggests to include EPEC strain B171 (serotype O111:NM) in our analysis. We agree that considering additional strains would be of value, however we believe that this is beyond the scope of this manuscript, which mainly focuses on the characterization of the structural features of the E2348/69 Tir effector. We are currently working on a broader comparative analysis among different Escherichia coli pathogenic strains, including B171, and we hope to share our findings with the community in the near future.
  
  **Minor comments**
  
  Statistic problem: Mann Whitney U Test (Wilcoxon Rank Sum Test) is a comparison of two independent samples with the underlying assumption is normally distributed or that the samples were sufficiently large. It is not certain that any of this assumption is correct. In addition, the effector are part of the whole proteome. Can it be then considered that both groups are independent?
  
  We thank the Reviewer for this remark, which allows us to clarify the choice of this particular test. Indeed the Mann Whitney U-test is a non-parametric test to compare two samples with the alternative hypothesis being that one of the two samples is stochastically greater than the other. As it is a nonparametric test samples are not required to be normally distributed, as it is for the Student t-test.
  
  Regarding the independence of the samples, when comparing the effectors collections to their corresponding proteomes, we did exclude the effectors sequences from the latter. We have clarified this point in the Supplementary Material and Methods section.
  
  Line 120 and 442: O127 not H127
  
  Thank you for pointing this out. It has now been corrected to O127.
  
  Line 212: positions 409 or 405?
  
  Yes, it should be 405. Thank you.
  
  Reviewer #3 (Significance (Required)):
  
  **Nature and significance:**
  
  Tir plays a major role during EPEC infection. It is a signalling platform that has been reported to interact with multiple proteins. Whereas the extracellular part has been well characterised and crystallised, the intracellular part has been proven so far to be difficult to study. Over the last decade, no progress has been made to explain how Tir works. This manuscript provides interesting information that shade some light on how the protein could work.
  
  **Existing literature:**
  
  The last research manuscript trying to highlight the structural function of Tir dates from 2007 (PMC1896257). This study is far more extended and in depth than any other previous work done.
  
  **Audience:**
  
  the Audience may probably limited to researcher working on the field of cellular microbiology and the function associated with bacterial effector in the host. This study could be also a useful tool to identify new effectors base on their "disorder".
  
  We thank the Reviewer for recognizing the importance of this study. We agree that our work highlights the pivotal role of disordered regions in bacterial effectors, thus enabling a better understanding of the molecular mechanisms used by pathogens to subvert the host-cell processes. We indeed believe that our work can stimulate further research on the characterization of intrinsically disordered effectors, and also beyond the cellular microbiology field, in order to gain a broader knowledge on the molecular dialogue at the host-pathogen interface, which is essential to design better therapeutic strategies.
  
  **Expertise:**
  
  I have been working on A/E pathogens for the last 15 years with a particular interest in Tir signalling. My domain of expertise is more in relation to cell signalling than crystallography or structural study.
  
  **Referee Cross-commenting**
  
  I agree with both reviewers. My comment about EPEC is more about the conclusion for some of the figures. I don't think they should conclude for the whole EPEC. The Tir variation among EHEC O157:H7 is low, but it is far more diverse for EPEC. Simply adding in the manuscript EPEC O127 should be enough.
  
  We thank the Reviewer for this comment. As mentioned above, we now state in the manuscript, in both Results and Discussion sections, that we used E2348/69 as a representative strain for EPEC.
  
  PeerReviewed
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https://medrxiv.org/cgi/content/10.1101/2021.10.05.21264054

1
1. sciscore 10 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.05.21264054: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Study participants and ethics statement: Blood samples were obtained from 143 dialysis patients and 48 healthcare workers under study protocols approved by the local Institutional Review Boards (Canton Ticino Ethics Committee, Switzerland).<br>Consent: All subjects provided written informed consent for the use of blood and blood components (such as PBMCs, sera or plasma).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">Statistical analysis: The study was designed to have 80% power to detect a minimum 25% difference in total incidence of cases with poor neutralizing antibody response (i.e., low or undetectable plasma antibody titers) or in average neutralizing titers between dialysis patients and healthy controls as well as within the HD subgroups.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines: Cell lines used in this study were obtained from ATCC (Vero E6 TMPRSS2) or ThermoFisher Scientific (Expi CHO cells, Expi293F™ and FreeStyle 293 cells)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vero E6 TMPRSS2 cells were grown in DMEM supplemented with 10% HyClone (FBS)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6 TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasma pseudovirus neutralization assay: Vero E6-TMPRSS2 were grown in DMEM supplemented with 10% FBS and seeded into white bottom 96 well plates (PerkinElmer, 6005688), as previously described35.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6-TMPRSS2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Production of recombinant glycoproteins: The SARS-CoV-2 RBD WT construct was synthesized by GenScript into phCMV1, with a sequence encoding an N-terminal mu-phosphatase signal peptide, an ‘ETGT’ linker, SARS-CoV-2 S residues 328-531, a linker sequence, an Avi tag, a twin Strep tag and a 8xHis-tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>phCMV1</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The SARS-CoV-2 stabilized Spike WT (D614G) construct was synthesized by GenScript into pCDNA with an N-terminal mu-phosphatase signal peptide, 2P stabilizing mutation28,29, a TEV cleavage site and a C-terminal foldon, 8x His-tag, Avi tag and C-tag30 and expressed in FreeStyle 293 cells following manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDNA</div><div>suggested: RRID:Addgene_105932)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Amino acid substitutions were introduced into the D614G pCDNA_SARS-CoV-2_S plasmid as previously described31.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCDNA_SARS-CoV-2_S</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed and visualized with Prism (Version 9.1.0).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were plotted and analyzed with GraphPad Prism software (version 9.1.0).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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: We detected the following sentences addressing limitations in the study:
  Our study has some limitations including a missing control group of patients matched by age, gender and comorbidities, without ESKD, while our control group was composed by younger individuals with few or no comorbidities. Another limitation was the low number of PD patients that did not allow to make subgroup analyses of risk factors of defective response. In addition, our patients were unbalanced in terms of type of mRNA vaccine received (BNT162b2 or mRNA-1273), although we had sufficient statistical power to make a comparison, even in a subgroup of patients matched by age, gender and comorbidity index. In conclusion, our study demonstrates, at the functional level, that mRNA vaccines induce a defective neutralizing antibody response against SARS-CoV-2 variants in dialysis patients, in particular in naïve HD patients immunized with BNT162b2. Our findings support the need of an additional boost, preferentially with a high-dose mRNA vaccine, in this population58-60, which, however, need to be continuously monitored with proper serological tests that measure not only the serum antibody levels, but also their neutralizing activity, either directly or indirectly through an avidity test. Finally, our data suggest that some patients may not respond efficiently even after an additional boost and, therefore, in case of SARS-CoV-2 infection, they should be considered for other therapeutic strategies, including early immunotherapy with monoclonal antibodies.
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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  SciScore for 10.1101/2021.10.05.463282: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Written informed consent was obtained from all patients.<br>IRB: This study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster, the Hong Kong East Cluster Research Ethics Committee, and the Kowloon West Cluster Research Ethics Committee (UW 13-265, HKECREC-2018-068, KW/EX-20-038[144-26]).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Male and female golden Syrian hamsters (Mesocricetus auratus) (aged 6–10 weeks) were purchased from the Chinese University of Hong Kong Laboratory Animal Service Centre through the HKU Laboratory Animal Unit</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">The hamsters were randomized from different litters into experimental groups.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Serially diluted plasma from healthy individuals or previously published monoclonal antibodies against HIV-1 (VRC01) were used as negative controls.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HIV-1</div><div>suggested: (bNAber Cat# bNAberID_1, RRID:AB_2491019)</div></div><div style="margin-bottom:8px"><div>VRC01</div><div>suggested: (bNAber Cat# bNAberID_1, RRID:AB_2491019)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Two consecutive staining steps were conducted: the first one used an antibody and RBD cocktail incubation of 30 min at 4 °C; the second staining involved staining with anti-His-APC and anti-His-FITC antibodies (Abcam) at 4 °C for 30 min to detect the His tag of the RBD.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-His-APC</div><div>suggested: (Miltenyi Biotec Cat# 130-101-320, RRID:AB_2747411)</div></div><div style="margin-bottom:8px"><div>anti-His-FITC</div><div>suggested: (Miltenyi Biotec Cat# 130-092-675, RRID:AB_1103226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For dIgA antibody production, plasmids of paired heavy chain (IgA1, IgA2) and kappa light chain together with a J chain were co-transfected into Expi293™ expression system (Thermo Fisher Scientific) at the ratio of 1:1:1 following the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgA1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IgA2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibodies produced from cell culture supernatants were purified immediately by affinity chromatography using recombinant Protein G-Agarose (Thermo Fisher Scientific) or CaptureSelect™ IgA Affinity Matrix (Thermo Fisher Scientific) according to the manufacturer’s instructions, to purify IgG and IgA, respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IgA</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">2 mL-fractions were collected, pooled, concentrated and evaluated by western blot using mouse anti-IGJ monoclonal antibody [KT109] (Abcam) and rabbit anti-human IgA alpha chain antibody (Abcam).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-IGJ</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-human IgA alpha chain antibody ( Abcam) .</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The differences in response units between ACE2 injection alone and prior antibody incubation reflect the antibodies’ competitive ability against ACE2 binding to the spike protein.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For identification and localization of SARS-CoV-2 nucleocapsid protein (NP) in organ tissues, immunofluorescence staining was performed on deparaffinized and rehydrated tissue sections using a rabbit anti-SARS-CoV-2-NP protein antibody together with an AF488-conjugated anti-rabbit IgG (Jackson ImmunoResearch, PA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2 nucleocapsid protein (NP</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-SARS-CoV-2-NP protein</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-rabbit IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After blocking with 0.1% Sudan black B for 15 min and 1% bovine serum albumin (BSA)/PBS at RT for 30 min, the primary rabbit anti-SARS-CoV-2-NP antibody (1:4000 dilution with 1% BSA/PBS) was incubated at 4°C overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2-NP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For identification of DC-SIGN expression, we stained the NT slices with rabbit anti-DC-SIGN primary antibody (Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies) according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-DC-SIGN</div><div>suggested: (IMGENEX Cat# DDX0208A488, RRID:AB_1929964)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For identification of ACE2 expression, the goat anti-ACE2 primary antibody (R&D) and Alexa Fluor 568 donkey anti-goat IgG (H+L) secondary antibodies (Invitrogen) according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-goat IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The infectious medium was replaced with fresh medium containing respective concentration of antibody after washing 3 times with PBS. 24 h later, the infected cells were imaged under fluorescence microscope after staining with AF488-conjugated anti-SARS-CoV-2 NP antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2 NP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were further permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit sera anti-SARS-CoV-2-N for 1 hour at RT before adding Alexa Fluor 488 goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (Life Technologies).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2-N</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After treatment with B8 antibodies at the dose of 3000 ng/ml/mL for 1 hour, HEK293T cells transfected with SARS-CoV-2 spike-GFP were added into the treated Vero-E6 TMPRSS2 cells and co-cultured for 48 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>B8</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The viral challenge experiments were then conducted in our Biosafety Level-3 animal facility following SOPs strictly, with strict adherence to SOPs Cell lines: HEK293T cells, HEK293T-hACE2 cells Vero-E6 cells, HK2 cells and Vero-E6-TMPRSS2 cells were maintained in DMEM containing 10% FBS, 2 mM L-glutamine, 100 U/mL/mL penicillin and incubated at 37 □ in a 5% CO2 setting 62</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-hACE2</div><div>suggested: RRID:CVCL_A7UK)</div></div><div style="margin-bottom:8px"><div>Vero-E6</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Vero-E6-TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, The pseudovirus was generated by co-transfection of 293T cells with pVax-1-S-COVID19 and pNL4-3Luc_Env_Vpr, carrying the optimized spike (S) gene (QHR63250) and a human immunodeficiency virus type 1 backbone, respectively 77</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The antibody-virus mixtures were subsequently added to pre-seeded HEK 293T-ACE2 cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293T-ACE2</div><div>suggested: RRID:CVCL_A7UK)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Mixtures were then transferred to 96-well plates pre-seeded with 1×104/well Vero E6 cells and incubated at 37°C for 24 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The HEK293T-CD209 cells were pre-treated with 10 ng/ml/mL of B8-dIgA or control dIgA and incubated for 6 h prior SARS-CoV-2 infection (MOI=0.05).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T-CD209</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Effects of B8 antibodies on SARS-CoV-2 mediated cell-cell fusion: Vero-E6 TMPRSS2 cells were seeded into 48-well plates and cultured overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero-E6 TMPRSS2</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After treatment with B8 antibodies at the dose of 3000 ng/ml/mL for 1 hour, HEK293T cells transfected with SARS-CoV-2 spike-GFP were added into the treated Vero-E6 TMPRSS2 cells and co-cultured for 48 hours.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, The pseudovirus was generated by co-transfection of 293T cells with pVax-1-S-COVID19 and pNL4-3Luc_Env_Vpr, carrying the optimized spike (S) gene (QHR63250) and a human immunodeficiency virus type 1 backbone, respectively 77</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pVax-1-S-COVID19</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>pNL4-3Luc_Env_Vpr</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequences were aligned using Clustal W in the BioEdit sequence analysis package (Version 7.2)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BioEdit</div><div>suggested: (BioEdit, RRID:SCR_007361)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Half-maximal (IC50) or 90% (IC90) inhibitory concentrations of the evaluated antibody were determined by inhibitor vs. normalized response -- 4 Variable slope using GraphPad Prism 6 or later (GraphPad Software Inc.)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The defocus value of each image, which was set from −1.0 to −2.0 μm during data collection, was determined by Gctf.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gctf</div><div>suggested: (GCTF, RRID:SCR_016500)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Sequential data processing was carried out on RELION 3.0 and RELION 3.1.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>RELION</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RBD-Fab maps were fitted onto the whole structure map using Chimera, then combined using PHENIX combine_focused_maps.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Model building and structure refinement: The spike model (PDB code: 6VSB) and the initial model of the B8 Fab generated by SWISS-Model were fitted into the EM density map, and further manually adjusted with Coot.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Coot</div><div>suggested: (Coot, RRID:SCR_014222)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The final structures were validated using Phenix.molprobity</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>molprobity</div><div>suggested: (MolProbity, RRID:SCR_014226)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">. UCSF Chimera, ChimeraX and PyMol were used for map segmentation and figure generation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PyMol</div><div>suggested: (PyMOL, RRID:SCR_000305)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">NP+ cells per field were quantified based on the mean fluorescence intensity (MFI) using the ZEN BLACK 3.0 and ImageJ (NIH).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ImageJ</div><div>suggested: (ImageJ, RRID:SCR_003070)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The fluorescence density of SARS-CoV-2 infected cells was acquired using a Sapphire Biomolecular Imager (Azure Biosystems) and then the MFI of four randomly selected areas of each sample was quantified using Fiji software (NIH).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Fiji</div><div>suggested: (Fiji, RRID:SCR_002285)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Re-analysis of published nasal brushing single-cell data: The preprocessed scRNA-seq data from nasal brushing samples of 2 healthy controls and 4 COVID-19 patients were downloaded from Gene Expression Omnibus (GEO) database with accession numbers GSE171488 and GSE164547.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gene Expression Omnibus</div><div>suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Quantification and statistical analysis: Statistical analysis was performed using PRISM 6.0 or later.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>PRISM</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr></table>
  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: We detected the following sentences addressing limitations in the study:
  One limitation is the insufficient amounts of HuNAbs distributed on the nasal mucosal surface for protection 47. Other reasons might include alternative entry pathways engaged by SARS-CoV-2 to evade HuNAbs. To this end, Liu et al. reported recently that antibodies against the spike N-terminal domain (NTD) induced an open conformation of the RBD and thus enhanced the binding capacity of the spike to the ACE2 receptor, leading to increased viral infectivity 66. Yeung et al. demonstrated nicely that SARS-CoV-2 could engage soluble ACE2 (sACE2) and then bind alternate receptors for viral entry, through interaction between a spike/sACE2 complex with the angiotensin II AT1 receptor, or interaction between a spike/sACE2/vasopressin complex with the AVPR1B vasopressin receptor, respectively 49. In this study, we found that, in the presence of potent neutralizing B8-dIgA1 or B8-dIgA2 antibodies, SARS-CoV-2 used the cellular receptor CD209 for capture or infection, which likely expanded the use of CD209+ cells as target cells, leading to enhanced NT infection and trans-infection. Interestingly, a preprint report suggests that cells expressing CD209 can be infected directly by SARS-CoV-2 through an interaction of the spike with the NTD instead of the RBD 67. This mode of action, however, was unlikely to explain our findings, because no enhancement of SARS-CoV-2 nasal infection was found in presence of control dIgA1 and dIgA2. Our results rather suggest that the direct binding of virus-b...
  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: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2021.10.05.463282
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https://biorxiv.org/cgi/content/10.1101/2021.10.03.462919

1
1. sciscore 08 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.03.462919: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IACUC: Animal work was performed at Lovelace Biomedical, with approval from the Institutional Animal Care and Use Committee.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Following 2-hr sample incubation, the plate was washed and SULFO-TAG (MSD) anti-hamster IgA (Brookwood Biomedical) detection antibody was added.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-hamster IgA</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">VeroE6 cell monolayers at ≥ 90% confluency in 96-well plates were rinsed with PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>VeroE6</div><div>suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Vaccine constructs: r-Ad-S is a rAd5 vector containing full-length SARS-CoV-2 S gene under control of the CMV promoter.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>rAd5</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">IgA MSD: S1 protein was biotinylated according to manufacturer’s instructions (EZ-link, Thermofisher), and was conjugated to a U-Plex MSD linker (Mesoscale Diagnostics).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Thermofisher</div><div>suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)</div></div></td></tr></table>
  Results from OddPub: Thank you for sharing your data.
  Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
  Our study does have limitations. Firstly, we do not measure mucosal T cell responses which are shown to play a role in limiting SARS-CoV-2 infection at mucosal sites19. Additionally, the SAR-CoV-2 challenge dose we used was above physiological dose likely to be picked up by an environmental exposure, as evidenced by the high viral RNA load in the nose at 1-day post infection in all index hamsters. It is likely that mucosal immunization would provide greater protection against SARS-CoV-2 transmission when lower doses of challenge virus are used. Our study was meant to be a stringent challenge to clearly identify advantages between vaccine groups. For practical reasons, such as the limitation of the number of aerosol transmission chambers, lower dose levels or evaluation at later time points were not done. Additionally, future work should include challenging mucosally-vaccinated hamsters with the Delta variant and other variants of concern. An orally-delivered, temperature-stable SARS-CoV-2 vaccine is ideal for global vaccination, where adequate storage and qualified health care providers maybe in short supply. IN delivery has some of the same advantages, but translating IN SARS-CoV-2 vaccination efficacy in humans has proven to be more difficult than in animals20,21. Implementing oral vaccine campaigns around the world has been done, as evidenced by the rotavirus and poliovirus vaccination efforts22,23. We have previously demonstrated that our SARS-CoV-2 clinical candidate vac...
  Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04563702</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Active, not recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Safety and Immunogenicity Trial of an Oral SARS-CoV-2 Vaccin…</td></tr></table>
  Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2021.10.03.462919
ecampusontario.pressbooks.pub ecampusontario.pressbooks.pub

Racism Set 2a

1
1. ChantelleG 08 Oct 2021
  
  in Public
  
  H5P TAG HERE
  
  wtf is a H5P tag?! If someone knows and reads his, please help a girl out and tell me?
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ecampusontario.pressbooks.pub/cs050racism/chapter/racism-set-2-overview/
www.medrxiv.org www.medrxiv.org

https://medrxiv.org/cgi/content/10.1101/2021.10.04.21264522

1
1. sciscore 07 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.10.04.21264522: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Ethical considerations: The Cuban Ministry of Public Health (MINSAP), the Independent Ethics Committee (IEC) for Studies on Human Subjects, at CENATOX and the Cuban National Regulatory Agency (Centre for State Control of Medicines and Medical Devices, CECMED), approved the trial and the procedures (CECMED, Authorization date: 13/10/2020, Reference number: 05.013.20BA).<br>Consent: Written informed consent was obtained from all volunteers.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Sixty Cuban volunteers aged 19-59 years, with body mass index 18.5-29.9 kg/m2, of both sexes, were recruited (Table 1).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Study design and participants: This phase I, randomized, double-blind clinical trial was carried out at the National Centre of Toxicology (CENATOX) in Havana, Cuba.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">Randomization and blinding: Stratified random blinded sampling was used to select the sample of the universe of Cuban citizens aged 19-59 years, which was proportionally divided in two age subgroups: 19-39 and 40-59 years to ensure a proper representation of each age subgroup.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Exclusion criteria were: history of COVID-19, SARS-CoV-2 PCR-positive tests or detection of antibodies anti-SARS-CoV-2, any severe disease or decompensated chronic disease, immunodeficiency, history of serious allergy, pregnancy, breastfeeding, and immunological treatment during the last 30 days.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Seroconversion rates for IgG anti-RBD antibodies (≥4-fold increase in antibody titres over pre-immunization titres) were calculated for each subject.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-RBD</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">sequence: 319-541 amino acid residues with a poly-histidine fusion tag at its C-terminus), expressed in CHO cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CHO</div><div>suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analyses were done using SPSS version 25·0; EPIDAT version 4.1,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPSS</div><div>suggested: (SPSS, RRID:SCR_002865)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">, Prism GraphPad version 6.0.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
  </footer>
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URL

medrxiv.org/cgi/content/10.1101/2021.10.04.21264522
www.biorxiv.org www.biorxiv.org

Functional fluorescently-tagged human profilin

1
1. Iratxe 07 Oct 2021
  
  in ASAPbio crowd review
  
  This review reflects comments and contributions by Ricardo Carvalho, Joachim Goedhart, Sónia Gomes Pereira, Pratima Gurung, Samuel Lord, Claudia Molina, Arthur Molines, Gregory Redpath, Mugdha Sathe, Sagar Varankar. Review synthesized by Ewa Sitarska.
  
  This preprint introduces a recombinant profilin that has a flexible linker to a genetically encoded fluorescent tag (either mApple or Halo). Fluorescent protein tagging is a popular and accepted method to study the properties of a protein of interest in solution and in cells. A careful analysis of the tagged protein relative to the untagged, native protein is crucial to understand whether the tagged protein faithfully reflects the behavior of the native protein. Therefore, studies like these are very valuable and the current manuscript is a good example of how such a study should be performed. The flexible linker presented here overcomes challenges observed in previous papers that found that linking a fluorescent protein to profilin disrupted some of its actin-related functions in cells and in vitro.
  
  This study is carefully conducted and nicely describes the properties of a fluorescent protein tagged profilin in a detailed manner. In particular, the authors use various in vitro assays as well as rescue experiments to demonstrate that their tagged version of profilin appears to behave similarly to wildtype profilin. The manuscript is written in a clear manner and was an enjoyable read. The comments below cover a couple of experiments, clarifications and questions for consideration to further add to the work. Regardless, this work is likely to contribute to the field, as anyone studying profilin is likely to try this construct in their future experiments.
  
  General comments:
  
  Why was mApple chosen as a tag (as opposed to the popular and best known fluorescent protein mEGFP)?
  
  The mApple is prone to photochromicity/photoswitching (https://doi.org/10.1038/nmeth.1209, https://doi.org/10.1038/nmeth.4074). This should be mentioned to warn future users of this fusion protein.
  
  It would be advisable to be consistent with the naming of ‘untagged profilin’ throughout the manuscript. Currently unlabeled, untagged, wild-type or rescue are used interchangeably.
  
  In Figures 4B, 5A there appears to be differences between untagged and tagged profilin in the images. Maybe a more representative image would be beneficial, where applicable.
  
  Depositing the plasmids from this paper at addgene.org would be beneficial for the public (the plasmids can be deposited under condition that these will be released only after publication of this work in a peer-reviewed journal).
  
  Title
  
  ‘Functional fluorescently-tagged human profilin’– suggest clarifying in the title and throughout the text that the fluorescent tags are genetically encoded.
  
  Abstract
  
  ‘high cellular concentrations (121 µM)’– This is a very precise number for such a general statement. It seems that the number is derived from a specific cell line, so it would be beneficial to present it as a number from this cell line or change it to an approximation (~100 µM).
  
  Introduction
  
  'Some profilin outcompetes actin bound'– suggest some rewording to clarify the fragment, for example, it could be mentioned whether it refers to F-actin or G-actin.
  
  Results- Design of tagged profilin
  
  ‘Profilin is considerably smaller than the smallest fluorescent tags’ – clarifying that it is a genetically encoded fluorescent tag would be of advantage. There are no smaller fluorescent proteins (FP) yet, but genetically encoded FP of a similar size exist, for example miRFP670nano is 17kDa. https://doi.org/10.1038/s41467-018-08050-8
  
  ‘Traditional direct labeling approaches are cytotoxic and disrupt actin-based functions’ – is there data showing the new fluorescent profilin side-by-side with one without a flexible linker (or other version used previously) to show that the latter disrupts profilin's functions? It’s not essential but it would strengthen this point and confirm the improvement over prior work.
  
  ‘Estimates of cellular profilin concentration are very high depending on the cell type’ – would be nice to provide a rough estimate at this point, similarly to the introduction part.
  
  ‘with an mApple fluorescent probe or as Halo-tagged single molecules’ - What is the meaning of 'single' here?
  
  ‘We cloned mApple- or Halo-tags fused to a ten amino acid flexible linker on the N-terminus of human profilin-1.’ – As in the introduction, it is stated that “Positioning a GFP-derived fluorescent tag on the C- or N-terminus disrupts PLP- and PIP-binding interactions, effectively rendering the fluorescent version flawed for critical measurements in cells”, it would be beneficial to state the rationale for tagging profillin at the N-terminus? Also, how was the linker composition and length determined and how is it related to other linkers used in compromised fusions of profilin?
  
  Figure 1
  
  In panel A, it would be helpful to indicate the N-terminus, as this is the side where the fluorescent protein is attached.
  
  In the legend, PFN1 is introduced for the first time and thus, it could be replaced with ‘tagged-profilin (PFN1)’.
  
  Results - mApple-profilin binds phosphoinositide lipids with similar affinity as untagged profilin
  
  ‘PIP’ - PIP is usually an abbreviation for Phosphatidyl Inositol Phosphate (which is a lipid). Phosphoinositide is the same thing, but is not abbreviated as PIP. Recommend using Phosphatidyl Inositol Phosphate = PIP. These types of lipids can be indicated as PIPs (without the addition of lipids).
  
  ‘Profilin also binds PI(3,5)P2 which regulates critical signal transduction events through intracellular vesicles to the early endosome’ - The prevailing consensus now is that PI(3,5)P2 is involved in late endosomal trafficking to the lysosome. This could be different for profilin specific purposes, but this statement could be updated with recent references to PI(3,5)P2.
  
  ‘Thus, mApple-tagged profilin retains functional interactions with two important PIP lipids.’ – Testing other phospholipids, including PIP3 (Lu et al., 1996 showed an interaction with profilin) and some negative controls would be beneficial. Covering most phosphoinositide species by using the phospholipid-binding dot blots would make this figure stronger.
  
  Figure 2
  
  In panel B, it would be easier to compare profilin and mApple-profilin binding affinity to each of the PIP lipids. For that, profilin and mApple-profilin samples could be run side by side in the same blot. The suggestion being that panel B includes profilin and mApple-profilin incubated with PI(3,5)P2, and panel C profilin and mApple-profilin incubated with PI(4,5)P2. For clarity, can it be specified in the figure legend or in the figure that the profilin-1 lane is the negative control pellet lacking the liposomes as well as that S and P stands for supernatant.
  
  Also, in panel B, what are the loading controls? The quantification and western-blots are unclear. For example, it is indicated that 1µM PFN1was used in this experiment, but the pellet band for mAp-PFN1 is not comparable to PFN1 pellet band. Despite this, quantification in 2D shows that they are similar. Please clarify and add the corresponding loading controls.
  
  Panel D: Please mention what is quantified here (supernatant, pellet or overall level),
  
  Panel D and E: the shades to pink may be tricky to distinguish, more contrasting colors and/or shapes might be useful.
  
  It is highly appreciated that the antibodies and dilutions are mentioned in the figure legend.
  
  Results - Direct visualization of fluorescent-profilin with polymerizing actin filaments
  
  ‘We used fluorescence anisotropy to measure the binding affinity between profilin and Oregon-Green (OG)-labeled monomeric actin (Figure S2).‘ - This appears confusing with the 3D results. If a change in fluorescence anisotropy with OG-actin is not detected, then is it okay to use OG-actin for bulk polymerisation assay. Maybe the OG-tag interferes only during fluorescence anisotropy, but not during fluorescence microscopy.
  
  ‘Several studies demonstrate that thymosin b4 (Tb4) competes with profilin to bind actin monomers‘ – It is worth mentioning that this refers to untagged/unlabeled actin monomers.
  
  Figure 3
  
  Please consider if it is relevant to compare the competitive and non-competitive data on the same graph.
  
  In panel A description, ‘10 nM GFP-thymosin b4 (GFP-Tb4) mixed with increasing concentrations of unlabeled actin.’ – Would ‘10nM unlabelled actin monomers in presence of increasing concentration of Τβ4’ be more appropriate?
  
  In panel D, the curves would be better visible when the y-axis runs from 0-30.
  
  In panel E, the mApple-Profilin samples show longer filaments, maybe quantification of the filament length could be performed?
  
  In panel G, are the errors bars based on the means of the technical replicates or on all aggregated data? The first option is preferred, as this plotting strategy was also used in 3F.
  
  In the panel description, ‘Data were quantified from four separate reactions (FOV) each.’ – could clarify whether the data derive from 4 totally independent reactions, or from the analysis of 4 different fields of view (FOV) from the same experimental procedure?
  
  In the panel description, ‘ns, not significantly different from 1 µM actin alone control; a, compared with control (p <0.05)‘ – such labeling may be confusing for the reader, it would be beneficial to state that the first ‘ns/a’ are related to the actin alone, and the second ‘ns’ are comparing labelled and unlabeled profilin or omit showing the statistical test in the plot, and show it in a table instead.
  
  Results - Fluorescent profilin stimulates formin-based actin filament assembly
  
  General: Based on the competition and interaction experiments it seems important to generate a dose-dependent inducible construct for profilin to govern the stoichiometry of interactions and study their relevance in the cells.
  
  ‘Similar to experiments in assessing only profilin-actin interactions (Figure 3F), we counted significantly fewer actin filaments in reactions containing actin and either profilin (Figure 4D)’ - In Figure 3F the plot shows around 45 actin filaments per FOV with 1 uM actin (20% oregon green actin). In Figure 4D the plot shows more than 100 actin filaments per FOV with 1 uM actin (10% alexa 647 actin). Surprisingly, the elongation rate are similar in Fig 3F and Fig 4D. Is the Oregon-Green actin known to be less efficient at nucleating filaments while retaining the same polymerization ability? If it is the case, it would be worth making a mention.
  
  ‘Thus, fluorescent profilin stimulates formin-mediated actin filament nucleation similar to the untagged version.’ - The data seem to suggest that the presence of profilin inhibits actin filament nucleation and polymerization, a clarification would be appreciated.
  
  ‘The ac-celerated rate of actin filament’ – Please change to ‘accelerated’.
  
  Results - Profilin directly binds tubulin dimers and enhances the growth rate of microtubules in vitro
  
  ‘microtubule stability index‘ – The values indicated on the y-axis for the plot in Fig 5E are confusing (0 / 25/ 50 / 75 / 1). Is the index expressed as a percentage and the max value supposed to be 100? Or is the index supposed to be the number of rescues per catastrophe?
  
  ’This suggests a mechanism where profilin accelerates microtubule polymerization by directly binding to tubulin dimers to promote microtubule assembly and then diffusing along the sides of the microtubule lattice to further stabilize microtubule growth.‘ - Microtubules are more stable the faster they grow (catastrophe frequency scales inversely with polymerization rate). In this condition where profilin increases polymerization rate by around 5x, it is unclear how much of the increased stability is due to the lattice binding. The fragment could be softened regarding the role of the transient lattice binding in microtubule stabilization.
  
  Figure 5
  
  In panel B, intensities are quite different, would it be possible to comment on this?
  
  In panels H an I, a black/magenta merge is tricky to see. Although it breaks consistency, a green/magenta or cyan/magenta merge may be more informative visually.
  
  Results - Profilin regulates the morphology of N2a cells through actin and microtubule crosstalk
  
  ‘We used quantitative western blots to determine the level of endogenous profilin as well as levels of profilin in CRISPR knockout cells following transfection with plasmids containing untagged profilin, mApple-profilin, or Halo-profilin.’ - The levels of profilin are quantified from a blot, which is a bulk measurement. The transfection of profilin will show substantial cell-to-cell variation (some cells may have much higher or lower levels than the measured average). Mentioning it and discussing its implications would be advisable.
  
  ‘We chose this parameter because N2a cells have unique actin filament and microtubule cytoskeletal features but do not efficiently perform other classic cell processes that require intact cytoskeletal crosstalk (i.e., migration or division).‘ – While looking at cell shape is one strategy, an additional experiment looking into a migration phenotype may strengthen this point. Another interesting experiment to strengthen this point and providing a direct measure of profilin function could be performing a pulse chase experiment using drugs to depolymerise actin/microtubules. In such an experiment, a distinct change in depolymerisation should be noted between WT, KO, and the profilin rescue cells. This could show that the mApple-profilin can substitute for WT profilin.
  
  ‘super resolution confocal microscopy to image fixed cells.‘ – how is super-resolution achieved here? Or is ‘super’ unnecessary here?
  
  ‘the ratio of endogenous cell area to other cell conditions’ - This metric is a ratio of areas and it is only valid as an assessment for shape if the cells from each condition cover similar areas. If it is not the case, then the two parameters (shape and area) are convoluted and the ratio measures both the difference in shape and in area covered. It would be good to provide the average area of the cells in each condition for clarification.
  
  ‘We also stained these cells for actin filaments (Figures 6H and 6I) and micro-tubules (Figure 6J and 6K) and used a similar morphology parameter to detect broad differences in cytoskeletal architecture.’ – Please clarify the reasons for using the cell area ratio metric for quantification of cell morphology. How is the quantification metric (area) used for sub-cellular network like actin and microtubule? F-actin stained with phalloidin looks different in endogenous PFN1 vs mApple-PFN1, but by using area metric there is no morphological difference. Microtubule, on the other hand, appear similar in all the cells.
  
  Figure 6
  
  In panel A, it does not seem like mApple-PFN1 and Halo-PFN1 reach endogenous levels. Maybe a quantification would be beneficial.
  
  In panel B, please report the number of independent replicates. Also, may be worth commenting on why tagged PFN1 rescue cells were not included?
  
  For panel E, please provide the error bars and the legend that contains the information from where the data derived or from how many independent experiments.
  
  In panel F, it may be advantageous to use red green and blue as colors for the overlay. This will generate unique colors for the different combinations of the three images.
  
  For the context of panel F, unprocessed images of the tagged profilin in living cells could be presented somewhere in the main text. They could be larger than the small panels here, and not be segmented into binary images. The point of the fluorescent profilin is that it can be used for live-cell imaging without substantially disrupting the typical profilin interactions. This should be confirmed by presenting live-cell images of the profilin construct. To avoid problems with high cytoplasmic concentrations of profilin drowning out any localization signal, maybe the fluorescent version could be expressed at a very low level or the Halo version could be used with a low concentration of fluorophore.
  
  In panel G, it is clear that the dots are from different cover slips, how many cells were analyzed per coverslip? Data could be shown from individual cells (not just their average). Also, please clarify if this quantification was made 24 hours after transfection as well?
  
  For panel I, actin morphology was calculated from actin filament signals similar to the cell morphology index. These calculations could be explained further in the methods. Does this mean that the actin morphology index is the ratio of actin area between the two conditions? Is the actin image somehow thresholded before taking the ratio?
  
  Panel L, is really appreciated and helpful to understand the "competition" between actin and microtubules for profilin. It would be also nice to represent the plasma membrane and the lipid-binding activity of profilin as well as binding to nucleation promoting factors (the proline-rich motifs of VASP and WASP), as this is mentioned throughout the paper.
  
  Discussion
  
  The fact that mApple and HaloTag both are entirely different and non-disturbing gives confidence that profilin can be fused with other tags, without losing functionality. Mentioning this in the discussion could give new insights for the readers.
  
  ‘Our genetic analyses in mammalian cells indicate that mApple-profilin and Halo-profilin are fully inter-changeable with the endogenous version.‘ - Authors have given the field an excelled tool which will be quite useful to study cellular functions of PFN1, its interaction with its several binding partners. Currently, cell shape is the metric used to determine if the tagged versions are fully inter-changeable with the endogenous version. Whether the tagged PFN1 can replace untagged PFN1 for other cellular functions will require further exploration. Also, high concentration of PFN1 will remain an issue even with the mApple-PFN1 developed here. Do the authors suggest mild over-expression as a strategy to go around the high concentration issue?
  
  ‘Based on localization experiments using the pan-formin inhibitor, SMIFH2, some interactions between profilin and the sides of micro-tubules are thought to be indirect.’ – suggest clarifying how SMIFH2 treatment leads to conclude that interactions between PFN1 and microtubules is indirect.
  
  Methods
  
  Statistical significance tests do not demonstrate that conditions are identical. That is, when two conditions are not statistically different, it is not possible to say that these are equal (https://doi.org/10.1053/j.seminhematol.2008.04.003). Suggest avoiding the use of "n.s." in graphs to indicate that the data are similar. It is clear from the data that (in many cases) the tagged and untagged profilin show similar properties. If equality needs to be demonstrated, recommend carrying out an equivalence test.
  
  ‘Different shades of data points show technical replicates.’ – Please rephrase to clarify, for example “Different colors represent biological replicates. Similar colored dots reflect technical replicates“. Does "technical replicate," mean repeated measurements within each independent experimental run? Or different experimental runs? If the shading is supposed to denote paired experiments (e.g. darkest shading in different conditions are from the same experimental run), that can be stated in the caption.
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Monetizing Innovation

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1. dandele93 06 Oct 2021
  
  in Public
  
  No one wants to sell his or her idea short. Minivations are products that tap neither a product concept’s full market potential nor its full price potential. Companies that fall into the minivation trap underexploit the market opportunity and the price they could have charged, thereby robbing themselves of profits. Minivations go down as undermonetized products cursed with a “what might have been” tag.
  
  Il caso del problema di #minivation è quello in cui il prodotto non raggiunge né il massimo del potenziale di mercato né il massimo del potenziale di prezzo. Le aziende che cadono in questa trappola sono aziende che danno al proprio prodotto un prezzo più basso di quanto sia effettivamente il suo valore ed in questo modo rinunciano a dei guadagni.
  
  [[Monetizing Innovation]] books pricing
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Notetaking In Semantic Triples

1
1. Henrycase 04 Oct 2021
  
  in Public
  
  I read your blog post with interest, as a researcher in literature and cultural studies I was looking for this kind of entry to… maybe end a feeling of loneliness as I’m going to switch for a tool mainly used by programmers? :) I do some programming (mostly copy/paste language for the moment tbh) but I felt the need to find a testimony from someone in my field to give me this last bit of force to make the jump and you gave it to me! I want more now :) would you have some ressources to share about knowledge management taking advantage of org-mode and computational thinking?
  
  I’ve been managing my personal archaic databases with a tag system and relational databases for 20 years including ten years of an overly complicated handwriting mode before years of Excel that ended in a frustrating stateless vagabondage. I came across Org-mode as I was engaging myself in databases programming and would love to have some keystone to help me getting started. Thanx a lot!
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https://biorxiv.org/cgi/content/10.1101/2021.09.30.462449

1
1. sciscore 04 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.09.30.462449: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: Mouse studies Ethical approval: All the experimental procedures were performed in accordance with the guide for the use of laboratory animals of the University of Sao Paulo and approved by the institutional ethics committee under the protocol number 105/2021.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">SARS-CoV-2 experimental infection and treatments: Female K18-hACE2 mice, 8-week-old, were infected with 2×104 PFU of SARS-CoV-2 (in 40 µL) by intranasal route.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">A total of 10 photomicrographs in 40X magnification per animal were randomly obtained using a microscope Novel (Novel L3000 LED, China) coupled to a HDI camera for images capture.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The virus was propagated and titrated in Vero E6 cells in a biosafety level 3 laboratory (BSL3) at the Ribeirão Preto Medical school</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: RRID:CVCL_XD71)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The viral inoculum was added to Vero cells in DMEM 2% FBS and incubated at 37 °C with 5% CO2 for 48 h.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For expression, BL21 cells transformed with plasmid were grown in ZYM-5052 to an OD600 of 0.6-0.8 at 37°C and 200 RPM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BL21</div><div>suggested: RRID:CVCL_M639)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">K18-hACE2 mice: To evaluate the effects of Pyronaridine in vivo, we infected the K18-hACE2 humanized mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J)(21, 65, 66).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>K18-hACE2</div><div>suggested: RRID:IMSR_GPT:T037657)</div></div><div style="margin-bottom:8px"><div>B6.Cg-Tg(K18-ACE2)2Prlmn/J</div><div>suggested: RRID:IMSR_JAX:034860)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PCR product was digested with NcoI and XhoI and cloned into pET28a (Novagen) in frame with a C-terminal his-tag coding sequence E. coli BL21 transformed with plasmids were grown in LB to an optical density (OD600) of 0.6 at 37 °C and 200 RPM.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pET28a</div><div>suggested: RRID:Addgene_114156)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The total septal area and total area were analyzed with the aid of the Pro Plus 7 software (Media Cybernetics, Inc., MD, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Pro Plus</div><div>suggested: (Image-Pro Plus, RRID:SCR_016879)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Prior to docking, ligand and protein were prepared with LigPrep (Schrödinger, 2017, USA) and Protein Preparation Wizard (Schrödinger, 2017, USA) using default parameters.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>LigPrep</div><div>suggested: (Ligprep, RRID:SCR_016746)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Figures were generated with ChimeraX.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ChimeraX</div><div>suggested: (UCSF ChimeraX, RRID:SCR_015872)</div></div></td></tr></table>
  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 did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
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  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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https://biorxiv.org/cgi/content/10.1101/2021.09.30.462420

1
1. sciscore 04 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.09.30.462420: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Patient recruitment: All donors have given written informed consent and analyses were approved by the Institutional Review Board of Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Berlin (study protocol number EA1/258/18), the Institutional Review Board of the Faculty of Medicine at Ludwig-Maximilians-Universität (LMU) Munich, Germany (20-371), as well as the ethics committee of the Innsbruck Medical University (1167/2020).<br>IRB: Patient recruitment: All donors have given written informed consent and analyses were approved by the Institutional Review Board of Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität Berlin, and Berlin Institute of Health, Berlin (study protocol number EA1/258/18), the Institutional Review Board of the Faculty of Medicine at Ludwig-Maximilians-Universität (LMU) Munich, Germany (20-371), as well as the ethics committee of the Innsbruck Medical University (1167/2020).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Diagnostic antibody testing: Initial serological testing of patient samples was performed using a solid phase immunoassay (SeraSpot®Anti-SARS-CoV-2 IgG, Seramun Diagnostica GmbH, Heidesee, Germany).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SeraSpot®Anti-SARS-CoV-2 IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing, bound antibodies were detected by incubation with horseradish peroxidase (HRP)-labeled anti-human IgG for 30 minutes at room temperature.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Second, the presence of SARS-CoV-2 S1-specific antibodies was analyzed using a commercially available anti-SARS-CoV-2-S1 IgG ELISA (EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany) according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-SARS-CoV-2-S1 IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After His-tagged RBD Beta was immobilized on anti-Penta His BLI sensors, sensors were first dipped into CS82 IgG (50 μg/ml), and then dipped into indicated IgG antibodies (12.5 μg/ml).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-Penta</div><div>suggested: (Bio-Rad Cat# MCA5995P, RRID:AB_2888689)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">His-tagged recombinant RBD Beta protein was produced in HEK cells (ACROBiosystems, SPD-C52Hp) and covalently labeled using CruzFluor488 (Santa Cruz Biotechnology, sc-362617) according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK293T cell-secreted RBD-Fc fusion proteins composed of the RBD-SD1 component of the SARS-CoV-2 spike S1 subunit (amino acids 319-591) and the constant region of rabbit IgG1 heavy chain (Fc) were immobilized onto 96-well plates via anti-rabbit IgG (Dianova, 711-005-152)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, Vero E6 cells (1.6 x105 cells/well) were seeded in 24-well plates and incubated overnight.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero E6</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Crystallization and structural determination: The RBD (residues 333-529) of the SARS-CoV-2 spike (S) protein (GenBank: QHD43416.1) and the Beta variant that carries three mutations on the RBD (K417N, E484K, and N501Y) were cloned into a customized pFastBac vector (37), and fused with an N-terminal gp67 signal peptide and C-terminal His6 tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pFastBac</div><div>suggested: RRID:Addgene_1925)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For expression and purification of the Fabs, heavy and light chains were cloned into phCMV3.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>phCMV3</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cloning was considered successful when sequence identity was >99.5% as verified by the cBASE module of BASE software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BASE</div><div>suggested: (BASE, RRID:SCR_010937)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After identification of public clonotypes, they were plotted in a Circos plot using the R package circlize (33).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Circos</div><div>suggested: (Circos, RRID:SCR_011798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">As the CoV-AbDab includes SARS-CoV-2 mAbs from other sources than humans, and against other epitopes than the RBD, the following selection criteria were used (nomenclature like in CoV-AbDab): Binds to: SARS-CoV-2,</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: (BioLegend Cat# 946101, RRID:AB_2892515)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Iterative model building and refinement were carried out in COOT (41) and PHENIX (42), respectively (table S4).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>COOT</div><div>suggested: (Coot, RRID:SCR_014222)</div></div><div style="margin-bottom:8px"><div>PHENIX</div><div>suggested: (Phenix, RRID:SCR_014224)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">1 and all statistical analyses were performed using GraphPad Prism (9.2.0).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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 34. 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.
  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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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biorxiv.org/cgi/content/10.1101/2021.09.30.462420
psychology.stackexchange.com psychology.stackexchange.com

What is the difference between confidence and overconfidence?

1
1. rasterpeanut 03 Oct 2021
  
  in Public
  
  we ask that question posters do some initial research before posting
  
  well with enough research(and the requisite time) answers to all except the most cutting-edge profound questions can be found without having to ask anything anywhere (except maybe google)... it is to get answers for questions more quickly than that, that questions are asked in sites like this one... if the question is not a duplicate in the site, instead of -1ing such non-technical questions why not create a category or tag called "non-technical" or something and not blame the question poster?
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rasterpeanut

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psychology.stackexchange.com/questions/1825/what-is-the-difference-between-confidence-and-overconfidence
blacklivesmatter.com blacklivesmatter.com

Home - Black Lives Matter

1
1. MBCeid100 02 Oct 2021
  
  in Public
  
  8 Years StrongTo commemorate BLM’s 8th anniversary, we put together an anniversary video. Watch it now. Black Lives Matter Condemns the Treatment of Haitian Migrants at the U.S./Mexico Border September 22, 2021 Black Lives Matter condemns the racist, anti-
  
  2) Articles are clearly separated - this is positive for the majority of users that do not have accessibility needs. Font size throughout is large, both of article title and article preview. This is positive for those with difficulty seeing/minor-major eyesight trouble. This is also positive for people that have ADHD, as a lack of clutter will make it an easier read.
  
  3) Image does not have an <alt> tag - this is negative regarding accessibility for the blind. *I have included 3) here along with 2) because when I highlight just the picture the functions that hypothes.is offers do not appear.
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www.medrxiv.org www.medrxiv.org

https://medrxiv.org/cgi/content/10.1101/2021.09.28.21264207

1
1. sciscore 01 Oct 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.09.28.21264207: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Consent: Written informed consent was obtained from all participants, and the trial was done in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice.<br>IRB: Study approval in the UK was done by the Medicines and Healthcare products Regulatory Agency (reference 21584/0424/001-0001) and the South Central Berkshire Research Ethics Committee (reference 20/SC/0145).</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">For some assays and where sample availability allowed, comparison was made with age- and sex-matched participants who were HIV negative, aged 18–55 years, enrolled into the main COV002 phase 2/3 randomised clinical trial, and randomly assigned (5:1) to receive either ChAdOx1 nCoV-19 or MenACWY by intramuscular vaccination.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After 2-hour incubation and a washing step, detection antibody (MSD SULFO-TAG™ Anti-Human IgG Antibody, 1/200) was added.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Human IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Assays were performed using Multiscreen IP ELISpot plates (Merck Millipore, Watford, UK) coated with 10 μg/mL human anti-IFNγ antibody and developed using SA-ALP antibody conjugate kits (Mabtech, Stockholm, Sweden) and BCIP NBT-plus chromogenic substrate (Moss Inc., Pasadena, MA, USA).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-IFNγ</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>SA-ALP</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After stimulation cells were stained with the anti-human antibodies contained in supplementary table 6.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A MULTI-SPOT® 96-well, 10 spot plate was coated with three SARS CoV-2 antigens (S, RBD, N), SARS-CoV-1 and MERS-CoV spike trimers, as well as spike proteins from seasonal human coronaviruses, HCoV-OC43, HCoV-HKU1, HCoV-229E and HCoV-NL63, and bovine serum albumin.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HCoV-229E</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>HCoV-NL63</div><div>suggested: RRID:CVCL_RW88)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mixtures were then transferred to 96-well, cell culture-treated, flat-bottom microplate containing confluent Vero cell monolayers in duplicate and incubated for further 2 hours, followed by the addition of 1.5% semi-solid carboxymethyl cellulose (CMC) overlay medium to each well to limit virus diffusion.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Participants with a history of laboratory-confirmed SARS-CoV-2 infection by anti-N protein IgG immunoassay (Abbott Architect, Abbott Park, IL, USA) at screening were excluded.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Abbott Architect</div><div>suggested: (Abbott ARCHITECT i1000sr System, RRID:SCR_019328)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Standardised EUs were determined from a single dilution of each sample against the standard curve which was plotted using the 4-Parameter logistic model (Gen5 v3.09, BioTek).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Gen5</div><div>suggested: (Gen5, RRID:SCR_017317)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The percentage of focus reduction was calculated and IC50 (reported as FRNT50) was determined using the probit program from the SPSS package.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPSS</div><div>suggested: (SPSS, RRID:SCR_002865)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data was analysed using FlowJo version 10 and Prism version 9.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div><div style="margin-bottom:8px"><div>Prism</div><div>suggested: (PRISM, RRID:SCR_005375)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Gating strategies are as shown in supplementary figure 1a and b Phylogenetic analysis: We used protein BLAST to download all human coronavirus S protein sequences from NCBI database.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BLAST</div><div>suggested: (BLASTX, RRID:SCR_001653)</div></div><div style="margin-bottom:8px"><div>NCBI</div><div>suggested: (NCBI, RRID:SCR_006472)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We used MAFFT to align all chosen human corona viruses, SARS-CoV, MERS-CoV and SARS-CoV-2 S protein sequences.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MAFFT</div><div>suggested: (MAFFT, RRID:SCR_011811)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We then calculated the pairwise distances between the sequences and built a neighbour joining tree using MATLAB.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MATLAB</div><div>suggested: (MATLAB, RRID:SCR_001622)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">We did all analyses using R (version 3.6.1 or later), and Prism 9 (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04400838</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Active, not recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Investigating a Vaccine Against COVID-19</td></tr></table>
  Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Results from scite Reference Check: We found no unreliable references.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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medrxiv.org/cgi/content/10.1101/2021.09.28.21264207
discuss.write.as discuss.write.as

Custom javascript in WriteFreely 0.81 - WriteFreely - Discuss Write.as

1
1. mrcolbyrussell 01 Oct 2021
  
  in Public
  
  I’m mostly interested in including an analytics script tag
  
  Don't do that.
  
  write.as antifeature
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discuss.write.as/t/custom-javascript-in-writefreely-0-81/348
www.bbc.com www.bbc.com

Bottled water has become liquid gold

1
1. helenyang 01 Oct 2021
  
  in Public
  
  Given that water is such a fundamental resource and a matter of life and death, and given that it is such an abundant commodity to many and so scarce to others,
  
  Water is something that humans cannot live without, but at the same time something that humans can get for nearly free. Putting a price tag on water effectively capitalizes on humans' need to live.
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bbc.com/news/business-11813975
Sep 2021
jon-e.net jon-e.net

Decentralized Infrastructure for (Neuro)science

1
1. sneakers.the.rat 30 Sep 2021
  
  in Public
  
  highlighted like this,
  
  Hello! this is an annotation! i promise!
  
  untagged new tag!
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sneakers.the.rat

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jon-e.net/infrastructure
www.lewiscarroll.org www.lewiscarroll.org

Annotated and Critical Editions, Letters and Diaries – Lewis Carroll Society of North America

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1. bauhouse 30 Sep 2021
  
  in Public
  
  My father was talking about how Lewis Carroll’s concrete poetry was related to his experience of suffering from migraine headaches.
  
  migraine Lewis Carroll Samuel Bau
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lewiscarroll.org/2021/09/14/matrix-4-well-take-the-red-pill/
beepb00p.xyz beepb00p.xyz

How to annotate literally everything | Mildly entertainingᵝ

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1. dfn 30 Sep 2021
  
  in Public
  
  Awesome collection. I've spent a lot of time looking into this myself. I'm a heavy Instapaper user, using this Chrome extension to export annotations to Roam (not Roam-specific): https://github.com/houshuang/instapaper-exporter-extension. However, it's really frustrating that Instapaper isn't better... Highlighting on iPhone is jarring, it often jumps randomly when I long-press, I can't capture images etc. And the API is useless - no way of getting more than the first 200 items, which is why the extension above scrapes their website... Looked into Pocket, but they don't even allow entering your own notes. I also recently looked into RSS - Feedly pro (not cheap) allows annotations and has an API - I'll experiment with the API later, but I might also just tag articles and have them sent to Instapaper... Wallabag looks cool, wish I was an iOS developer so I could add the stuff I want :)
  
  The way to export Instapaper annotations to Roam without using Readwise as a middleware
  
  Roam
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dfn

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beepb00p.xyz/annotating.html
www.medrxiv.org www.medrxiv.org

https://medrxiv.org/cgi/content/10.1101/2021.09.27.21264163

1
1. sciscore 30 Sep 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.09.27.21264163: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: The S3 study, the COSCA study and the RECoVERED study were approved by the medical ethical review board of the Amsterdam University Medical Centers (<br>Consent: All participants provided written informed consent.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All S constructs were verified by Sanger sequencing and subsequently produced in HEK293F cells (ThermoFisher) and purified as previously described22.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293F</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudoviruses were produced by co-transfecting the SARS-CoV-2-S expression plasmid with the pHIV-1NL43 ΔEnv-NanoLuc reporter virus plasmid in HEK293T cells (ATCC, CRL-11268), as previously described48.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T</div><div>suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Shortly, HEK293T/ACE2 cells, kindly provided by Dr. Paul Bieniasz48, were seeded at a density of 20,000 cells/well in a 96-well plate coated with 50 μg/mL poly-L-lysine one day prior to the start of the neutralization assay.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK293T/ACE2</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">They were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned in a pPPI4 expression vector containing a hexahistidine (his) tag with Gibson Assembly (ThermoFisher)22.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pPPI4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudovirus construction: The WT, D614G, Alpha, Alpha E484K, Beta and Gamma pseudovirus S constructs were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned using SacI and ApaI in the pCR3 SARS-CoV-2-SΔ19 expression plasmid48 using Gibson Assembly (ThermoFisher)c</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCR3</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pseudoviruses were produced by co-transfecting the SARS-CoV-2-S expression plasmid with the pHIV-1NL43 ΔEnv-NanoLuc reporter virus plasmid in HEK293T cells (ATCC, CRL-11268), as previously described48.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHIV-1NL43 ΔEnv-NanoLuc</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The inhibitory concentration (IC50) and neutralization titers (ID50) were determined as the NAb concentration and serum dilution at which infectivity was inhibited by 50%, respectively, using a non-linear regression curve fit (GraphPad Prism software version 8.3).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr></table>
  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: We detected the following sentences addressing limitations in the study:
  There are several limitations of our study. First, our study includes substantially more female than male participants, reflecting the gender distribution among HCW at our institute. Second, the age distribution in the four groups is not identical. In particular, the AZD1222 group is, on average, considerably older as a consequence of restrictive use of the AZD1222 vaccine in individuals aged 60-64 years in the Netherlands. As immune responses tend to become weaker with higher age, this is a relevant factor when considering the weaker responses in the AZD1222 group. Finally, the samples we tested were taken at the expected peak of immunity. It will be relevant to study the durability of the neutralizing antibody responses after vaccination with each of these vaccines. Some studies suggest that immunity induced by adenovirus vaccines might be more durable than immunity from mRNA vaccines8,45. We have only analyzed known VOCs and VOIs and cannot predict how our results apply to future variants. One consideration is that current VOCs were probably selected based on increased fitness and/or transmissibility, while future variants may very well be selected based on escape from immunity when more and more people are vaccinated or have experienced COVD-19. Such escape variants may be more resistant to neutralizing antibodies induced by current vaccines than the VOCs and VOIs studied here and may render vaccines even less effective at preventing infection. However, while circulating ...
  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: We did not find any issues relating to colormaps.
  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.
  Thank you for including a protocol registration statement.
  Results from scite Reference Check: We found no unreliable references.
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  shops were to be visited; some new part of the town to be looked at;
  
  An artist's depictions of Bath in the early 19th century in context of Austen's life and work
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1. alanoeg 29 Sep 2021
  
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  areas of action of this Recommendation
  
  The OEGlobal 2021 conference was themed around the five action areas. For "raw material" to anchor here, you can find sessions related to each action area via these links:
  
  Building Capacity
  
  Developing Supportive Policy
  
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  oeglobal21
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https://medrxiv.org/cgi/content/10.1101/2021.09.24.21263853

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1. sciscore 29 Sep 2021
  
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  SciScore for 10.1101/2021.09.24.21263853: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">IRB: All subjects were recruited following protocols approved by local Institutional Review Boards (IRBs) or from NIH NIAID IRB, as indicated below: Chile: IRB: Comité Ético Científico Facultad de Medicina Clínica Alemana Universidad del Desarrollo, Santiago, Chile Protocol: 2020-41 Title: Infección por Sars CoV-2 y enfermedad Covid-19: caracterización epidemiológica, clínica, virológica e inmunológica (SARS-CoV-2 infection and COVID-19: Epidemiologic, clinical, virologic and immunologic characterization) Pavia, Italy: IRB: Ethics Committee of the Fondazione IRCCS Policlinico San Matteo, Pavia Protocol: 20200037677 Title: Analisi comprensiva della risposta immunitaria innata ed adattativa durante</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 antibody testing: SARS-CoV-2 anti-spike and anti-nucleocapsid antibody testing was performed via luciferase immunoprecipitation systems assay, as previously described [6].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SARS-CoV-2</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-spike</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>anti-nucleocapsid</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PBMC pools were Fc blocked (Human TruStain FcX, BioLegend) and stained with Totalseq-C human ‘hashtag’ antibodies (BioLegend), washed with staining buffer (2% BSA in PBS).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human ‘hashtag’</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The fluorescence-labeled antibody cocktail against human CD45 (APC/Cyanine7), CD3 (AF488), CD19 (APC), CCR7 (BV786), CD95 (BV650), IgD (PerCP-Cy5.5) and CD27(PE/Cyanine7; all antibodies obtained from Biolegend) were added at the end of blocking and incubated for 20 minutes at 4°C in the dark.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>human CD45</div><div>suggested: (BD Biosciences Cat# 563716, RRID:AB_2716864)</div></div><div style="margin-bottom:8px"><div>CD3</div><div>suggested: (SouthernBiotech Cat# 8200-30, RRID:AB_2796425)</div></div><div style="margin-bottom:8px"><div>CD19</div><div>suggested: (BD Biosciences Cat# 563333, RRID:AB_2738141)</div></div><div style="margin-bottom:8px"><div>CCR7</div><div>suggested: (Creative Diagnostics Cat# CPBT-66983GM, RRID:AB_2528776)</div></div><div style="margin-bottom:8px"><div>BV786</div><div>suggested: (BD Biosciences Cat# 740991, RRID:AB_2740614)</div></div><div style="margin-bottom:8px"><div>CD95</div><div>suggested: (BioLegend Cat# 305642, RRID:AB_2632622)</div></div><div style="margin-bottom:8px"><div>BV650</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>PerCP-Cy5.5</div><div>suggested: (BD Biosciences Cat# 560612, RRID:AB_1727457)</div></div><div style="margin-bottom:8px"><div>CD27</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Because multiple samples from different timepoints for each donor were collected and could not be demultiplexed by this method alone, ‘hashtag’ antibodies (Biolegend) were used to uniquely label the different time points.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>‘hashtag’</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The data of several proteins that directly bind with secondary antibodies detected through buffer incubation without any serum were excluded (such as IGHG1, IGHG3 and so on) alongside the controls (such as Rhodamine+IgG64, Anti-human IgG, GST 10ng/ul etc.).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IGHG1</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IGHG3</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>Anti-human IgG</div><div>suggested: (LSBio (LifeSpan Cat# LS-C23907-500, RRID:AB_900919)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Multiplex particle-based anticytokine autoantibody screening assay and functional evaluation: Plasma samples were screened for autoantibodies against IFN-α, IFN-β, IFN-ω and IFN-γ in a multiplex particle-based assay [31], in which differentially fluorescent magnetic beads were covalently coupled to recombinant human proteins (2.5 ug/reaction).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>IFN-α, IFN-β</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>IFN-ω</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the comparison of pHC with pCOVID-19, the classification was then repeated after the exclusion of allergic pHC, with similar results.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCOVID-19</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These analyses were completed with IBM SPSS Statistics v.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SPSS</div><div>suggested: (SPSS, RRID:SCR_002865)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">27 and GraphPad Prism version 9 [7].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Trained with Python sklearn library’s RandomForestClassifier object, using parameters: n_estimator=2000, random_state=42 for data set.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Python</div><div>suggested: (IPython, RRID:SCR_001658)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The statistical analysis of SOMAscan® results was performed using R Studio (R Core Team, 2020) [11], also using a specifically developed webtool for basic data plotting and analysis [12].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>SOMAscan®</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To assess the difference in the overall predictive importance (derived by GENIE3) of each variable with and without IVIG treatment, we summed the interaction strengths associated with each predictor-target pair for a given predictor variable in either treatment condition (steroid treatment applied in both conditions).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GENIE3</div><div>suggested: (GENIE3, RRID:SCR_000217)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting values were visualized using the Complexheatmap package in R.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Complexheatmap</div><div>suggested: (ComplexHeatmap, RRID:SCR_017270)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All sequencing data were then processed with Burrows–Wheeler Aligner (BWA) and the Genome Analysis Toolkit (GATK</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BWA</div><div>suggested: (BWA, RRID:SCR_010910)</div></div><div style="margin-bottom:8px"><div>Genome Analysis Toolkit</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>GATK</div><div>suggested: (GATK, RRID:SCR_001876)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Raw fastq files were trimmed using Trimmomatic v0.39 [15] and mapped to the hg38 human reference genome using BWA-MEM v07.17.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Trimmomatic</div><div>suggested: (Trimmomatic, RRID:SCR_011848)</div></div><div style="margin-bottom:8px"><div>BWA-MEM</div><div>suggested: (Sniffles, RRID:SCR_017619)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">PCR Duplicates were marked using Samblaster v0.1.2.5 [16] and GATK4 v4.1.9.0 was used to perform BAM recalibration, and HLA*LA [17] was used to call HLA genotypes.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Samblaster</div><div>suggested: (SAMBLASTER, RRID:SCR_000468)</div></div><div style="margin-bottom:8px"><div>GATK4</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting CDR3 sequences were collapsed and filtered to quantify the absolute abundance and frequency of each unique CDR3 region with Adaptive Biotechnologies’ pipeline [19]</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Biotechnologies’</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">10x Genomics 5’ Single cell gene expression, cell surface protein tag, TCR and BCR libraries were pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics 5’ v1.1 user guide. d) Bulk RNA sequencing and single cell sample demultiplexing: For each sample, 100,000-500,000 cells were processed in Trizol using the miRNAeasy micro kit (Qiagen, Germantown, MD) and standard RNA sequencing libraries were generated using Illumina Truseq library preparation kits.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Genomics</div><div>suggested: (UTHSCSA Genomics Core, RRID:SCR_012239)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The sequencing reads were adapter and quality trimmed and then aligned to the human genome using the splice-aware STAR aligner and SNP calls were generated using the previously published protocol [23].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>STAR</div><div>suggested: (STAR, RRID:SCR_004463)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lowly expressed genes were removed for each cell type individually using the filterByExpr function from edgeR [26]</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>edgeR</div><div>suggested: (edgeR, RRID:SCR_012802)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Differentially expressed genes were identified using the limma voom [27] workflow which models the log of the cpm (counts per million) of each gene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>limma</div><div>suggested: (LIMMA, RRID:SCR_010943)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Enriched gene sets were identified using the pre-ranked GSEA algorithm implemented in the FGSEA R package [https://www.biorxiv.org/content/10.1101/060012v3].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FGSEA</div><div>suggested: (fgsea, RRID:SCR_020938)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pseudobulk gene counts were normalized with the varianceStabilizingTransformation function from DEseq2 (doi:10.1186/s13059-014-0550-8) for the score calculation.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>DEseq2</div><div>suggested: (DESeq2, RRID:SCR_015687)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For BCR data, V(D)J sequencing contigs from 10x CellRanger output was processed using Immcantation v2.7.0 toolbox (https://immcantation.readthedocs.io/en/latest/index.html).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Immcantation</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">BCR sequence genotype inference and mutation load quantification were performed with reference to the pipeline from Mathew et al. [29] using the TIgGER package [30] and SHazaM package [28].</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>TIgGER</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>SHazaM</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Autoantibody analysis: Autoantibody analysis was performed using HuProt™ v4.0 human protein microarrays and processed by CDI Laboratories (Baltimore, MD)</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HuProt™</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Beads were washed again, resuspended in assay buffer, and analyzed on a BioPlex X200 instrument.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BioPlex</div><div>suggested: (BioPlex, RRID:SCR_016144)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cells were acquired on a BD LSRFortessa cytometer, gated on CD14+ monocytes, and analyzed with FlowJo software.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>FlowJo</div><div>suggested: (FlowJo, RRID:SCR_008520)</div></div></td></tr></table>
  Results from OddPub: Thank you for sharing your code and data.
  Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
  This study has some limitations. Only a few children with severe pCOVID-19 were included in the study, and therefore the results obtained may not apply to rare cases of life-threatening disease in children. We did not have longitudinal samples available for all MIS-C patients. However, the number of patients included in the study was sufficient to detect early and late signatures of the disease. Finally, while an increasing number of cases of pCOVID-19 due to the delta variant have been recently reported [62], almost all samples were collected prior to the emergence of this variant. Therefore, the impact of the delta variant on innate and adaptive immune responses in children with pCOVID-19 and MIS-C remains to be studied. Despite these limitations, this study has helped identify novel age-, time- and treatment-related immunopathological signatures that characterize MIS-C and pCOVID-19.
  Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT03394053</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">The Mechanistic Biology of Primary Immunodeficiency Disorder…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT03610802</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Send-In Sample Collection to Achieve Genetic and Immunologic…</td></tr></table>
  Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
  Results from JetFighter: We did not find any issues relating to colormaps.
  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.
  Thank you for including a protocol registration statement.
  Results from scite Reference Check: We found no unreliable references.
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1
1. ihicks411 29 Sep 2021
  
  in Public
  
  The FBI said it has stopped using the "Black Identity Extremist" tag and acknowledged that white supremacist violence is the biggest terrorist threat this country faces. https://trib.al/OepGw2S
  
  This article initially looked compelling and true. The name is catchy and so I assumed The Root was a community organizer for black justice but after scrolling over the check I learned it is a digital magazine platform that shares thought provoking articles from various black perspectives. I used the consensus/check for other coverage and found out there is no actual news on this and the link in the tweet shared is directly to the blog post on their page.
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www.biorxiv.org www.biorxiv.org

https://biorxiv.org/cgi/content/10.1101/2021.09.24.461616

1
1. sciscore 28 Sep 2021
  
  in SciScore
  
  SciScore for 10.1101/2021.09.24.461616: (What is this?)
  Please note, not all rigor criteria are appropriate for all manuscripts.
  Table 1: Rigor
  <table><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Ethics</td><td style="min-width:100px;border-bottom:1px solid lightgray">Field Sample Permit: Animal studies were carried out at Galveston National Laboratory at University of Texas Medical Branch at Galveston, Texas, an AAALAC accredited (November 24, 2020) and PHS OLAW approved (February 26, 2021) high-containment National Laboratory, based on a protocol approved by the Institutional Animal Care and Use Committee at UTMB at Galveston.<br>IACUC: Animal studies were carried out at Galveston National Laboratory at University of Texas Medical Branch at Galveston, Texas, an AAALAC accredited (November 24, 2020) and PHS OLAW approved (February 26, 2021) high-containment National Laboratory, based on a protocol approved by the Institutional Animal Care and Use Committee at UTMB at Galveston.<br>IRB: All animal procedures and operations were approved by the ethical committee of Wuhan Institute of Virology, Chinese Academy of Sciences.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Sex as a biological variable</td><td style="min-width:100px;border-bottom:1px solid lightgray">Rhesus macaques were randomly divided into control group, low-dose (10 mg/kg of 2G1) and high-dose (50 mg/kg of 2G1) groups with one male and one female in each.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Randomization</td><td style="min-width:100px;border-bottom:1px solid lightgray">Rhesus macaques were randomly divided into control group, low-dose (10 mg/kg of 2G1) and high-dose (50 mg/kg of 2G1) groups with one male and one female in each.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Blinding</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Power Analysis</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr><tr><td style="min-width:100px;margin-right:1em; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Cell Line Authentication</td><td style="min-width:100px;border-bottom:1px solid lightgray">not detected.</td></tr></table>
  Table 2: Resources
  <table><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Antibodies</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Biotin labeled RBD (Sino Biological), PE labeled streptavidin (ThermoFisher) and 7AAD (BD) Single memory B cells with potential SARS-CoV-2 antibody secretion were sorted out by gating 7AAD-, CD19+, CD27+, IgG+, and RBD+ using a BD Aria III cell sorter with fluorescence-activated cell sorting modules.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>CD27+ , IgG+</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The transfection efficiency was examined by flow cytometry using S1-mFc recombinant protein (Sino Biological) as primary antibody and FITC-AffiniPure Goat Anti-Mouse IgG (Jackson) as secondary antibody.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-Mouse IgG</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antigen-binding ELISA: Enzyme-linked immunosorbent assays (ELISA) were applied to study the binding ability of antibodies with SARS-CoV-2 RBDs (Sino Biological) and S trimers (AcroBiosystems).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Antigen-binding ELISA</div><div>suggested: (Ling-Chu Hung / Animal Health Research Institute, Council of Agriculture, Executive Yuan, Taiwan Cat# 12-Hung-03 m& 20 ORF3 7D3, RRID:AB_2827541)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After pipetting off the unbound antibodies, plates were washed 4 times with PBST and further incubated with 100 μL per well of goat anti-human IgG (Fc specific)-Peroxidase antibody (1: 5000 dilution, Sigma) for 1 h at 37°C.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>anti-human IgG ( Fc specific)-Peroxidase</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2 competition ELISA: For experiments involving the competitive binding of antibodies to SARS-CoV-2 RBD or S trimer, recombinant hACE2-Fc protein was first biotinylated using EZ-Link Sulfo-NHS-Biotin (ThermoFisher) as the instruction described.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Sulfo-NHS-Biotin</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Antibody-Dependent Cellular Phagocytosis (ADCP): In ADCP experiment, CD14+ monocytes (Allcells) were cultured and differentiated for 7 days to obtain macrophage cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Antibody-Dependent Cellular Phagocytosis ( ADCP)</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">After washing, a Goat Anti-human Fc HRP (Sigma) was used as secondary antibody with 1:6000 dilutions.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Anti-human Fc HRP</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Cell Lines</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, HEK-293T cells (ATCC) with 70% - 80% confluence in a 10 cm dish were co-transfected with 12 μg of plasmid pHIV-puro encoding RRE and ACE2 genes, 8 μg of plasmid psPAX2 encoding gag and pol, and 4 μg of plasmid VSV-G encoding G glycoprotein of vesicular stomatitis virus(VSVG) using Lipofectamine 3000 Reagent (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">500 μL of filtered lentivirus supernatant was added in a 24-well plate with Jurkat cells (ATCC).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Jurkat</div><div>suggested: TKG Cat# TKG 0209, RRID:CVCL_0065)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The culture medium of ACE2-293T cells was removed and then replaced by the antibody-pseudovirus mixture.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>ACE2-293T</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The mixture was then added into a 96-well plate covered with Vero cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Vero</div><div>suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This recombinant S protein was overexpressed using the HEK 293F mammalian cells (Invitrogen) at 37°C under 5% CO2 in a Multitron-Pro shaker (Infors, 130</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>HEK 293F</div><div>suggested: RRID:CVCL_6642)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Experimental Models: Organisms/Strains</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Pharmacokinetic study and toxicity test: For the pharmacokinetic study, BALB/c mice were tail intravenously injected with 2G1 (15, 30, or 60 mg/kg), or equivalent volume of PBS.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BALB/c</div><div>suggested: None</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Recombinant DNA</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">mAb preparation: Heavy chains and light chain genes were inserted separately into pcDNA3.4 and amplified in E. coli DH5α.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pcDNA3.4</div><div>suggested: RRID:Addgene_131198)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">In brief, HEK-293T cells (ATCC) with 70% - 80% confluence in a 10 cm dish were co-transfected with 12 μg of plasmid pHIV-puro encoding RRE and ACE2 genes, 8 μg of plasmid psPAX2 encoding gag and pol, and 4 μg of plasmid VSV-G encoding G glycoprotein of vesicular stomatitis virus(VSVG) using Lipofectamine 3000 Reagent (Invitrogen).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHIV-puro</div><div>suggested: None</div></div><div style="margin-bottom:8px"><div>psPAX2</div><div>suggested: RRID:Addgene_12260)</div></div><div style="margin-bottom:8px"><div>VSV-G</div><div>suggested: RRID:Addgene_138479)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">S protein over-expression cells: The coding sequence for full-length wild-type S protein (GenBank: QHD43416.1) from Met1 to Thr1273 was inserted into plasmid pHIV-puro1.0, followed by an internal ribosome entry site (IRES) and puromycin resistance gene.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pHIV-puro1.0</div><div>suggested: None</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Expression and purification of S protein: The prefusion S extracellular domain (1-1208 a.a) (Genbank ID: QHD43416.1) was cloned into the pCAG vector (Invitrogen) with six proline substitutions at residues 817, 892, 899, 942, 986 and 98739, a “GSAS” substitution (instead of “RRAR”) at residues 682 to 685 and a C-terminal T4 fibritin trimerization motif followed by one Flag tag.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>pCAG</div><div>suggested: RRID:Addgene_74288)</div></div></td></tr><tr><th style="min-width:100px;text-align:center; padding-top:4px;" colspan="2">Software and Algorithms</th></tr><tr><td style="min-width:100px;text=align:center">Sentences</td><td style="min-width:100px;text-align:center">Resources</td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The resulting bulk transfected population was sorted on a BD FACSJazz Cell Sorter (BD) with the BD FACS™ Sortware.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>BD FACS™</div><div>suggested: (BD FACS Aria II, RRID:SCR_018091)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Data were analyzed with GraphPad Prism Version 9.0.0 and EC50 values were determined using a four-parameter nonlinear regression.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>GraphPad Prism</div><div>suggested: (GraphPad Prism, RRID:SCR_002798)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">SARS-CoV-2 RBD (Sino Biological), S trimer (AcroBiosystems), mutated RBDs (Sino Biological), and mutated S trimers (AcroBiosystems) were coated onto High Binding ELISA 96-Well Plate (BEAVER).</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>AcroBiosystems</div><div>suggested: (ACRObiosystems, RRID:SCR_012550)</div></div></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The defocus values were estimated with Gctf43. Particles for S in complex with 2G1 were automatically picked using Relion 3.0.644–47 from manually selected micrographs.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>Relion</div><div>suggested: (RELION, RRID:SCR_016274)</div></div></td></tr></table>
  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 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 17. 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.
  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.
  <footer>
  About SciScore
  SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
  </footer>
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biorxiv.org/cgi/content/10.1101/2021.09.24.461616
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AustP/Gitso: Gitso is a frontend to reverse VNC connections. It is meant to be a simple two-step process that connects one person to another's screen.

1
1. TylerRick 27 Sep 2021
  
  in Public
  
  Disclaimer I really have no desire to maintain this project, as it's not mine to begin with. I was looking for something like Gitso but it didn't quite have what I wanted. After making my changes I thought I might as well put this up on GitHub for others who wanted something similar. So if you have issues, you're better off forking the project and fixing them yourself.
  
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Reply to the reviewers

Referee #2

Evidence, reproducibility and clarity

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